CRYOPRESERVATION STRATEGY FOR ADIPOSE TISSUE...

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CRYOPRESERVATION STRATEGY FOR ADIPOSE TISSUE AND ADIPOSE TISSUE DERIVED STEM CELLS BY DEVELOPING NON- TOXIC FREEZING SOLUTIONS Dissertation submitted in partial fulfillment of the degree of Doctor of Philosophy in Biotechnology and Medical Engineering by Sirsendu Sekhar Ray (Roll Number: 510BM408) based on research carried out under the supervision of Prof. (Mrs.) Krishna Pramanik And Prof. Sunil Kumar Sarangi June, 2016 Department of Biotechnology and Medical Engineering National Institute of Technology Rourkela

Transcript of CRYOPRESERVATION STRATEGY FOR ADIPOSE TISSUE...

CRYOPRESERVATION STRATEGY FOR ADIPOSE TISSUE AND

ADIPOSE TISSUE DERIVED STEM CELLS BY DEVELOPING NON-

TOXIC FREEZING SOLUTIONS

Dissertation submitted

in partial fulfillment of the degree of

Doctor of Philosophy

in

Biotechnology and Medical Engineering

by

Sirsendu Sekhar Ray

(Roll Number: 510BM408)

based on research carried out

under the supervision of

Prof. (Mrs.) Krishna Pramanik

And

Prof. Sunil Kumar Sarangi

June, 2016

Department of Biotechnology and Medical Engineering National Institute of Technology Rourkela

Dedicated to

My Parents

Declaration of Originality

I, Sirsendu Sekhar Ray, Roll Number -510BM408 hereby declare that this dissertation entitled

''Cryopreservation strategy for adipose tissue and adipose tissue-derived stem cells by developing

non-toxic freezing solutions” represents my original work carried out as a doctoral student of NIT

Rourkela.To the best of my knowledge, it contains no material previously published or written by

another person, nor any material presented for the award of any other degree or diploma of NIT

Rourkela or any other institution. Any contribution made to this research by others, with whom I

have worked at NIT Rourkela or elsewhere, is explicitly acknowledged in the dissertation. Works

of other authors cited in this dissertation have been duly acknowledged under the section

''Bibliography''. I have also submitted my original research records to the scrutiny committee for

evaluation of my dissertation.

I am fully aware that in the case of any non-compliance detected in future, the Senate of NIT

Rourkela may withdraw the degree awarded to me based on the present dissertation.

June 30, 2016 Sirsendu Sekhar Ray

NIT Rourkela

Acknowledgements

I avail this opportunity to express my indebtedness, deep gratitude and sincere thanks to my

advisors Prof. Krishna Pramanik and Prof. Sunil Kumar Sarangi. I appreciate all their

contributions of guidance, ideas, and funding to make my Ph.D. experience productive and

stimulating. Under their expert guidance, I successfully overcame many difficulties and learned a

lot. Without them, this thesis would not have been materialized. I can only say proper thanks to

them through my future work.

I would like to extend a special thanks to Dr. Ajit Samal, Dr. Nirved Jain and Dr. Manoj Khanna

for their constant support and help to complete this work. I express my sincere thanks to Prof. M.

K. Gupta, Head, Biotechnology & Medical Engineering Department and members of Doctoral

Scrutiny Committee (DSC) Prof. R.K.Sahoo, Prof. S. Das , Prof. A. Biswas, Prof. S. Bhutia and

all the faculty member of Biotechnology & Medical Engineering Department for their suggestions

and constructive criticism during the preparation of the thesis.

My special thanks to my collegues Prof.Kunal Pal , Prof.Indranil Bannerjee and Prof. Suprotim

Giri for their motivation and support. I take this opportunity to thank Rahman, Shahensha, Sagar,

Nimal, Krishan, Somaraju, Joseph, Narendra, Priyanka, Rik, Sweta, Abinaya, Bhism and

Akalabya for their help and constant support towards the completion of the thesis work. I would

like to express my gratitude to my research group and students, whom I guided for their B.Tech

and M.Tech thesis completion. I also would like to thank Department of Biotechnology and

Department of Science and Technology, Govt.of India for funding the projects on

cryopreservation of stem cells.

At last, words are not enough to say thank you to my Baba, Ma, Dada, Didi, Boudi, and

Dabababu for their patience, support and endurance towards the completion of the thesis. My

special thanks also to my Father in laws and Mother in laws for their motivation. I greatly

indebted to my wife, Sonali for her everlasting support.

Abstract The present research focuses on the development of a Me2SO and serum-free non-toxic freezing

solution from natural cryoprotective agents (CPAs) in a suitable carrier media for preserving

adipose tissue and adipose tissue-derived stem cells (hADSCs) with long shelve-life. The

efficiency of the various hydrocolloids and organic osmolytes as CPAs and individual PBS

components such as NaCl, Na2HPO4, KCl and KH2PO4 were evaluated to select the potential

CPAs and carrier media. Among these, trehalose and NaCl were found to be the most efficient

extracellular CPAs and carrier media. The freezing solution comprising of 160mM NaCl/90mM

trehalose achieved adipose tissue viability of 84%. The efficiency of the freezing solution (89%

viability) was increased by the addition of curcumin as antioxidant. The cryopreservation

efficiency was further improved by optimizing the control freezing parameters, which resulted in

93% cell viability. Thus, the formulated freezing solution comprising of 160mM NaCl/90mM

trehalose/1mg/ml curcumin has been proven to have the ability in maintaining the viability for a

long time and provides the isolated hADSCs from cryopreserved adipose tissue with desired

proliferation and differentiation potential.

An effort has also been given to isolate hADSCs from adipose tissue and develop a

cryopreservation strategy for their preservation by formulating an effective freezing solution

similar to adipose tissue. A cell viability of 81% was achieved with 10%PVP/0.9%NaCl/60mM

ectoin. The cryopreservation efficacy of the formulated freezing solution was optimized by

following Taguchi orthogonal design method thereby optimal composition of the freezing solution

was obtained as 160 mM NaCl/10% PVP/90mM ectoin/100µg/ml catalase, providing the post-

thaw viability of 85%. The viability was further enhanced to 89% in control rate freezing with the

optimized condition at -1°C/min. The freezing solution has the ability to maintain cell viability,

proliferation and differentiation capability of hADSCs for long storage time.

Thus, it has been demonstrated that the formulated serum-free and non-toxic freezing solutions

comprising of 160mM NaCl/90mM trehalose/1mg/ml curcumin and 160 mM NaCl/10%

PVP/90mM ectoin/100µg/ml catalase are effective for long-term preservation of adipose tissue

and hADSCs respectively. These solutions may provide effective cryopreservation strategy for the

supply of these products for clinical application in future.

Keywords: Cryopreservation; Adipose tissue; Adipose tissue derived stem cells; Non-toxic

freezing solution.

List of Contents

Certificate of Examination I

Supervisor’s Certificate II

Declaration of Originality IV

Acknowledgement V

Abstract VI

Contents VIII

List of figures XII

List of tables’ XVII

List of abbreviations XVII

Chapter 1 Introduction 1-10

1.1 Background and significance of study 2

1.2 Cryopreservation of cells and tissues 3

1.2.1 Principle of cryopreservation 3

1.2.2 History of cryopreservation 3

1.3 Important factors involved in cryopreservation 4

1.3.1 Ice formation in a cell suspension 4

1.3.2 Rate of cooling 4

1.3.3 Rate of thawing 5

1.3.4 Ion transport 5

1.3.5 Generation of free radicals 5

1.3.6 Cytoskeleton and cell membrane changes 5

1.3.7 Apoptosis and necrosis 6

1.4 Storage systems for cryopreservation 6

1.4.1 Mechanical freezers 6

1.4.2 Cryogenic freezers 7

1.5 Strategy of cryopreservation 7

1.5.1 Optimization of freezing solution 7

1.5.2 Optimization of thermodynamics 8

1.6 Cryoprotectant and toxicity 9

1.7 Adipose tissue 9

1.8 Importance and application of adipose tissue 9

1.8.1 Lipofilling 9

1.8.2 Production of stem cells 9

1.9 Cryopreservation of adipose tissue 9

1.10 Cryopreservation of adipose tissue derived stem cells 9

Chapter 2 Literature review 11-24

2.1 Cryopreservation of adipose tissue 12

2.1.1 Viability analysis of cryopreserved adipose tissue 14

2.1.2 Isolation of mesenchymal stem cells from cryopreserved

adipose tissue

15

2.2 Cryopreservation of mesenchymal stem cells 16

2.2.1 Effect of freezing on functionality of adipose-derived stem cells 23

Chapter 3 Scope and objectives 25-29

Chapter 4 Materials and methods 30-38

4.1 Chemicals and culture media 31

4.1.1 Cryoprotective agents 31

4.1.2 Carrier media 31

4.1.3 Antioxidants and inhibitors 31

4.1.4 Cell culture and differential media 31

4.1.5 Viability assessment 32

4.2 Cryopreservation of adipose Tissue 32

4.2.1 Collection and processing of adipose tissue 32

4.2.2 Preparation of freezing solution 32

4.2.3 Cryopreservation experiment 33

4.2.4 Adipose tissue viability assessment 33

4.2.5 Morphological characterization 35

4.2.6 Isolation of hADSCs from cryopreserved adipose tissue 35

4.3 Cryopreservation of adipose-derived stem cells 35

4.2.1 Isolation and culture of hADSCs 35

4.3.2 Immunophenotypic characterization of hADSCs 36

4.3.3 Preparation of freezing solution 36

4.3.4 Cryopreservation experiment 36

4.3.5 hADSCs viability assessment 37

4.3.6 Cytoskeleton analysis 37

4.3.7 Differentiation potential assessment 37

4.3.8 Proliferation kinetics 38

4.4 Statistical analysis 38

Chapter 4 &5 Results and discussion 39-129

Chapter 5 Cryopreservation of adipose tissue 40-97

5.1 Screening of potential cryoprotectants and carrier media towards

the formulation of freezing solution for cryopreservation of

adipose tissue

41

5.1.1 Screening of extracellular CPAs 42

5.1.2 Screening of intracellular CPAs 47

5.1.3 Screening of ionic compounds 52

5.1.4 Formulation and evaluation of freezing solution for adipose

tissue cryopreservation

58

5.2 Improvement of Adipose tissue viability by the addition of anti-

oxidants in freezing solution

63

5.2.1 Screening of antioxidants 63

5.2.2 Improvement of 160mM NaCl/90mM trehalose freezing

solution supplemented with antioxidants

68

5.3 Effect of signaling pathway inhibitors 74

5.4 Control rate freezing of adipose tissue using formulated freezing

solution

78

5.4.1 Effect of cooling rate 78

5.4.2 Effect of Seeding 82

5.5 Long-term viability of adipose tissue by controlled rate freezing

using formulated freezing solution

87

5.5.1 Morphological assessment of cryopreserved adipose tissue 90

5.5.2 Survival of stem cells in cryopreserved adipose tissue 94

Chapter 6 Cryopreservation of adipose tissue derived stem cells 98-129

6.1 Screening of potential cryoprotectants and carrier media towards

the formulation of freezing solution for cryopreservation of

adipose tissue-derived stem cells

99

6.1.1 Morphological and immunophenotypic characterization of

hADSCs

99

6.1.2 Evaluation of hydrocolloids 100

6.1.3 Evaluation of PVP in combination with PBS components as

carrier media

102

6.1.4 Improvement of the developed freezing solution by addition

of organic osmolytes

104

6.1.5 Cytoskeletal analysis 105

6.1.6 Differentiation Potential 106

6.2 Optimization of freezing solution composition to improve

cryopreservation outcome of adipose tissue-derived mesenchymal

stem cells

108

6.2.1 Formulation of Freezing solution 108

6.2.2 hADSCs viability assessment by Trypan blue dye exclusion

assay

109

6.2.3 Taguchi statistical analysis 110

6.2.4 Flow cytometry 113

6.2.5 MTT Assay 115

6.2.6 Validation of Taguchi results 116

6.3 Evaluation of signaling pathway inhibitors for cryopreservation of

hADSCs

117

6.3.1 Trypan blue dye exclusion 118

6.3.2 MTT assay 118

6.4 Optimization of controlled rate freezing parameters for

cryopreservation of hADSCs using the developed freezing solution

120

6.4.1 Effect of cooling rate 120

6.4.2 Effect of seeding temperature 122

6.5 Effect of long-term storage on cryopreserved hADSCs using the

developed freezing solution

125

6.5.1 Trypan blue dye exclusion assay 125

6.5.2 MTT assay 126

6.5.3 Morphology of cryopreserved hADSCs 126

6.5.4 Cytoskeleton distribution of cryopreserved hADSCs 127

6.5.5 Proliferation kinetics 128

6.5.6 Differentiation ability 128

Chapter 7 Summary & Conclusion 130-36

Bibliography 137

List of publications 156

CV 157

List of figures

Figure 5.1 Oil release indicating the efficiency of extracellular cryoprotectants in maintaining

viability of the cryopreserved adipose tissue. 43

Figure 5.2 Metabolic activity of the cryopreserved adipose tissue treated with trehalose

and sucrose in varying concentrations measured by XTT assay. 44

Figure 5.3 Evaluation of the efficiency of trehalose and sucrose as CPAs for the

cryopreservation of adipose tissue by measuring extracellular activity of

glycerol-3-phosphate-dehydrogeage (G3PDH) enzyme. 45

Figure 5.4 Cellular viability in terms of malondialdehyde production of cryopreserved

adipose tissues using trehalose and sucrose as CPAs was assessed by

TBARS assay. 47

Figure 5.5 Screening of intracellular cryoprotectants based on oil ratio analysis. The

higher efficiency of ectoin is evident from its lower oil release than other

CPAs. 49

Figure 5.6 Evaluation of organic osmolytes in various concentrations for

cryopreservation of adipose tissue by XTT assay. 50

Figure 5.7 Effect of ectoin and hydroxyectoin as intracellular CPAs on the

cryopreservation of adipose tissue by the assessment of G3PDH enzyme

activity. 51

Figure 5.8 Viability assessment of post thaw adipose tissues using ectoin and

hydroxyectoin as CPAs by TBARS assay. 52

Figure 5.9 Screening of ionic compounds for cryopreservation of adipose tissue by oil

ratio analysis. 54

Figure 5.10 Evaluation of selected ionic compounds for cryopreservation of adipose

tissue by XTT assay. 55

Figure 5.11 Evaluation of selected ionic compounds for cryopreservation of adipose

tissue by the extracellular G3PDH activity assay. 56

Figure 5.12 Viability of cryopreserved adipose tissue in presence of different ionic

compounds in freezing solution as carrier media was measured by TBARS

assay. 57

Figure 5.13 Post-thaw viability of cryopreserved adipose tissue in freezing solutions

formulated from trehalose and ectoin in NaCl as carrier media by oil ratio

analysis 59

Figure 5.14 Cellular viability of cryopreserved adipose tissue in freezing solutions

prepared from trehalose and ectoin in NaCl carrier media by XTT assay 60

Figure 5.15 Viability of cryopreserved adipose tissue in freezing solutions prepared from

trehalose and ectoin in NaCl carrier media assessed by G3PDH activity 61

Figure 5.16 Viability of cryopreserved adipose tissue in freezing solutions prepared from

trehalose and ectoin in NaCl carrier media measured by TBARS assay. 62

Figure 5.17 Screening of antioxidants for cryopreservation of adipose tissue measured by

oil ratio. 64

Figure 5.18 Evaluation of curcumin and carnitine for cryopreservation of adipose tissue

measured by XTT assay. 65

Figure 5.19 Performance evaluation of curcumin and carnitine for cryopreservation of

adipose tissue measured by G3PDH assay 66

Figure 5.20 Evaluation of curcumin and carnitine for cryopreservation of adipose tissue

measured by TBARS assay. 67

Figure 5.21 Oil ratio analysis indicate the viability of cryopreserved adipose tissue using

curcumin and carnitine in freezing solution. 69

Figure 5.22 The viability assessment of cryopreserved adipose tissue using curcumin and

carnitine as antioxidants in freezing solution. 70

Figure 5.23 G3PDH assay indicate the viability of cryopreserved adipose tissue using

curcumin and carnitine in freezing solution. 71

Figure 5.24 TBARS assay indicate the viability of cryopreserved adipose tissue using

curcumin and carnitine in freezing solution. 72

Figure 5.25 Effect of Rho kinase and caspases inhibitors supplemented with the most

effective 160mM NaCl/90mM trehalose/1mg/ml curcumin freezing solution

on the viability of cryopreserved adipose tissue by oil ratio. 75

Figure 5.26 XTT assay indicates the viability of cryopreserved adipose tissue using Rho

kinase and caspase inhibitors supplemented with the formulated freezing

solution. 76

Figure 5.27 G3PDH assay indicates the viability of cryopreserved adipose tissue using

Rho kinase and caspases inhibitors supplemented with freezing solution. 77

Figure 5.28 TBARS assay indicates the effect of Rho kinase and caspases inhibitors on

the viability of cryopreserved adipose tissue using

160mMNaCl/90mMtrehalose/1mg/ml curcumin freezing solution. 77

Figure 5.29 Oil ratio analyses indicates the effect of cooling rate on the cryopreservation

of adipose tissue using 160mM NaCl/90mM trehalose/1mg/ml curcumin

freezing solution 79

Figure 5.30 XTT assay showing the effect of cooling rate on the viability pattern of

cryopreserved adipose tissue using 160mM NaCl/90mM trehalose/1mg/ml

curcumin freezing solution. 80

Figure 5.31 G3PDH assay indicates the effect of cooling rate on the cryopreservation of

adipose tissue using 160mM NaCl/ 90mM trehalose/1mg/ml curcumin

freezing solution. 81

Figure 5.32 Effect of cooling rates on the post thaw viability adipose tissue

cryopreserved in 160mM NaCl/90mM trehalose/1mg/ml curcumin freezing

solution assessed by TBARS assay. 82

Figure 5.33 Measured oil ratio showing the effect of seeding temperature on the

cryopreservation of adipose tissue using 160mM NaCl/90mM

trehalose/1mg/ml curcumin freezing solution. 83

Figure 5.34 Effect of seeding temperature on the cryopreservation of adipose tissue using

160mM NaCl/90mM trehalose/1mg/ml curcumin freezing solution evaluated

by XTT measurement. 84

Figure 5.35 G3PDH assay showing the effect of seeding temperature on the post thaw

viability of adipose tissue cryopreserved in 160mM NaCl/90mM

trehalose/1mg/ml curcumin freezing solution. 85

Figure 5.36 The viability pattern for post thaw adipose tissue evaluated by TBARS assay

indicating -7°C as the seeding temperature that provided maximum tissue

viability with producing minimum lipid peroxidation products 86

Figure 5.37 Measured oil ratio showing the effect of 160mM NaCl/90mM

trehalose/1mg/ml curcumin freezing solution on long-term viability of

cryopreserved adipose tissue 88

Figure 5.38 Assessment of long-term viability of cryopreserved adipose tissue measured

by XTT assay. 88

Figure 5.39 G3PDH assay shows the efficiency of 160mM NaCl/90mM

trehalose/1mg/ml curcumin freezing solution on long-term viability of

cryopreserved adipose tissue. 89

Figure 5.40 TBARS assay shows the viability pattern of adipose tissue cryopreserved in

the developed 160mM NaCl/90mM trehalose/1mg/ml curcumin freezing

solution during the 90 days period of storage 90

Figure 5.41 CLSM images of fresh and post thaw cryopreserved (90 days) adipose tissue

90

Figure 5.42 CLSM images showing the structural integrity of fresh (a) and cryopreserved

adipose tissue of 90 days storage (b) using PI as fluorescence dye. 91

Figure 5.43 CLSM of fresh (a) and cryopreserved (90 days) adipose tissue (b) using PI

indicating dead endothelial cells. 92

Figure 5.44 CLSM images of adipose tissue using curcumin in PBS (a) and curcumin in

2.5% Me2SO. 93

Figure 5.45 Shows the structural and functional integrity of SDs treated (a), fresh (b) and

cryopreserved (90 days) adipose tissue (c) using 0.1% curcumin/ 2.5%

Me2SO. 94

Figure 5.46 Phase contrast microscopic images shows gradual change of round to

fibroblastic cell morphology of hADSCs isolated from fresh (a) and

cryopreserved (90 days) adipose tissue (b) observed upon culture in DMEM

containing 10% FBS. 95

Figure 5.47 Proliferation kinetics of hADSCs isolated from fresh and cryopreserved

adipose tissue of 3months storage. 96

Figure 4.48 Assessment of differentiation potential of hADSCs isolated from fresh (a, b)

and cryopreserved (90 days storage) adipose tissue (c, d). 97

Figure 6.1 Flowcytometric analysis showing the expression of positive CD90 (99%),

CD73 (89%), and CD105 (98%) markers and negative HLA-DR (0.5%),

CD34 (1.2%) and CD45 (2%) markers representing the cells are of

mesenchymal stem cells in characteristics 100

Figure 6.2 Effect of types of hydrocolloids and their concentrations on the viability of

cryopreserved hADSCs assessed by Trypan blue assay. 101

Figure 6.3 Effect of selected hydrocolloids on the viability of cryopreserved hADSCs

assessed by MTT assay. 102

Figure 6.4 Effect of PBS and its individual ionic components as carrier media on the

viability of cryopreserved hADSCs in 10% PVP assessed by Trypan blue

assay (a) and MTT assay (b). 103

Figure 6.5 Effect of organic osmolytes on the viability of cryopreserved hADSCs. The

Trypan blue assay (a) and MTT assay (b) results revealed that

10%PVP/0.9%NaCl freezing supplemented with organic osmolytes improve

cell viability. 105

Figure 6.6 CLSM images of frozen hADSCs in 10%PVP/0.9%NaCl/60mM ectoin

freezing solution. 106

Figure 6.7 Osteogenic and adipogenic differentiation potentiality of hADSCs

cryopreserved in 10%PVP/0.9%NaCl/60mM ectoin freezing solution (a) and

(b). 107

Figure 6.8 Illustrates Main effect plot for mean of S/N ratios. S/N ratio increases with

increase in the concentration of NaCl (A) and ectoin (C). PVP (B) and

catalase (D) showed the highest S/N ratio at a concentration of 10% (w/v)

and 100µg/ml respectively. 111

Figure 6.9 Flow cytometry analysis of cryopreserved hADSCs using PI 114

List of tables

Table 2.1 Literature review of adipose tissue cryopreservation 15

Figure 6.10 MTT assay of cryopreserved hADSCs. 115

Figure 6.11 Trypan blue assay results showing the effect of signaling pathway inhibitors

supplemented with the formulated freezing solution 118

Figure 6.12 MTT assay showing the post thaw viability of cryopreserved hADSCs using

signaling pathway inhibitor supplemented with the developed freezing

solution. 119

Figure 6.13 Trypan blue assays showing the effect of cooling rate on post thaw viability

of hADSCs cryopreserved in 160mM NaCl/ 10%PVP/90mM

ectoin/100µg/ml catalase freezing solution. 121

Figure 6.14 Effect of cooling rate on post thaw viability of hADSCs cryopreserved in

160mM NaCl/ 10%PVP/90mM ectoin/100µg/ml catalase freezing solution

assessed by MTT assay. 122

Figure 6.15 Trypan blue assay showing the effect of seeding temperature on post thaw

viability of hADSCs cryopreserved in the formulated 160mM

NaCl/10%PVP/90mM ectoin/100µg/ml catalase freezing solution. 123

Figure 6.16 Effect of seeding temperature on post thaw viability of hADSCs

cryopreserved in 160mM NaCl/ 10%PVP/90mM ectoin/100µg/ml catalase

freezing solution assessed by MTT assay. 124

Figure 6.17 Long-term viability study on cryopreserved hADSCs using 160mM

NaCl/10% PVP/90mM ectoin/100µg/ml catalase freezing solution assessed

by Trypan blue assay. 125

Figure 6.18 Long-term viability study on cryopreserved hADSCs using 160mM

NaCl/10% PVP/90mM ectoin/100µg/ml catalase freezing solution assessed

by MTT assay. 126

Figure 6.19 Morphological changes of cryopreserved (90 days storage) hADSCs in

160mM NaCl/10% PVP/90mM ectoin/100µg/ml catalase freezing solution

as observed under phase contrast microscope. (Scale bar 100µm) 127

Figure 6.20 Cytoskeleton distribution of cryopreserved (90 days storage) hADSCs in

160mMNaCl/10% PVP/90mMectoin/100 µg/mlcatalase freezing solution.

127

Figure 6.21 Proliferation kinetics of cryopreserved (90 days storage) hADSCs in 160mM

NaCl/10% PVP/90mM ectoin/100 µg/ml catalase freezing solution. 128

Figure 6.22 Differential ability of frozen (90 days storage) hADSCs in 160mM

NaCl/10% PVP/90mM ectoin/100µg/ml catalase freezing solution.(Scale bar

100µm) 129

Table 2.2 Literature review isolation of stem cells from cryopreserved

adipose tissue

17

Table 2.3 Literature review on cryopreservation of adipose tissue derived

stem cells

18

Table 5.1 Comparison of viability using different freezing solution 86

Table 5.2 Comparison of viability between fresh and cryopreserved adipose

tissue (3months)

97

Table 6.1 Four control factors and three levels of concentration 109

Table 6.2 Formulation of freezing solutions based on Taguchi L9 (3(4))

array

109

Table 6.3 Trypan blue dye exclusion assay of post-thawed hADSCs 110

Table 6.4 Response table for mean viability of cryopreserved hADSCs 112

Table 6.5 ANOVA analysis of the four factors 112

Table 6.6 Table 6.6 Comparison of hADSCs viability cryopreserved in

various freezing solution

129

List of abbreviations

hADSCs Human adipose tissue derived stem cells

MSCs Mesenchymal stem cells

PVP Polyvinylpyrrolidone

MC Methylcellulose

TG Tragacanth gum

AG Acacia gum

DMEM Dulbecco’s Modified Eagle’s Medium

FBS Fetal bovine serum

HS Human serum

PBS Phosphate Buffered Saline

XG Xanthum gum

IN Inulin

CG Carrageenan

CMC Carboxymethylcellulose

Me2SO Dimethyl sulphoxide

FACS Flow activated cell sorter

LN2 Liquid nitrogen

DNA Deoxyribonucleic acid

E Ectoin

HE Hydroxyectoin

T Trehalose

ROS Reactive oxygen specifies

CFU Colony forming unit

Caspase Cysteinyl aspartic acid-protease

ROCK Rho kinase

Da Dalton

PE Phycoerythrin

FITC Fluorescein Isothiocyanate

RNA Ribonucleic acids

RBC Red blood cells

XTT 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-

Carboxanilide

MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide

G3PDH Glycerol 3 Phosphate dehydrogenase

PI Propidium iodide

CD Cluster differentiation

PVA Polyvinyl alcohol

EGTA ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid

IGF Insulin growth factor

BMP Bone morphogenic protein

HES Hydroxyethyl starch

CLSM Confocal laser scanning microscope

IBMX Isobutylmethylxanthine

OD Optical density

PC Pectin

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Chapter 1

Introduction

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1.1 Background and Significance of study

Lipofilling is an important surgical procedure in reconstructive and aesthetic surgery that

is used to fill up the depressed region or scar of a patient body. The procedure involves the

surgical removal of excess adipose tissue from one region and injecting or placing it back

to the desired location. Because of the resorption of the adipose tissue with time, the

procedure requires repetition for good contour build up at the desired location, which

increases the patient morbidity and decreases the comfort of both doctor and patient [1].

One of the solutions to this problem is to cryopreserve the adipose tissue at the time of the

first harvest and inject the tissue as per the requirement over the time.

Similar to the need of adipose tissue in reconstructive procedures, there is also a lot of

demand for mesenchymal stem cells (MSCs) for tissue engineering and stem cell therapy

applications. Tissue engineering and stem cells based therapy have the potential to

alleviate the suffering of millions of people worldwide who are affected by various tissue

and blood-related diseases such as diabetes, Parkinson diseases, osteoarthritis, bone,

cartilage, tendon, muscle, neuronal, etc. Besides lipofilling, adipose tissue can also cater

the demand of adipose tissue-derived mesenchymal stem cells (hADSCs) for the above

purpose [2]. hADSCs are used for tissue regeneration because they differentiate into

different cell types including osteoblasts, adipocytes, chondrocytes, myoblasts, etc.

Therefore, to reduce patient morbidity during collection and for advancements towards

clinical applications, developing an effective cryopreservation strategy is of utmost

importance for preserving both adipose tissue and hADSCs with long shelve-life.

Cryopreservation is a complex process, which often leads to cryoinjury mediated cell

death, and therefore, the successful cryopreservation requires suitable freezing solution for

specific cells and tissues [3]. Cryopreservation using a conventional freezing solution

containing dimethyl sulfoxide (Me2SO) and fetal bovine serum (FBS) has several

detrimental effects including, the acute and chronic toxicity to patients and genotoxicity to

preserved cells [4]. Furthermore, the most commonly used DMEM carrier media having

multicomponent constituents creates a more complex and unpredictable cell environment

at sub-zero temperature because of ice crystallization [5]. Hence, the development of a

Me2SO and serum-free freezing solution in a suitable carrier media for cryopreserving

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both adipose tissue and hADSCs is inevitable to meet the growing demand of successful

lipofilling operation and hADSCs for therapeutic and tissue regeneration applications.

1.2 Cryopreservation of cells and tissues

1.2.1 Principle of cryopreservation

Cryopreservation is defined as the maintenance of cells and tissues at temperatures below

–80°C and in freezing condition whereas hypothermic storage includes maintenance at

temperatures above 0°C but below 32°C [6]. Both these preservation methods are based

on the principle of low-temperature effect on the metabolism of a living system.

Metabolism which involves both anabolism and catabolism is essentially controlled by

thermal energy governed molecular mobility and activity. At lower thermal energy,

molecular motion and activity slowed down. In fact, a decrease of 10°C equals a 3%

decrease in metabolic activities [7]. By slowing down the biophysical processes and

reducing the metabolic activity, the living systems can be preserved for a very long time.

Hence, cryopreservation can preserve cells and tissues for a longer duration of time than

hypothermic preservation without alteration of their characteristics.

1.2.2 History of cryopreservation

In 1776, Spallanzani reported the maintenance of sperm motility even after low-

temperature exposure [8]. Subsequently, Mantegazza in 1866 suggested the need of sperm

cryobanks [9]. However, the real works on cryopreservation started after the discovery of

glycerol as cryoprotective agents by Polge and colleagues in 1949 [10]. James Lovelock

proposed that damage to cells occur because of osmotic stress [11]. In 1960, Peter Mazur

demonstrated the significance of solute concentration effect in cryopreservation and

proposed that slow freezing could allow sufficient time to permit water to leave the cells

and thus could avoid intracellular ice formation [12]. In 1972, the strategy for the

maintenance of embryos in freezing condition was developed and in-vitro fertilization was

adopted [13]. Routine cryopreservation of various cells and tissues are common in

laboratory setting and numbers of both profit and non-profit banking systems exist

worldwide.

4 | P a g e

1.3 Important factors involved in cryopreservation

1.3.1 Ice formation in a cell suspension

During freezing, aqueous cell suspension tends to undercool or supercool until the

crystallization of water molecules starts because of heterogeneous nucleating agents and

then ice crystals form [14]. The supercooling effect is because of reduction of freezing

point by the solutes necessary for the osmotic equilibrium of cells. Furthermore, the

intracellular solute concentration is higher than the extracellular solute concentration and

thermal conduction through lipid bilayer hinders the intracellular ice formation. Thus,

extracellular freezing usually precedes the intracellular freezing, as the intracellular

solution tends to supercool more than the extracellular solution. The ice formation process

involves the progressive growth of the ice crystals extracellularly initially and separation

of remaining unfrozen solution from frozen water. This progressive ice formation process

increases the concentration of solutes in unfrozen solution progressively resulting in

osmosis out of water from intracellular to the extracellular environment [15]. The loss of

water and consequent shrinkage induces stress to the structure and components of the cells

resulting loss of viability of cells. Furthermore, both intracellular and extracellular

hexagonal strong ice crystals also damage the structure of cells mechanically resulting

lysis of the cells [16].

1.3.2 Rate of cooling

A rate of cooling is a very important factor that affects cell survival during freezing. At the

slow cooling rate, extracellular ice formation proceeds much earlier than intracellular ice

formation and hence more solute concentration related injury occurs to cells whereas, at a

rapid cooling rate, a higher amount of intracellular ice formation occurs causing damage to

cells. Hence, an optimal cooling rate is necessary during cryopreservation [17]. Each cell

type has its characteristic optimum cooling rate, which is determined by the water

permeability of the cell and the freezing solution composition. The optimal cooling rate

usually lies between 0.3°C and 10°C per min.

Another important factor during cooling is latent heat of fusion [18]. The phase change

from liquid water to crystalline ice is an exothermic process, thus increases the localized

temperature and deregulates the ice nucleation process. Dysregulated ice nucleation

process causes injury to cells and tissues. To minimize the damage controlled nucleation

in the form of seeding is required.

5 | P a g e

When the rate of cooling is sufficiently high, liquid water, rather than crystallizes into ice,

changes into the highly viscous glassy state. The process is called vitrification [19].

Vitrification techniques were successfully applied to a variety of complex biological

materials such as gametes, kidney and liver for effective cryopreservation.

1.3.3 Rate of thawing

Though less critical than the rate of cooling, the rate of warming also has a significant

impact on the viability of cells. During thawing, the reverse of cooling occurs in which the

extracellular ice melts leads to hypotonic solution formation [20]. Hypotonicity results in

swelling of cells and loss of viability. Although rapid warming is better for most types of

cells and tissues, slow warming was reported to increase the viability of embryos and

RBCs.

1.3.4 Ion transport

The intracellular and the extracellular ionic distribution are different. Sodium and chloride

are the most common extracellular and potassium and phosphates are most common

intracellular ions [21]. This distribution of ions is maintained by membrane pumps and

principally by Na+K+ATPase pump [22]. At low temperature, when the pump activity

decreases, cells swell because of the inward drive of extracellular water resulted in

colloid-osmotic lysis of the cells. Furthermore, the functionality of ions pumps is affected

by temperature changes leading to ionic imbalances in low temperature. Moreover, ionic

imbalances of hydrogen ions cause pH to fall intracellularly resulted in alteration of

molecular structure [23].

1.3.5 Generation of free radicals

Metabolic activity in the respiratory chain of mitochondria is one of the last events to

cease at cryotemperature. Furthermore, ionic imbalances resulted in an increase in

intracellular calcium concentration, which activates the free radical generation [24]. The

generated free radicals remained unchecked because of cold denaturation of free radical

scavenging enzymes such as superoxide dismutase and catalase [25]. This unrestricted

formation of free radicals causes damage to a variety of cellular components such as lipid

membranes, proteins, DNA and RNA.

1.3.6 Cytoskeleton and cell membrane changes

Solute concentration dependent shrinkage during cooling and hypotonicity-induced

swelling of cells impart stress to the skeleton of cells maintained by microtubular systems.

6 | P a g e

Furthermore, cold denaturation is also known to depolymerize the microtubules causing

additional damage to the cytoskeleton [26]. The normal cytoskeleton is required for

maintaining the structural and functional integrity of cells.

During cooling, phospholipids of cell membrane undergo an abrupt change from a

disordered fluid to a highly ordered hexagonal lattice [27]. However, as cholesterol,

another lipid of the cell membrane is still in fluid phase, phase separation of the membrane

occurs, with redistribution of membrane proteins from the organized membrane to the

disorganized liquid phase of the membrane. The consequence of the resulting packing

fault induces an alteration in membrane permeability resulting in imbalances in ionic

distribution and damage to cells.

1.3.7 Apoptosis and necrosis

Necrosis and apoptosis are two distinct ways in which cells may die. Necrosis is caused by

the general failure of cellular homeostatic regulation following injury induced by a variety

of deleterious stimuli whereas apoptosis is programmed cell death, which is a regulated

process distinguishable from necrosis by numerous morphological biochemical and

physiological features. Cryoinjury can lead to both necrosis and apoptosis-mediated cell

death [28].

1.4 Storage systems for cryopreservation

Storage below -120°C is advisable to stop the metabolic activity completely and avoid the

recrystallization-induced damage in the temperature range of -80°C to -100°C. For this

purpose, two types of storage containers are usually used: Mechanical freezers and

cryogenic freezers.

1.4.1 Mechanical freezers

Mechanical freezers use a recirculating refrigerant via heat exchanger coils that exchanges

heat from air circulating within the freezer to reduce the temperature [29]. Mechanical

freezers usually operate at a temperature range of -20°C to -80°C.Although such storage

containers can be used for a short period, it has few advantages. Mechanical freezers

provide more uniform temperature distribution from top-to-bottom; pose fewer

contamination risks to samples and less risk to workers.

7 | P a g e

1.4.2 Cryogenic freezers

Cryogenic freezers usually cool the temperature of storage container through direct

application of LN2, which has a temperature of -196°C [30]. At this temperature, samples

can be preserved for years as it provides a wider safety zone of -120°C. However, because

of the risk of contamination, most of the biobanks prefer storing the samples at vapor

phase of LN2, which provide a temperature of -150°C, although the margin of safety is

compromised.

1.5 Strategy of cryopreservation

1.5.1 Optimization of freezing solution

Carrier media

Carrier media is usually composed of various ionic solutes and buffers to provide optimal

osmotic equilibrium of cells and tissues for cryopreservation. The role of carrier media

becomes important in sub-zero temperature [31]. The improper composition may lead to

ionic disequilibrium and loss of viability of cryopreserved cells and tissues.

Cryoprotectants

Cryoprotectants are chemically diverse additive compounds to carrier media that are able

to protect cells against the stresses of freezing and thawing. They are usually highly

soluble in water and have low or no toxicity to the tissues and cells. Usually,

cryoprotectants protect the frozen cells by one or more of the following mechanisms a)

suppressing high salt concentrations b) reducing cell shrinkage at a given temperature d)

reducing the fraction of the solution frozen at a given temperature e) minimizing

intracellular ice formation [32]. Cryoprotectants are divided broadly into two types based

on whether they permeate cells; membrane-permeating or low molecular weight such as

glycerol, dimethyl sulfoxide (Me2SO) and membrane non-permeating such as sucrose and

polyvinylpyrrolidone (PVP). Cell membrane permeating or intracellular cryoprotectants

are low molecular weight compounds that are more effective in minimizing cell damage in

slowly frozen biological systems whereas non-membrane permeating, or extracellular

cryoprotectants are high molecular weight compounds that are usually more effective at

protecting biological systems cooled at rapid rates [33]. Some of these non-permeating

cryoprotective agents such as trehalose have direct protective effects on the cell

membrane. Combinations of cryoprotectants may result in additive or synergistic

enhancement of cell survival.

8 | P a g e

Ice blockers: Antifreeze proteins act by preferential adsorption to the prism face of

internal planes of ice in such a manner, that ice crystal growth perpendicular to the prism

face is inhibited. By synthesizing this antifreeze protein certain animals and plants survive

in extremely cold climates [16].

Organic osmolytes

Organic Osmolytes are small solutes that are usually synthesized by cells to counteract the

stresses such as hyperosmolarity, anhydrobiosis, thermal and pressure stresses. They

protect the cells by acting as antioxidants (e.g. polyols, taurine), providing redox balance

(e.g. glycerol) and stabilizing the macromolecules such as proteins, DNA and RNA ( e.g.

ectoin and hydroxyectoin) [34]. Osmolytes act as a protectant of low temperature-induced

stress and increase the viability of cryopreserved cells and tissues.

Antioxidants

As already discussed, the free radicles that generate during cryoprocess may damage the

cryopreserved cells and tissues. Antioxidants counteract the free radicle mediated damage

by chelating or neutralizing those free radicles resulted in increased viability of

cryopreserved cells and tissues. Antioxidants such as Vit C, Vit E, carnitine and curcumin

have already been used as additive to freezing solutions in cryopreservation of various

cells and tissues [35].

Caspase and Rho kinase inhibitors

Cryopreservation-induced apoptosis is usually mediated by a caspase-dependent pathway.

Thus, the inhibitor of caspases such as Z-VAD-FMK is known to decrease the apoptosis

mediated loss of viability in post-thawed cells and tissues [36]. Rho kinase is mainly

involved in regulation of shape and movement of cells by acting on the cytoskeleton.

Thus, Rho kinase inhibitor such as Y-27632 improved the post-thaw viability of cells by

stabilizing the cytoskeleton [37].

1.5.2 Optimization of thermodynamics

Controlled rate freezer

Controlled rate freezer offers the widest control options for a freezing protocol. As the

controlled-rate freezer allows complex, fully controlled temperature versus time profiles to

be created, protocols can be designed that are appropriate to the cell type and

cryoprotectant concentration [38]. Slow (<0.1°C/min) to rapid (>50°C/min) cooling rate

can be achieved and controlled by the controlled rate freezer. Additional steps such as for

9 | P a g e

manual seeding can be added to the profile. With control rate freezer, it is possible to

optimize cryoprocess for various cell types.

1.6 Cryoprotectant and toxicity

Cryoprotectants are reported to have adverse effects on cells due to chemical toxicity, or

they could be a result of osmotic stress during the addition of the cryoprotectant before

freezing and during dilution of the cryoprotectant after thawing. Cryopreservation using

conventional 10% Me2SO supplemented with FBS as freezing solution is warranted with

several detrimental effects including genotoxicity of preserved cells, the acute and chronic

toxicity of patients. Hence, development of an effective freezing solution free from

Me2SOand serum is inevitable.

1.7 Adipose tissue

Adipose tissue or adipose organ is derived from the mesodermal germ layer. The main

cellular component of adipose tissue is adipocytes, which is lipid-filled. Also, other cells

like stem cells, endothelial cells, smooth muscle cells, immune cells also exist in the

tissue. White adipose tissue is the dominant form of adipose tissue in body compared to

brown adipose tissue and constitutes 15 - 20% of the mass of males and 25 - 30% in

females. Adipocytes, filled with lipid droplets, store energy for the body. Also, adipose

tissue secretes many hormones and signaling molecules and thus acts as an endocrine

gland [39].

1.8 Importance and application of adipose tissue

1.8.1 Lipofilling

Liposuction has become one of the most common procedures in plastic surgery. Most of

the time the goal is to remove excessive fat to improve the body contour. In such cases,

the aspirated fat can be transferred to various acceptor sites, e.g., to the thorax after a

breast segmentectomy or mastectomy. Furthermore, multiple reports show that autologous

fat grafting can enhance healing and improve scar quality in mature burn wounds, chronic

ulcers and skin areas affected by radiation therapy. Lipofilling can also be used for soft-

tissue augmentation in hand or facial rejuvenation when injected in volume lacking areas

[40]. However, the main drawback of this lipofilling technique remains the

unpredictability of the outcome, mainly due to resorption of injected tissue. This increases

the need for more grafting operation after initial procedures, which increases the morbidity

and expenditure of patients [41].

10 | P a g e

1.8.2 Production of stem cells

Stem cells are a population of cells possessing self-renewal, long-term viability and

multilineage potential [42]. Albeit embryonic stem cells have potent ability to differentiate

into various cells lineages, their practical use is limited due potential problems of cell

regulation and ethical considerations [43]. In contrast, MSCs are derived from the

autologous origin and neither have ethical nor immunological problems [44]. The MSCs

can be isolated from various organs such as fetal liver, umbilical cord blood, adipose

tissue, and bone marrow.

Isolation of mesenchymal stromal cells from adipose tissue has become prominent in

recent years as it is abundantly available and the frequency of obtaining MSCs is relatively

high i.e. 1 fibroblastoid like colony (CFU-F) in every 100 colonies formed. This ratio is

very high when compared to MSCs obtained from bone marrow i.e. 1 in 100000 CFU

[45]. Furthermore, the MSCs from adipose tissue can differentiate into adipogenic,

osteogenic, myogenic, neurogenic and chondrogenic lineages and these cells are

genetically stable when compared to bone marrow-derived mesenchymal stem cells [46].

Moreover, they are immunocompatible and immunosuppressant, which increases their

potential as therapeutics for a number diseases and disorders [47].

1.9 Cryopreservation of adipose tissue

A possible solution in addressing the problem of resorption after lipofilling is storage of

the remaining adipose aspirates after the initial liposuction. Studies have been done to

determine whether cryopreservation is a valid option to store adipocytes. Theoretically, if

resorption occurs, the previously aspirated adipose tissue could be thawed and injected.

This procedure could be performed on an outpatient basis and under local anesthesia. The

main advantage of this method would be that it eliminates the need for more liposuction

procedures under general anesthesia and, consequently, that it reduces hospitalization

time, costs and operative or anesthetic risks.

1.10 Cryopreservation of adipose tissue-derived stem cells

The advancement in mesenchymal stem cell-based therapeutics calls for an effective

cryopreservation strategy for preservation of human adipose-derived stem cells with long

selve-life. Cryopreserved hADSCs will not only cater the emergent and elective

requirement of hADSCs but also will provide clinically relevant cell numbers for

therapeutic purposes.

11 | P a g e

Chapter 2

Literature review

12 | P a g e

2.1 Cryopreservation of adipose tissue

With increased awareness of aesthetic appeal, lipofilling has grown in popularity as

dermal fillers and increasingly being used in reconstructive and aesthetic surgery.

However, because of lipoatrophy in lipofilling, the procedure often needs to be repeated

increasing the morbidity of the patient. Cryopreservation of left over adipose tissue at the

time of first lipofilling surgery emerged as a possible solution to improve the patient

compliance [48]. However, cryopreservation of cells and tissues often requires

optimization of cryoprocess parameters in the presence of suitable freezing solution to

prevent low temperature induced damage to cells and tissues [49].

Among the cryoprocess parameters, storage temperature, cooling and thawing rate are

important factors that influence the viability of cryopreserved adipose tissue. To observe

the significance of storage temperature on the viability of cryopreserved adipose tissue,

Lidagoster MI et al., in 2000 implanted the adipose tissue in mice model after preservation

at -16°C and 1°C. They observed the signs of inflammation and higher loss of tissue

viability in the grafted cryopreserved adipose tissue compared to fresh adipose tissue that

indicates that storage temperature has a significant influence on the cryopreservation of

adipose tissue [50]. Subsequently, Wolter TP et al. compared the viability of

cryopreserved fats stored at -20°C and -80°C for different duration. They concluded that -

80°C is a preferable temperature than -20°C [51]. However, Erdim M et al. demonstrated

that 4°C is enough to maintain the viability of stored fats for 2 weeks without any need of

freezing solution [52]. In contrasts, Son D et al. in 2010 showed that lipoaspirates retain

inadequate viability, if stored at -15°C and -70°C [53]. Therefore, the consensus is that

storage temperature has significance influence on the viability of cryopreserved adipose

tissue and to prevent ice-related damage at sub-zero temperature, it is preferable to store

the tissue at less than -70°C.

To find out the suitable cooling rate for adipose tissue cryopreservation, Pu LLQ et al. in

2005 demonstrated that slow cooling is preferable than fast cooling. They first cooled

down the samples to -30°C with a constant decline of 1°C/min and then they held it at that

temperature for 10min and transferred it to -196°C (Liquid nitrogen) [54]. Therefore,

keeping in view that the not much work has been done to find out optimal cooling rate and

the importance of rate of cooling in maintaining the viability of cryopreserved tissue,

further experiments need to be performed to find out the ideal rate of cooling and optimize

the cryopreservation process for adipose tissue. Similar to cooling rate, the thawing rate of

13 | P a g e

cryopreserved adipose tissue is another important factor that controls viability of post-

thawed tissue. Hwang SM et al. compared the viability of adipose tissue in different

thawing condition such as natural thawing at 25℃ for 15min; natural thawing at 25C for

5min, followed by rapid thawing at 37℃ in a water bath for 5min; and rapid thawing at

37℃ for 10min in a water bath [55]. They demonstrated that rapid thawing at 37°C for

10min in a water bath was ideal for the thawing the cryopreserved adipose tissue.

A freezing solution is usually composed of cryoprotectants that protect the cell membrane

and macromolecules of cells and tissues from ice-induced damage. To find out the

significance of cryoprotectant in the cryopreservation of adipose tissue, in 2004, MacRae

JW et al. preserved adipose tissue with and without cryoprotectants. They concluded that

at -196°C, the samples preserved with cryoprotectants showed increased viability of

adipose tissue than the samples preserved without the cryoprotectants proving the

necessity of cryoprotectant in the cryopreservation of adipose tissue [56]. Subsequently,

Wolter TP et al. evaluated various cryoprotectants such as polyvinylpyrrolidone, glycerol,

dextran and hydroxyethyl starch and stored the tissue at -20°C and -80°C. They revealed

that storing at -20°C lead to the death of adipose tissue with or without cryoprotectants;

however cryoprotectants provided slight protection of adipose tissue at -80°C resulting in

improved viability outcome [51]. To find out most effective cryoprotectant for adipose

tissue cryopreservation, Moscatello DK et al. compared the effect of Me2SO with PVP and

glycerol and demonstrated that Me2SO is a better cryoprotectant for adipose tissue than

other cryoprotectants [57]. Later on, Pu LLQ et al. in a series of experiments showed that

among the polyols, trehalose is a better cryoprotectant than other polyols and upon

combination with Me2SO showed the improved viability of cryopreserved adipose tissue

[58-65]. The adipose tissue cryopreserved with Me2SO (0.5M), and trehalose (0.2M)

maintained volume, weight and fatty tissue structure of injected free grafts more than the

tissues cryopreserved without the cryoprotectants. However, another experimental study

by the same group suggested that 0.35M trehalose is the optimal concentration for

cryopreservation of adipose tissue. 0.35M trehalose provided similar viability outcome of

free fat grafts injected in nude mice compared to free fat grafts preserved with

Me2SO(0.5M) and trehalose (0.2M). Thus, 0.35M trehalose can replace the toxic Me2SO

containing the freezing solution for cryopreservation of adipose tissue. To correlate the

effect of harvesting technique with effects of cryoprotectants, they harvested fats with

Coleman technique and cryopreserved that with Me2SO (0.5M) and trehalose (0.2M).

14 | P a g e

They observed that the fat retained normal histology albeit with a lower concentration of

glycerol 3 phosphate dehydrogenase enzyme. However, Li BW et al. when compared the

fat preserved with normal saline and preserved with hydroxyethyl starch at -20, -80 and -

196 °C, found no differences in cell viability once injected into nude mice [66].

Thus, it can be concluded that cryoprotectants protect the adipose tissue from the ice

related injury during cryopreservation. However, there are studies, which pointed

otherwise. Most of the studies establish Me2SO and trehalose as preferred cryoprotectants.

However, ideal concentrations are not conclusive with reports of different concentrations

of trehalose as optimal concentration, and further research and investigation would be

preferable. Furthermore, the role of normal saline and other buffers as carrier medium is

also controversial needing more research on that topic.

2.1.1 Viability analysis of cryopreserved adipose tissue

Because of the complexity of adipose tissue, viability measurement becomes complex.

There is no consensus on the reliable and accurate methods to measure the viability of

adipocytes in adipose tissue and different methods were used in different experimental

studies to measure the viability of adipose tissues. In 2005, Lei H et al. demonstrated that

glucose transportation test could be used to measure the viability of adipose tissue along

with histology examination [67]. Suga H et al. suggested that single tests might not be

sufficient to measure the viability of adipose tissue rather a combination of glycerol 3

phosphate dehydrogenase, XTT and adipocyte staining using fluorescent dyes such as Nile

Red, Hoescht 33342 and propidium iodide (PI) may be required to predict the viability of

adipose tissue reliably [68]. Fluorescent staining, XTT and Glycerol 3 Phosphate

Dehydrogenase (G3PDH) assay provided good correlations between the number of viable

adipocytes and resulting values and G3PDH assay is a specific test for the viability of

adipocytes in adipose tissue. In addition, many other tests such as oil ratio analysis, MTT,

Trypan blue assay were also used to measure the viability of adipose tissue. Therefore, it

can be concluded that the combination of tests is required to assess the viability of adipose

tissue reliably.

15 | P a g e

Table 2.1: Literature review of adipose tissue cryopreservation

Article Carrier media/

Cryoprotectant

Cooling/Thawing

rate

Temperatur

e (°C)

Duration Conclusion

Wolter TP

[51]

DMEM/HES, Glycerol,

Dextran, PVP

1°C/min to -20C or -

80C/Rapid thawing

-20/-80 30 days Addition of cryoprotectants

improved viability

Pu LLQ

/Cui X [48,

54, 58-61,

65]

0.9% NaCl/3.3% Me2SO

0.9% NaCl/7.5%

trehalose or.9%

NaCl/3.3% Me2SO+7.5%

Trehalose

In methanol bath till -

30°C then hold 20 min

and transferred to LN2

/Rapid thawing

-196 20/30

min

0.9% NaCl/3.3% Me2SO+7.5%

Trehalose

Son D [53] Surgical fluid/NA NA/Slow thawing -15/-70 56 days Most cells died immediately

Li BW [66] 0.9% NaCl/HES -1°C/min till -20°C

and hold for 20 min

before transferring to

freezers/Rapid

thawing

-20/-80/-196 2/7 days No difference in viability between

-20°C/-80°C/-196°C; HES no

benefit compared to normal saline

Hwang SM

[55]

0.9% NaCl/ No

cryoprotectants

NA/different thawing

rate

-20 NA Rapid thawing for 10 min in a

37°C water bath is better

Lee HJ[69] 0.9% NaCl/No

cryoprotectants

NA/Rapid thawing 4 22

months

No difference between 0.9% NaCl

preserved and fresh tissue

16 | P a g e

2.1.2 Isolation of mesenchymal stem cells from cryopreserved adipose tissue

Successful strategy for cryobank of adipose tissue is not only important for the supply of

adipose tissue but also important for the supply of adipose tissue-derived mesenchymal

stem cells when the need arises. Thus, in 2006, Matsumoto D et al. evaluated the effect of

storage temperature on the retrievability of adipose- derived stem cells from cryopreserved

adipose tissue. They concluded that adipose-derived stem cell yield was significantly

reduced by preservation at room temperature for 24h and by preservation at 4°C for 2 or 3

days [70]. Furthermore, the adipose-derived stem cell yield from cryopreserved fat was

much lower than that of freshly aspirated fat when preserved at -80°C for 1 month.

However, in the same year, Pu LLQ et al. cryopreserved human adipose tissue with 0.5M

dimethyl sulfoxide and 0.2M trehalose in LN2 and retrieved 90% of stem cells from the

cryopreserved tissue [71]. However, they observed a latency of hADSCs growth with the

retrieved hADSCs after two weeks of culture. To find out the significance of

cryoprotectants on the retrievability of hADSCs from cryopreserved adipose tissue, Lee JE

et al. in 2010 cryopreserved adipose tissue in -20°C and -80°C for 1 year and concluded

that no stem cells were viable in tissue cryopreserved without cryoprotectants. However,

stem cells survived successfully for 1 year in adipose tissue stored with 10% Me2SO and

differentiated into adipocytes [69]. Contrasting the findings of Lee JE et al., Kim JB et al.

did not observe any adherent hADSCs when human adipose tissues were preserved at -

20°C and -80°C in the presence of 10% Me2SO [72]. However, they observed that stem

cells isolation is possible when the adipose tissue was stored in at -190°C (LN2) in the

presence of 10% Me2SO. They further reported that the growth rate of the stem cells is

low when the tissue was preserved in 10% Me2SO + 10% FBS compared to 10% Me2SO

+ 90% FBS. hADSCs isolated from adipose tissue preserved with 10% Me2SO + 90%

FBS exhibits similar growth pattern compared to stem cells isolated from fresh tissue. In

2013, Choudhery MS et al. demonstrated that stem cells from fresh and cryopreserved

tissues with 10% Me2SO displayed similar fibroblastic morphology [73].

Cryopreservation did not alter expression of phenotypic markers, the proliferative

potential of MSCs, the differentiation capability of MSCs. Devitt SM et al. in 2014

reported that longer cryopreservation negatively impacts initial live adipose-derived stem

cell isolation; however, this effect is neutralized with continued cell growth [74].

Furthermore, patient age does not significantly affect stem cell isolation, viability, or

growth.

17 | P a g e

Table 2.2: Literature review isolation of stem cells from cryopreserved adipose tissue

Article Carrier

media/Cryoprote

ctant

Cooling /Thawing rate Temperature

(°C)

Duration Conclusion

Pu LLQ [71] 0.9% NaCl/ 0.5M

Me2SO and 0.2M

trehalose

In methanol bath till -30°C,

then hold 20 min and

transferred to LN2

/Rapid thawing

-196 20min Good yield of hADSCs,

however, there is a latency of

cell growth

Matsumoto D

[70]

Proprietary

formulation

1°C/15 min till -80C/Rapid

thawing

25/4/-80 1 month No/low yield of hADSCs

Lee JE [69] DMEM/10%

Me2SO

NA -20C/-80 1 year No yield without Me2SO

JB Kim[72] DMEM/ 10%

Me2SO +80% HS

NA -20/-70/- 196 7 days No yield at lower

temperature; latency of

growth with 10% FBS but

not with 80% FBS

Choudhery

MS[73]

DMEM/10%

Me2SO

NA -196 1 month Good yield; no latency of

growth; retain differentiation

ability

Devitt SM[74] No

cryoprotectants

NA -70 3 years Good yield; latency of

growth initially but not with

subsequent passaging

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Table 2.3: Literature review on cryopreservation of adipose tissue derived stem cells

Article Cooling /Thawing rate Carrier

media/Cryoprotectant

Temperatu

re (°C)

Durati

on

Viability

Goh BC [87] NA DMEM/10% Me2SO

+80% FBS

-196 1

month

81% with 5x105 cells/ml

Oishi K

[88]

Rapidly cooled NA/ 10% Me2SO

-80 7 days 70% with 10% Me2SO

Liu G [89] Cooling rate o f −0.5°C /min from 4 to

−20°C. The vials were then put into a

−80°C freezer. After 24h at −80°C,

the cells were transferred into a LN2

DMEM/10% Me2SO

+20% FBS

-196 2 wks NA

Rossa AD

[90] -20°C for 30min,then at -80°C for 1 h

and transferred to LN2

NA / 4% Me2SO

+6% trehalose+90% FBS

-196 1 year 92.5% in 1 month, 84.6%

% in 6 months and 70%

after 1 year with solution 2

Thirumala S

[91]

Frozen overnight in a −80°C freezer

inside ethanol jacketed container and

transferred to LN2

DMEM/ 1% MC or 10% PVP

+ 80% of either HS or FCS

-196 2 wks 54% with 10% PVP and

DMEM a, 63% with 10%

Me2SO ,37% with MC

Thirumala S

[92]

Frozen overnight in a −80°C freezer

inside ethanol jacketed container and

transferred to LN2.

DMEM/1% MC + 10% of

either HS or FCS

-196 2 wks 84% with 2% Me2SO ;

84% with 10% Me2SO

+80% HS/FBS

Thirumala S

[93]

−80°C freezer inside ethanol jacketed

container and transferred to LN2.

DMEM/ 10% PVP + 80% of

either HS or FCS

-196 2 wks 70% with 10% PVP

Dariolli R

[94]

NA DMEM/10% Me2SO

+10% FBS

-196 1 year 90–95%

James AW

[95]

With and without Mr.Frosty overnight

and then transferred to LN2

NA/ 10% Me2SO

+90% FBS

-196 2 wks 96%

Miyamato

Y [96] -1°C/min to -80°C DMEM/10% Me2SO

+1% sericin+0.1M maltose

-80 1-4

wks

95%

Ginani F 18h at -20°C, and then, storage at - 10% Me2SO -80 30 90%

19 | P a g e

[97] 80°C +20% FBS days

Minonzio G

[98]

From 4°C to 0°C in 6min, then hold

for 15min at 0 °C. From 0°C to −2 °C

in 9min and then hold for 2min at −2

°C. From −2°C to −35°C in 25.5min

and finally from −35°C to −100°C in

13 min.

5% albumin solution in

cryobag/5% Me2SO

-196 6

month

s

89.6%

Fernandez

MLG [99]

Kept at −80°C overnight and

transferred to LN2 (−196°C) the next

day

DMEM/ 10% Me2SO

+ 80% FBS

-196 90

days

5% Me2SO

gives 75%

And all others near about

80%

Yong KW

[100-102]

24h at −80 °C, then transferred to LN2 DMEM/ 5% Me2SO

+ 20% FBS

-196 90

days

90% with 10% Me2SO

/ 90% FBS

López M

[103]

0°C and cooled to -7°C at 2°C/min.

After seeding of extracellular ice and

holding at -7°C for 10 min, the straws

were cooled to -70°C at 1°C/min and

then plunged into liquid nitrogen

DMEM in ½-cc straws /

0.1mM EGTA+ 0.25 M

trehalose+2% PVA and 5%

ficoll + 5% Me2SO

+5% EG+3mM reduced

glutathione+5mM ascorbic

acid 2-phosphate

-196 NA 90.0% and 98.7%

Irioda AC

[104]

First stage (Program 3–15 min)

reaches the temperature −30°C; in the

end of step 2 (Program 5–45 min), the

temperature is −60°C; finally, in the

last stage, step 3 (Program 9-10min),

the temperature is −110°C and then

transferred to LN2

DMEM/10% Me2SO

+ 80% of FBS

-196 20

days

74.99%

20 | P a g e

2.2 Cryopreservation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent adult stromal cells with capability of

self-renewal and differentiation to mesoderm or non-mesoderm derived tissues [75].

Because of their unique properties such as easy isolation and culture, immunotoleranancy

to allogenic transplantation and differential ability, it emerged as one of the leading

candidates for cell therapy in regenerative and tissue engineering [76]. Because of

diversity of adult stem cells, the Mesenchymal and Tissue Stem Cell Committee of the

International Society for Cellular Therapy proposed the minimal criteria to define human

MSCs as: (i) MSCs must be plastic–adherent when maintained in standard culture

conditions (ii) MSCs must express CD105, CD73 and CD 90 and lack expression of

CD45, CD34, CD14 or CD11b, CD79alpha or CD19 and HLA-DR surface molecules and

(iii) MSCs must also demonstrate tri-lineage differentiation into osteocytes, adipocytes

and chondrocytes in vitro [77].

MSCs from bone marrow, cord blood and dental pulp are cryopreservable by slow

freezing protocols utilizing Me2SO and FBS as cryoprotectants is in use by many groups

[78-82]. Many of the alternative cryoprotectant formulations have also attempted to

remove Me2SO and animal serum from the cryoprotectant solution; both to reduce cost

and to improve clinical utility through reducing toxicity and possibility of zoonotic

infections. Human serum and human serum albumin was tried as an alternative to FBS.

However, this too is costly and introduces the risk of transmission of human pathogens.

Attempts were also given to reduce or eliminate Me2SO in freezing solution by replacing

it with polyethylene glycol, polylysine, glycerol, methylcellulose and trehalose [83-85].

Therefore, these laboratory studies indicate that there is a potential for the development of

xeno-free and serum-free freezing solution for cryopreservation of MSCs.

The incidence of mesenchymal stem cells in various tissues is extremely low, ranging

from around 0.00003% of nucleated cells in cord blood to 0.001–0.01% of nucleated cells

in the marrow, though this decreases with age. However, adipose tissue has been shown to

have a higher proportion of MSCs (approximately 2% in the stromal vascular fraction)

offering an advantage over MSCs from another tissue source [86].

Choice of the storage container is important in ensuring proper thermodynamics and

volume of cell suspension for the appropriate cost-benefit ratio of cryopreservation.

Usually, for biobanking of stem cells cryovials are the most commonly used container and

21 | P a g e

straws are used for preservation of germ cells. Cryobags are mainly used for large-scale

cryopreservation. Although most of the study used cryovials for the storage of hADSCs,

Minonzio G et al. used 25ml cryobags and López M et al. preserved hADSCs in ½-cc

straws [98, 103].

The significance of carrier medium is evident from the role of extenders in the outcome of

sperm cryopreservation. Inappropriate extenders as carrier medium lead to loss of post-

thaw sperm viability and functionality. However, not much work has been carried out to

optimize the carrier medium for mesenchymal stem cell cryopreservation. Almost all the

study for cryopreservation of hADSCs used DMEM/F-12 as carrier media except in one

study where 5% albumin solution was used as carrier medium [98].

Concentrations of cells are a very important factor that influences the outcome of a

preservation strategy. Goh BC et al. in 2007 demonstrated that for cryopreservation of

adipose-derived stromal cells in a cryovial at -196°C in the presence of 10% Me2SO+80%

FBS freezing media, the optimum concentrations of cells should be 5x105 cells/ml [87].

However, the optimal concentration of cells depends on upon the type of storage

container, the stage of cells and the freezing solution composition. Therefore, the

concentrations of cells differ from study to study. Out of 16 studies mentioned, 8 studies

conducted their study with 1x106 cells/ml and 1 study used 5x105 cells/ml. Most of the

other studies used cell concentration higher than 1x106 cells/ml.

Cryoprotectants are a very important component of freezing solution, which prevents

freezing related damage to biological samples. Almost 50% of total studies, Me2SO with

or without FBS was used for the cryopreservation of hADSCs, whereas another 40%

studies were conducted to reduce or eliminate Me2SO from freezing solution. To reduce

the concentration of Me2SO, in 2009, Rossa AD et al. reported that 4% Me2SO+6%

trehalose+90% FBS is sufficient in maintaining more than 90% viability of hADSCs even

after 1 month of storage. However, the viability reduces to 70% after 1 year [90].

Furthermore, in 2014 Minonzio G et al. demonstrated that 5% Me2SOand 5% human

albumin freezing solution gave viability of 90% [98]. In an another recent study, López M

et al. optimized a freezing solution cocktail by the addition of antioxidants glutathione and

ascorbic acid, extracellular cryoprotectants polyvinyl alcohol (PVA) and ficoll, reduced

concentration of Me2SO and a calcium chelator EGTA along with trehalose [103]. The

optimized solution (0.1mM EGTA + 0.25M trehalose + 2% PVA and 5% ficoll + 5%

22 | P a g e

Me2SO + 5% EG + 3mM reduced glutathione + 5mM ascorbic acid 2-phosphate) gave

viability of more than 98%. Thirumala S at al. in a series of the study revealed that 10%

PVP, which gave viability of 70% could replace Me2SO [91, 93]. In another significant

finding of their study, 2% Me2SO gave the remarkable cryoprotective ability to preserve

hADSCs [92]. Recently, Yong KW et al. also observed that 5% Me2SO maintained a high

cell viability (75%) comparable to those preserved in standard cryomedium (10%

Me2SO + 90% FBS) [100-102]. Miyamato Y et al. reported that supplementation of 1%

sericin and 0.1M maltose with 10% Me2SO gave more than 95% viability of

cryopreserved hADSCs [96].

As with other biological samples, cooling rate during cryopreservation is an important

factor that determines the viability of post-thawed hADSCs. Except the study by Oishi K

et al. in 2008, where they cooled the samples rapidly, all other study used the slow cooling

method. Liu Q et al. kept the samples at -20°C freezer to achieve a cooling rate of

0.5°C/min and transferred it to -80°C freezer to expose the samples to -1°C/min till -80°C.

They kept the samples for 24h in -80°C freezer before transferring them to LN2. Rossa AD

et al. also followed similar cooling protocol. In all the three studies by Thirumala S et al.,

they used an ethanol-jacketed container to freeze the cells overnight in a -80°C freezer

before transferring them to LN2. In the ethanol-jacketed container, ice nucleation was

observed around −5°C, and a cooling rate of ∼1.1°C/min was achieved at a temperature of

−40°C. Subsequently, the cooling rates drop to 0.3°C/min and 0.1°C/min, before reaching

−80°C. James AW et al., Yong KW et al. and Fernandez MLG et al. also followed similar

cooling protocol with slight modifications. Minonzio G et al. and Irioda AC et al. used a

control rate freezer to achieve predictable and reliable cooling rate. Minonzio G et al.

cooled the cryobags from 4°C to 0°C in 6min (hold for 15min), from 0 °C to −2°C in 9

min (hold for 2min), from −2°C to −35°C in 25.5min and finally from −35°C to −100°C in

13min before transferring them to LN2. Irioda AC et al. took 15 min to reach -30°C, then

45min to reach -60°C and finally 10min to reach -110°C before transferring the samples to

LN2. All the study used rapid thawing rate at -37°C water bath.

23 | P a g e

2.2.1 Effect of freezing on functionality of adipose-derived stem cells

Retention of adhesion to plastics, proliferation ability, differentiation potential, surface

marker expression and chromosomal normality is essential for further application of

cryopreserved hADSCs. Out of total 16 studies mentioned, 10 studies performed

proliferation kinetics of frozen hADSCs. Out of 10 studies, 6 studies reported normal

proliferation kinetics, whereas four studies reported delay or accelerated proliferation of

frozen cells. Three studies also conducted colony forming unit assay with frozen stem

cells with one study each reported normal, decrease and increased colony formation. All

studies reported normal morphology of cryopreserved hADSCs except one by James AW

et al. The authors reported the presence of unusual morphology of the frozen cells upon

culture and proliferation. Oishi K et al., in 2008 showed that the proliferation rate of

hADSCs decreases if frozen with 10% Me2SO. However, in the same year, Liu Q et al.

demonstrated that frozen cells maintain their capability of proliferation compared to

freshly cultured cells. Again, in 2009, Rosa AD et al. reported that there is an initial delay

in the proliferation of frozen hADSCs till passage 2 and from passage 3 the growth

characteristics of frozen cells are similar to fresh cells. However, James AW et al. showed

that there is a significant impairment of cell attachment and proliferation of hADSCs

frozen with 10% Me2SO and 90% FBS. Contrary to James AW et al., in the same year,

Dariolli R et al. reported that that the growth characteristics of porcine hADSCs are not

influenced by long-term preservation with 10% Me2SO. Miyamato Y et al. reported higher

proliferation rate of hADSCs after cryopreservation with 10% Me2SO + 1% sericin +

0.1M maltose. Ginani F et al. also reported normal growth characteristics of hADSCs after

freezing with 10% Me2SO. However, Minonzio G et al. showed that frozen adipose

stromal cells maintain normal proliferation but higher the colony forming units if

preserved with 5% Me2SO and 5% human albumin solution. Contrasting with Minonzio G

et al., Irioda AC et al. demonstrated that there is a decrease in colony forming unit by

frozen cells preserved using 10% Me2SO and 80% FBS.

Out of 16 studies, 15 studies differentiated the frozen cells to osteogenic and adipogenic

lineages, out of which, 13 studies reported normal differentiation with frozen hADSCs.

Among the other two study, Goh BC et al. demonstrated that although there is a decrease

in the osteogenic differentiation, adipogenesis remains normal after storing the stem cells

in LN2 for 1 month in the presence of 10% Me2SO and 80% FBS. However, even the

decrease in osteogenesis is non-significant compared to the fresh cells. In both in-vitro and

24 | P a g e

in vivo study, James AW et al. showed that passage 1 cells frozen with 10% Me2SO +

90% FBS in LN2 for 2 weeks showed the deleterious effect on both osteogenic and

adipogenic differentiation. However, they demonstrated that IGF-1 and BMP-4 rescued

the deleterious effect on osteogenic differentiation in vivo.

Out of 16 studies, 5 studies conducted the expression of surface markers after freezing

hADSCs. Four of which reported similar surface marker expression as fresh cells,

whereas, in one study by Irioda AC et al. there was a significant reduction in expression of

CD49d expression in cells frozen with 10% Me2SO and 80% FBS.

Five studies evaluated the effect of freezing on chromosome and gene expression. Dariolli

R et al. demonstrated that long-term cryostorage using 10% Me2SO do not influence

karyotype and senescence cues. Ginani F at al. reported no morphological changes in the

nuclei of frozen hADSCs (nuclear fragmentation or the presence of pyknotic nuclei).

Yong KW et al. observed significantly higher (p<0.05) expression level in stemness

genes including OCT-4, REX-1, SOX-2 and NANOG of cryopreserved hADSCs

compared to fresh hADSCs, indicating the frozen cells have greater ability to maintain

their stemness properties. They also observed normal levels of the tumor suppressor

markers p53, p21, p16 and pRb, hTERT, telomerase activity and telomere length in frozen

cells compared to fresh cells.

25 | P a g e

Chapter 3

Scope &Objective

26 | P a g e

Cryopreservation using Me2SO as potential CPA is the most well known and efficient

method for preserving cells and tissues. However, keeping in view of the harmful effect of

this conventional freezing solution, recent research has focussed in the search of potential

non-toxic CPAs of natural origin for developing freezing solutions. Therefore, the present

dissertation work has been undertaken to explore potential natural CPAs and suitable

carrier media thereby develop a freezing solution that can be used as an effective

cryopreservation strategy for preserving much needed adipose tissue and adipose tissue

derived stem cells.

Adipose tissues harvested by liposuction are important tissue source, the large quantity of

which is required for reconstructive and aesthetic surgery for making fat grafts. However,

the absorption rate of adipose tissue grafts result in poor clinical outcome requiring

repeated harvesting procedures, which leads to not only surgical risk but also increase in

hospital expenditure. Once it is harvested, cryopreserving spare adipose tissue with high

survival rate for long time can avoid the problem of repeated harvesting. Besides fat

grafts, adipose tissue is considered as a promising source for providing mesenchymal stem

cells for various therapeutic and upcoming tissue-engineering applications. As a result,

there is a growing demand worldwide for adipose tissue and hADSCs for the various

clinical applications. For catering the great demand of adipose tissue and hADSCs, as

important tissue and cell source, the development of an appropriate strategy for preserving

such clinical products with long shelve-life without change in their desired property is of

paramount importance. Keeping in view of their actual use when need arises,

cryopreservation with Me2SO and serum in freezing medium is conventionally used.

However, the use of these components in cryopreservation soluting is harmful to the

patient by causing cell death.

In the context, developing a freezing solution, which is devoid of Me2SO and serum is one

of the key challenges as promising cryopreservation strategy for adipose tissue and

hADSCs.

Therefore, the main aim of the present dissertation work is the development of effective

cryopreservation strategies by exploring suitable non-toxic natural CPAs and carrier media

for cryopreservation of adipose tissue and hADSCs with maintained cells viability,

structural and functional ability for long-term basis.

27 | P a g e

The specific objectives of the present research work are as follows.

i. Screening of potential natural cryoprotective agents, carrier media and

antioxidants to formulate an effective freezing solution

ii. To evaluate the performance of the formulated freezing solution for preserving

adipose tissue and hADSCs

iii. To optimize the controlled rate freezing for cryopreservation of adipose tissue and

hADSCs

iv. To evaluate the long-term preservation of adipose tissue and hADSCs using the

developed freezing solutions

Scope of work

The specific scope of this present research work is as follows-

A. CRYOPRESERVATION OF ADIPOSE TISSUE

I. Screening of CPAs and carrier media

In this phase, efforts will be given to explore the potential hydrocolloids and natural

organic osmolytes as extracellular and intracellular CPAs as well as individual PBS

components such as NaCl, Na2HPO4, KCl and KH2PO4 as possible carrier media for the

formulation of freezing solution. The cryopreservation experiments with adipose tissue

will be performed to evaluate the effectiveness of the individual CPAs and individual PBS

components (ionic compounds) as carrier media. The screening of CPAs and carrier media

will be done by measuring oil ratio as a preliminary screening criteria and final selection

will be based on XTT, G3PDH and TBARS assays.

II. Formulation and evaluation of freezing solutions

Different batches of freezing solutions will be prepared by using the selected CPAs and

carrier media with varying concentrations. The freezing solutions will be tested for their

performance towards cryopreservation of adipose tissue by conducting series of

experiments in Mr. Frosty. The most effective freezing solution cocktail will be selected

based on a battery of assays as mentioned above.

III. Improvement of cryopreservation by the addition of anti-oxidant

The oxidative stress mediated cellular damage is reported to be a critical phenomenon in

low temperature storage, which is expected to be higher in case of adipose tissue than

other body tissues. The supplement of suitable antioxidants in freezing solution is reported

28 | P a g e

to improve the cryopreservation outcome of other tissues. Therefore, efforts will be given

to explore suitable antioxidants by screening method and most potential antioxidant will

be selected on the basis of their performance evaluattion by conducting cryopreservation

experiments using the formulated freezing solution supplemented with various

antioxidants. The optimal composition of freezing solution containing the most effective

antioxidant is expected to give maximum post-thaw tissue viability.

IV. Control rate freezing of Adipose Tissue using the formulated freezing solution

An effort will be given for further improvement of the cryopreservation efficiency by

performing cryopreservation of adipose tissue using a control rate freezer . The cooling

rate has a profound influence in cryopreservation of cells and tissues. Therefore, the

optimal cooling rate for achieving maximum cell viability in controlled rate freezing will

be established by performing cryopreservation experiments at varying cooling rates.

V. Assessment of long-term storage viability

The maintenance of tissue viability for a long time is the most vital aspect in

cryopreservation. Therefore, the efficiency of the formulated freezing solution towards

maintaining tissue viability will be evaluated by conducting control rate freezing

experiments with cryopreserved adipose tissue with long storage time. The structural and

functional integrity of cryopreserved adipose tissue will the assessed by morphological

characteristics as well as ability of frozen adipose tissue to provide hADSCs with retained

metabolic activity, proliferation, and differentiation ability.

B.CRYOPRESERVATION OF ADIPOSE TISSUE DERIVED STEM CELLS

I. Evaluation of hydrocolloids as CPAs

Effort will be given to isolate hADSCs from adipose tissue and develop a cryopreservation

strategy for their preservation by formulating an effective freezing solution. Similar to

adipose tissue, the efficiency of various hydrocolloids and natural organic osmolytes as

extracellular and intracellular CPAs will be evaluated for their efficiency in maintaining

viability of adipose tissue by performing cryopreservation experiments. Based on the

viability results measured by Trypan blue and MTT assays, the most effective freezing

solution achieving maximum cell viability will be selected.

29 | P a g e

II. Evaluation of PVP in combination with PBS components as carrier media

PBS as carrier media consists of NaCl, Na2HPO4, KCl and KH2PO4. The specific role of

these components as carrier media in cryopreservation of stem cells has not been explored

so far. Therefore, efforts will be given to investigate the effect of individual ionic

component on the cryopreservation outcome achieved using formulated freezing solution.

III. Improvement of cell viability by addition of organic osmolytes

An attempt will be made for further improvement of the efficiency of the formulated

freezing solution as mentioned above by the addition of ectoin and hydroxyectoin as the

potential organic osmolytes.

IV. Optimization of the freezing solution composition

The cryopreservation efficacy of the formulated freezing solution will be optimized by

following Taguchi orthogonal design methodology and thus an optimal composition of the

freezing solution providing the maximum post-thaw viability is expected to achieve.

V. Control rate freezing of hADSCs and optimization of freezing parameters

To achieve optimal cooling rate in controlled rate freezing, the cryopreservation

experiment will be carried out at different cooling rate. Further, the effect of long-term

storage on the viability of hADSCs frozen in the formulated cryopreservation solution will

be assessed to ensure the ability of the developed freezing solution in maintaining viability

for a long time. Besides viability study, the structural and functional integrity of post thaw

hADSCs will also be verified.

30 | P a g e

Chapter 4

Materials and methods

31 | P a g e

4.1 Chemicals and culture media

4.1.1 Cryoprotective agents

Various extracellular cryoprotective agents such as trehalose, mannitol, dextran, raffinose,

sucrose, arabitol, polyvinylpyrrolidone, methylcellulose, carboxymethylcellulose, inulin,

guar gum, xanthan gum, acacia gum, tragacanth gum, hydroxyethyl starch, stachyose were

purchased from Himedia Pvt. Ltd (Mumbai, India). The intracellular cryoprotectants such

as ectoin, hydroxyectoin, betaine and Me2SO was procured from Sigma Chemical Co. (St.

Louis, MO, USA).

4.1.2 Carrier media

To formulate carrier media different ionic compounds like NaCl, KCl, Na2HPO4, KH2PO4,

MgCl2 and MgSO4 used in the thesis works were purchased from Himedia Pvt Ltd

(Mumbai, India). MilliQ water was used for the preparation of carrier media and freezing

solution.

4.1.3 Antioxidants and inhibitors

Curcumin, carnitine, lipoic acid, Vit E, quercetin, gallic acid, and ellagic acid was used as

antioxidants for cryopreservation of adipose tissue and was bought from Himedia Pvt Ltd

(Mumbai, India). Catalase, which was used for cryopreservation of adipose-derived stem

cells was procured from Sigma Chemical Co. (St. Louis, MO, USA). Caspase inhibitor Z-

VAD-FMK and Rho kinase inhibitor Y-27632 were also used of Sigma Chemical Co. (St.

Louis, MO, USA).

4.1.4 Cell culture and differential media

For isolation of culture of hADSCs, collagenase type I, Dulbecco’s modified Eagle

medium (DMEM), fetal bovine serum (FBS), and 0.25% Trypsin/EDTA solution were

procured from Gibco (BRL, USA). For immunophenotypic analysis, BD FACS lysis

buffer, CD 34-PE, CD45-FITC, CD73-APC, CD90-FITC, CD105- PerCP/cy5.5 and HLA-

DR-PerCP/cy5.5 were purchased from BD pharminogen (Becton Dickenson, San Jose,

CA, USA). For cytoskeletal and nucleus staining Phalloidin-TRITC, Phalloidin-Alexa

Fluor 488, Hoechst and DAPI were procured from Invitrogen, USA. Triton X-100,

formaldehyde and bovine serum albumin (BSA) are from Himedia Pvt. Ltd (Mumbai,

India). For differentiation and staining of hADSCs, dexamethasone, β-glycerophosphate,

indomethacin insulin, Isobutylmethylxanthine (IBMX), Oil Red O and Alizarin Red were

32 | P a g e

procured from Sigma-Aldrich (St Louis, MO, USA). L-ascorbate was obtained from

Himedia Pvt. Ltd (Mumbai, India). All the tissue culture and plastic wares were from

Tarsons Product Pvt. Ltd. (Kolkata, India).

4.1.5 Viability assessment

Trypan blue, MTT, XTT, mercaptoethanol, pyridine, thiobarbituric acid (TBA) assay,

acetic acid, sodium dodecyl sulfate (SDS) and Tris-HCl was procured from Himedia Pvt.

Ltd (Mumbai, India). PMS (N-methylphenazonium methyl sulfate) and

Tetraethoxypropane was obtained from Sigma-Aldrich (St Louis, MO, USA). Propidium

iodide (PI) was purchased from Invitrogen, USA. Glycerol 3 phosphate dehydrogenase

(G3PDH) assay kit (MK426) was procured from Takara Bio Inc, Japan.

4.2 CRYOPRESERVATION OF ADIPOSE TISSUE

4.2.1 Collection and processing of adipose tissue

Adipose tissue of twelve patients was collected from Jeewan Memorial Hospital, Raipur,

Chhattisgarh and Cosmetic Surgery Clinic, Kolkata India and processed in the stem cell

laboratory of the Department of the Biotechnology & Medical Engineering of the Institute

with the approval of the Institute Ethical Committee vide No.

NITRKL/IEC/FORM/2/25/4/11/004 dated 25.04.2011. The collected adipose tissue was

washed and centrifuged with normal saline at 3000rpm for 5min. After centrifugation, the

upper layer and lower layer consisting of oils and aqueous cell debris respectively was

pipetted out and discarded. The middle layer consisting of adipose tissue was used for the

cryopreservation experiment.

4.2.2 Preparation of freezing solution

Freezing solutions containing polyols, organic osmolytes and antioxidants in various

concentrations individually were prepared in PBS for selection of the best extracellular

and intracellular cryoprotectant and antioxidants for cryopreservation of adipose tissue.

Freezing solutions containing 80mM-240mM concentration of ionic compounds was also

prepared in MilliQ water for the screening of the best carrier media. The selected

extracellular and intracellular cryoprotectant, carrier media and antioxidants were

combined in various combinations to obtain the desired Me2SO-free, serum free and

DMEM free freezing solution. The developed freezing solution was also combined with

10nM Z-VAD-FMK or 100mM Y-27632. Five ml of the prepared freezing solution in

33 | P a g e

various phases was added to 5gm of adipose tissue in a 15ml centrifuge tube for

cryopreservation.

4.2.3 Cryopreservation experiment

After equilibrating with freezing solutions at 4°C for 1h, the sealed tubes containing fat

tissues and freezing solutions were placed in -80°C freezer dipped in isopropanol

containing container. All the samples were stored for 48h before thawing. For

optimization of cryoprocess thermodynamic, controlled rate freezer (Kryo360-17, Planer

Inc, UK) was used. To find out the optimal cooling rate, samples were cryopreserved in

varying cooling rate such as 0.5°C/min, 1°C/min, 2.5°C/min, 5°C/min from 4°C to -120°C

followed by preservation in LN2 for 48h. Furthermore, seeding at -5°C, -7°C, and -9°C

was also introduced to find out the optimal seeding temperature. To evaluate the long-term

efficacy of the developed freezing solution and cryopreservation strategy, adipose tissue

was also stored for 90 days in LN2. The tissues were thawed rapidly by immersing the

tubes in a water bath at 37°C. Each batch of experiments was repeated three times.

4.2.4 Adipose tissue viability assessment

Oil ratio

After thawing the cryopreserved tissue were centrifuged at 1500rpm for 10min. Three

distinct layers including oil, tissue and aqueous part were formed. The oil ratio was

calculated by the formula reported by Matsumoto D et al. [70] as follows-

Oil ratio=Oil volume / (Oil volume+ Fat volume)

XTT assay

XTT assay shows the metabolic activity of cells and tissues. In brief, after centrifuge, the

fat was resuspended in phosphate-buffered saline to a total of 7.5ml. The XTT ((2,3-Bis-

(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) and PMS (N-

methylphenazonium methyl sulfate; electron coupling reagent) were mixed at a ratio of

50:1. 2.5ml of this resultant mixed reagent was added to the fat sample and was incubated

for four hours at 37°C. After incubation and centrifugation at 1500rpm for 10min, 1ml of

the aqueous part was transferred to quartz cuvette to measure the optical absorbance using

a spectrophotometer. The absorbance was measured at a test wavelength of 450nm and a

reference wavelength of 650nm. Percentage (%) viability was calculated as

34 | P a g e

% viability= (OD of frozen sample / OD of fresh sample) X 100

G3PDH assay ,

G3PDH enzyme activity was measured by following the protocol of G3PDH assay kit

(Takara Bio Inc; MK426). Briefly, the cryopreserved tissues were centrifuged at 1500rpm

for 10min to segregate the adipose tissue from the aqueous fraction. 200µl of aqueous

fraction was added with 200µl of dilution buffer containing 1mM mercaptoethanol to form

a sample solution. 25µl of the sample solution was added to 100µl of substrate solution

containing (NADH and dihydroxyacetone phosphate) for spectrophotometric assay. The

optical absorption at 340nm was measured for 10min and the change in optical density

(∆OD) from the linear position of the curve was obtained. The enzyme activity is

calculated by using the following formula.

G3PDH activity (Units

ml) =

∆OD340 × A(ml) × Dilution ratio of test sample

6.22 × B(ml) × C(cm)

Where,

ΔOD 340: Decrease in the absorbance at 340nm per min

A (ml): Total reaction volume

B (ml): The volume of enzyme solution (diluted sample) added

C (cm): Optical path length of the cell used

6.22: Millimolar absorption coefficient of NADH molecules

TBARS assay

The TBARS assay was performed following the protocol mentioned by Włostowski T et

al. [105]. In brief, the cryopreserved tissue samples were homogenized in a buffer solution

containing 50mM Tris-HCl (pH 7.4) and 1.15% KCl followed by centrifugation at

1500rpm for 10min. The supernatant was used for the assay. To 200µl of the tissue

homogenate, 200µl of 8.1% (w/v) SDS (sodium dodecyl sulfate), 1.5ml of 20% acetic

acid, 1.5ml of 0.8% TBA (thiobarbituric acid) and 600µl distilled water were added and

vortexed. The reaction mixture was heated at 95°C for 1h in a water bath and cooled.

Then 1ml sample was added to 5ml of butanol/ pyridine mixture (15:1 v/v) and vortexed.

After centrifugation at 14000rpm, the absorbance of the organic phase was determined at

532nm. Calibration curve was prepared using Tetraethoxypropane. The results were

expressed as TBA-reactive substances (nM/g wet wt.).

35 | P a g e

4.2.5 Morphological characterization

Morphology of fresh and cryopreserved adipose tissue after long-term storage was

assessed by confocal laser scanning microscope (CLSM; Leica Microsystem, Germany) in

both phase contrast and fluorescence mode. For fluorescence staining, PI was used.

Adipose tissue was incubated with 100ug/ml of PI for 15 min and observed under CLSM

(Ex-488nm; Em-600-650nm). Furthermore, keeping in view the fluorescence property of

curcumin, the curcumin-containing freezing solution was explored for staining the adipose

tissue. 0.1% (w/v) curcumin was dissolved in PBS and subsequently incubated with fresh

adipose tissue in room temperature for 30min. CLSM with excitation at 458nm and

emission filter at 500-520nm was set up to image the adipose tissue. Later on, 2.5%

Me2SO was added along with the curcumin to stain the adipose tissue homogenously.

Furthermore, to observe dead cell characteristics in the presence of curcumin adipose

tissue was treated with 0.25% sodium dodecyl sulfate (SDS) and stained with 0.1% (w/v)

curcumin/ 2.5%Me2SO.

4.2.6 Isolation of hADSCs from cryopreserved adipose tissue

hADSCs were isolated from cryopreserved adipose tissue and morphological

characteristics, proliferation kinetics and differentiation ability was assessed by following

protocols mentioned in the next section. The results were compared with hADSCs isolated

from fresh adipose tissue.

4.3 CRYOPRESERVATION OF ADIPOSE-DERIVED STEM CELLS

4.3.1 Isolation and culture of hADSCs

The stem cells were isolated by collagenase digestion method. In brief, 5gm adipose tissue

was washed with PBS and incubated with 1mg/ml collagenase I at 37°C for 30min. The

digested adipose tissue was centrifuged to separate out the stromal cells as a pellet. The

pellet was washed with PBS and suspended in DMEM medium containing 10% FBS. The

cells were incubated at 37°C with 5% humidified CO2.The non-adherent cells were

discarded after 24h. The expansion medium was changed twice weekly until 80%

confluence was achieved and then the cells were trypsinized and passaged further for

proliferation.

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4.3.2 Immunophenotypic characterization of hADSCs

The expression of the specific surface antigen by the cells was evaluated by flow

cytometry analysis (FACS Calibur, Becton Dickinson and Co, San Jose, CA, USA). The

cells of third passage were trypsinized and stained with human monoclonal antibodies

against CD90, CD73, CD105, CD34, CD44 and HLA-DR. In brief, the trypsinized cells

were washed with PBS and 2% FBS and kept in dark at 4°C for 1h after adding 10µl

antibodies. The cells were washed with PBS and 2% FBS before being resuspended in

PBS for flow cytometry analysis.

4.3.3 Preparation of freezing solution

Hydrocolloids in PBS were screened in different concentration for selection of the best

extracellular cryoprotectant. Subsequently, to replace PBS as carrier media, isotonic

solutions of NaCl, Na2HPO4, KCl and KH2PO4 was combined with the selected

cryoprotectant and was evaluated for its cryopreservation efficacy. Furthermore, ectoin

and hydroxyectoin in different concentration as intracellular cryoprotectant were added to

the above solutions and evaluated. The developed freezing solution containing suitable

extracellular and intracellular cryoprotectant in the selected carrier media was further

optimized and improved by adding catalase. For the development of an optimal freezing

solution, Taguchi’s orthogonal design method was applied. To this end, four factors

namely NaCl, PVP, ectoin, catalase as freezing media components and three levels of their

different concentrations were selected (Table 6.1) to prepare the freezing solutions with

varying composition (Table 6.2). To further improve the efficacy of the developed

freezing solution 10nM Z-VAD-FMK or 100mM Y-27632 was added to it. hADSCs from

different passages (P3-P6) were transferred into 1.5ml cryovials with the prepared freezing

solution at a concentration of 1x106 cells/ml for cryopreservation experiment.

4.3.4 Cryopreservation experiment

After equilibrating the hADSCs with freezing solutions for 1h at 4°C, The sealed vials

containing a freezing solution and hADSCs were placed in Mr. Frosty (Nalgene, New

York, U.S.A) and kept in -80°C freezer. After 48h, the cells were thawed in a water bath

and the post-thawed cells were diluted gradually by adding 9ml prewarmed media and

centrifuged at 3000rpm for 10min. The supernatant was discarded and the pelleted cells

were resuspended in DMEM and 20% FBS for culture. To optimize the cooling rate and

seeding temperature, controlled rate freezer was used following the methods used for

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adipose tissue cryopreservation in the previous section. Furthermore, hADSCs were also

stored in LN2 for 90 days to evaluate the long-term efficacy of the developed freezing

solution.

4.3.5 hADSCs viability assessment

Trypan blue assay

Ten µl Trypan blue (0.4 w/v %) was added to 10µl post-thawed cell suspension for cell

viability assessment. The cell viability was measured by counting cells in a

hemocytometer and the stained cells were considered dead cells.

MTT assay

Cell viability was also measured by the MTT assay. Cells (5 x 104cells /well) were

incubated at 37°C in a CO2 incubator in a 6-well plate. After 24h, 4µl MTT (500µg/ml)

per 100µl media was added and incubated for 3h at 37° C. The formazan crystals were

solubilized with Me2SO and the absorbance of the solution was recorded at 570nm.

Flow cytometry study

To assess the viability of hADSCs, the cells were labeled with PI. In brief, the cells were

incubated with PI (100µg/ml) for 15min and then subjected to flow cytometry analysis by

using FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA). For each sample,

10,000 events were acquired from the region defined for MSCs and analyzed by BD

FACSDiva Software 6.0 (Becton Dickinson, San Jose, CA) for cell viability assessment.

4.3.6 Cytoskeleton analysis

For cytoskeleton analysis, 24h cultured hADSCs were fixed with 2% paraformaldehyde in

PBS at room temperature for 5min. After that, the cells were treated with 2% BSA and 1%

glycine and were permeabilized with 0.5% Triton X-100 for 15min. For actin staining and

nuclear staining, samples were incubated with TRITC-Phalloidin/Alexa-fluoro 488-

Phalloidin and DAPI/Hoechst respectively at 37°C for 30min in dark and samples were

observed under CLSM.

4.3.7 Differentiation potential assessment

The fresh and cryopreserved cells were plated in a 6-well plate and cultured using DMEM

in 20% FBS supplemented with differentiation media. The osteogenic differentiation

media consists of 10mM β-glycerophosphate, 100nM dexamethasone and 0.2mM L-

ascorbate and adipogenic differentiation media comprises of 50µm indomethacin, 300nM

insulin, 100nM dexamethasone, and 500µM IBMX. The osteogenic and adipogenic

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potential was measured by Alizarin red on the 21st day and Oil Red O staining on the 14th

day.

4.3.8 Proliferation kinetics

5×103 cells/well were seeded into 6-well plates with 2ml of growth medium and incubated

at 37ºC and 5% CO2. After trypsinization, the cell number counted from day 1 to 9 day.

The number of viable cells was counted using automated cell counter with cells stained

with 0.4% Trypan blue.

4.4 Statistical analysis

The experimental data were analyzed statistically using IBM SPSS Statistics 20.0

software. The results are expressed as mean ± standard error. The following equation was

used for the S/N ratio calculation during optimization of hADSCs cryopreservation by

Taguchi’s method

𝑆

𝑁= −10 ∗ log (𝛴 (

1

𝑌2)/𝑛

Where Y is the measured data and n is the number of data

In addition to S/N ratio, response table of mean and statistical analysis of variance

(ANOVA) was employed to determine the effect of parameters on hADSCs viability.

39 | P a g e

Chapter 5 - 6

Results &Discussion

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Chapter 5

Cryopreservation of adipose tissue

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5.1 Screening of potential cryoprotectants and carrier media towards the

formulation of freezing solution for cryopreservation of adipose tissue

Autologous fat tissue usually harvested by liposuction is used in reconstructive and

aesthetic surgery due to its abundant availability, relatively easy harvesting and

biocompatibility of fat tissue [106]. However, the implanted fat tissue is not persistent due

to its high absorption rate that results in frequent graft failure clinically, thereby, demands

additional grafts leading to repeated harvesting of fat or adipose tissue, which increases

cost and patient morbidity [107-110]. Preserving additional tissue at sub-zero temperature,

keeping in view of its supply as additional grafts is an effective technique to avoid

repeated harvesting. Besides tissue graft, there is a growing demand of hADSCs that can

be obtained from adipose tissue as a potential source for the tissue engineering and

therapeutic applications [111-117].

Successful cryopreservation strategy requires a suitable freezing solution comprising of

CPAs and carrier media. The use of conventional cryoprotectant such as Me2SO is

harmful due to its toxic effects [118, 119]. Although there are reports of using

extracellular CPAs, the efficacy of non-toxic intracellular CPAs in cryopreservation of

adipose tissue has not been reported till date. Furthermore, evaluation of organic

osmolytes such as ectoin and hydroxyectoin as intracellular CPAs can be of much

interesting [120].

Keeping the above in view, the present study focuses on the evaluation of a number of

extracellular and intracellular CPAs as well as ionic compounds as possible carrier media

towards the development of a freezing solution for preservation of adipose tissue.

Furthermore, an attempt was also given to formulate an optimized freezing solution

cocktail from the combination of the potential non-toxic extracellular and intracellular

CPAs in the selected carrier medium. The preliminary screening of CPAs and carrier

media was assessed based on oil ratio analysis, and the potential components were finally

selected through the further assessment such as XTT, G3PDH and TBARS assays. The

result and discussion on the various experimental results generated from the above study

are presented in this chapter.

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5.1.1 Screening of extracellular CPAs

Oil ratio

The efficiency of the various extracellular CPAs such as sucrose, trehalose as sugars and

PVP, dextran and HES as hydrocolloids, in PBS carrier medium was evaluated by

measuring oil ratio as an indicator of damaged or degenerated adipocytes. The experiment

was also performed with different concentration of CPA solutions in the range of 30-

150mM with the aim of finding optimal concentration of CPAs for cryopreservation of

adipose tissue.

The oil ratio in Fig 5.1 indicates that among the extracellular CPAs, trehalose shows

higher viability by providing the least damaged adipocytes in cryopreserved tissue than

other CPAs used in the study. Besides the type of CPAs, variation in the viability of

frozen adipose tissue was also observed with varied concentration of CPAs. Though not

much statistically significant change in viability was shown with varied concentration,

trehalose at 90mM concentration showed least oil ratio (0.047 ± 0.002) indicating the

highest viability at this concentration, followed by sucrose (30mM, 0.069 ± 0.010) and

mannitol (30mM, 0.077 ± 0.015).

The superior performance of trehalose may be due its excellent membrane stabilizing

property and trehalose also retains hydrophilic shell around proteins and nucleic acids,

thereby prevents cryoinjury mediated cell damage [121-123]. the protective role of

trehalose in adipose tissue cryopreservation was also reported by Pu LLQ et al, though a

higher concentration (300mM) of trehalose was demonstrated as the optimal concentration

for cryopreservation of adipose tissue [124].

Among the hydrocolloids, PVP and dextran, which are established cryoprotectants for

mesenchymal and hematopoietic stem cells, provided less cell viability as shown by

higher oil ratio [81, 125, 126]. Similarly, HES, which was reported as an efficient CPA for

RBCs, was not favorable to adipose tissue preservation [127-130]. The similar finding

with HES was also reported elsewhere [66]. The raffinose families of oligosaccharides

(RFOs) such as raffinose and stachyose, which are alpha-galactosyl derivatives of sucrose,

has shown good cryoprotectant ability for mouse sperms, but these were found to be

inefficient in the present study [131]. Although arabitol has shown promise in

cryopreservation of rat embryo, the tissue viability obtained with arabitol was lower to the

viability obtained with trehalose and sucrose [132].

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Therefore, trehalose and sucrose were selected for further study by XTT, G3PDH and

TBARS assay.

Figure 5.1: Oil release indicating the efficiency of extracellular cryoprotectants in

maintaining viability of the cryopreserved adipose tissue. Among the CPAs, trehalose is the

most efficient achieving maximum cell vitality as shown by the least oil release. A substantial

viability is also shown by sucrose. PBS was used as carrier media.

XTT Assay

XTT assay used as functional assay has an advantage over commonly used MTT assay

due to the solubility of XTT cleavage products in water [133]. The absorbance of cleavage

products in XTT assay correlates well with the metabolic activity of adipose tissues with

higher absorbance that represents higher metabolic activity and thus higher viability of

adipose tissue.

As observed from XTT results (Fig 5.2), trehalose has shown superior metabolic activity

achieving higher adipose tissue viability to sucrose as CPA irrespective of level of

concentrations. Compared with respect to the tested CPA concentration, XTT cleavage

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product was increasing with increase in concentration up to 90mM and then a declining

trend was observed with trehalose though the variation was not statistically significant

between 30mM to 120mM. Whereas metabolic activity was noticed more or less same,

when adipose tissue was cryopreserved using sucrose as CPA. Among different

concentrations of sucrose, 60mM sucrose (69.4 ± 5.8%) achieved slightly higher viability

than other concentrations of sucrose. However, the difference between 30mM and 60mM

sucrose is not significant (p<0.05) as also revealed by oil ratio analysis. Therefore,

trehalose with 90mM concentration has been proven to be superior CPA than sucrose.

Figure 5.2: Metabolic activity of the cryopreserved adipose tissue treated with

trehalose and sucrose in varying concentrations measured by XTT assay. Trehalose

has shown superior metabolic activity achieving higher adipose tissue viability than

sucrose at all concentration levels. However, among the concentration range,

trehalose performed the best at 90mM concentration

G3PDH assay

G3PDH assay involving adipose specific enzyme is a relatively simple method chosen in

this study to assess the cellular function of the frozen adipose tissue. The cell membrane

integrity is detected by this assay through the marking of diffusion of intracellular

elements through the damaged adipose tissue membrane. The extracellular G3PDH level

was measured and a higher G3PDH level represents lower tissue viability [134].

45 | P a g e

The analysis of G3PDH (Fig 5.3) level measured in the washing solution of cryopreserved

adipose tissue shows that a higher level of enzyme activity was observed when sucrose

was used as CPA at all concentrations than the level of enzyme activity shown by

trehalose. Furthermore, among the different concentration of trehalose level tested,

trehalose at 90mM was found to be more effective than either lower or higher

concentration providing higher membrane integrity and thus higher cellular function (2.25

± 0.10 U/ml) of cryopreserved adipose tissue. Whereas a gradual increase in G3PDH

enzyme level with an increase in CPA concentration was depicted by sucrose when used

as CPA, though the difference in enzyme activity is not statistically significant. Unlike

trehalose, the enzyme level representing the adipose tissue viability obtained with sucrose

at 30mM concentration (2.57 ± 0.26 U/ml) is found to be best among other concentrations.

Figure 5.3: Evaluation of the efficiency of trehalose and sucrose as CPAs for the

cryopreservation of adipose tissue by measuring the extracellular activity of glycerol-

3-phosphate-dehydrogeage (G3PDH) enzyme. A comparatively lower enzyme

activity representing the higher tissue viability was shown by trehalose than sucrose.

90mM was shown to be the most favorable concentration for trehalose giving

maximum adipose tissue viability

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TBARS assay

TBARS assay is one of the most commonly used tests for assessing the lipid peroxidation

of cells and tissues [135]. Free radical mediated cryo-injury is one of the well-established

models of cells and tissue damage in cryopreservation [136]. Free radicals formed in this

process participate in a cascade of reaction with lipid of the cells and tissues and

peroxidize them to form lipid peroxidation products or malondialdehyde. Therefore, it was

hypothesized that TBARS assay might be a useful test to quantify malondialdehyde in

post-thawed tissue, thereby would provide an indirect indication of the viability of the

adipose tissue.

The malondialdehyde level measured by TBARS assay (Fig 5.4) was also observed to be

least with 90mM trehalose (321 ± 7 nM/g wet wt) representing the superiority of the CPA

over other CPAs. Although, in the concentration range of 30mM to 150mM, the difference

between malondialdehyde production in trehalose and sucrose cryopreserved adipose

tissue is statistically insignificant (p<0.05), similar to G3PDH assay the trend suggests

trehalose is a superior cryoprotectant for adipose tissue cryopreservation than sucrose.

Among the various concentrations of sucrose, 30mM sucrose gave least TBARS

production (345 ± 15 nM/g wet wt) indicating the superiority of 30mM sucrose over other

concentrations of it. The malondialdehyde level of fresh adipose tissue was found to be

142 ± 11 nM/g wet wt. of the tissue.Thus,there was also significant increase in the level of

malondialdehyde in the cryopreserved tissues than the fresh tissues indicating the

significanceof free radical mediated damage in cryopreserved tissues.

47 | P a g e

Figure 5.4: Cellular viability in terms of malondialdehyde production of

cryopreserved adipose tissues using trehalose and sucrose as CPAs was assessed by

TBARS assay. The results indicate that 90mM trehalose is superior to sucrose for

cryopreservation of adipose tissue.

Overall, all the results taken together suggest that trehalose is a better cryoprotectant than

sucrose for adipose tissue cryopreservation with 90mM trehalose as the best concentration

level achieving the highest viability. In the case of sucrose, we could not get uniform

results in all the assays; however, the trends suggest a lower concentration of sucrose,i.e.,

30mM is better than its higher concentrations. Thus, 90mM trehalose was selected as

extracellular CPA for the preparation of freezing solution cocktail for adipose

cryopreservation.

5.1.2 Screening of intracellular CPAs

Organic osmolytes such as betaine, ectoin and hydroxyectoin are known to protect cells

and tissues from osmotic and thermal stresses [137]. Ectoin has also reported being an

excellent intracellular cryoprotectant for embryo and others [138]. Thus, like extracellular

CPAs, organic osmolytes in PBS solution was also evaluated as intracellular CPAs for

48 | P a g e

their efficacy in cryopreservation of adipose tissue through the analysis of oil ratio, XTT,

G3PDH and TBARS assays, which are described below.

Oil ratio

The analysis of the results of oil ratios (Fig 5.5) indicate betaine showed a higher oil ratio

than the other intracellular CPAs representing the lower viability of adipose tissue when

cryopreserved in betaine. The overall viability trend follows

ectoin>hydroxyectoin>betaine. The higher efficiency of ectoin has also been reported

earlier when other tissues cryopreserved in freezing solution containing ectoin [139-143].

A substantial tissue viability was also obtained using hydroxyectoin as CPA. Furthermore,

no significant statistical change in oil ratio value was observed between the concentration

levels of both ectoin and hydroxyectoin in the range of 90mM to 150mM. Therefore,

90mM was selected as the most favorable concentration of ectoin and hydroxyectoin.

However, there was a statistically significant difference in tissue damage between the

organo-osmolites and the conventional 10% Me2SO as CPA, which is evident from

decreased oil ratio value (0.042 ± 0.006) measured with the later. Therefore, ectoin and

hydroxyectoin were selected as the potential organo-osmolites for further study and 10%

Me2SO as a control.

49 | P a g e

Figure 5.5: Screening of intracellular cryoprotectants based on oil ratio analysis. The

higher efficiency of ectoin is evident from its lower oil release than other CPAs. The

overall viability trend follows ectoin>hydroxyectoin>betaine. No statistically

significant change in oil ratio was observed between the concentration levels for both

ectoin and hydroxyectoin the range of 90-150mM. Therefore, 90mM was selected as

the most favorable concentration of ectoin and hydroxyectoin. The viability was

lower than 10% Me2SO used as a control.

XTT assay

The results in Fig 5.6 indicate that the control 10% Me2SO (80.2 ± 3.2%) gave higher

viability than organic osmolytes (p<0.05). However, with an increase in the concentrations

of organic osmolytes, the viability of the post-thawed adipose tissues increased. In the

concentration range of 90mM-150mM, 150mM ectoin (69.8 ± 3%) achieved the highest

viability of tissues, which is slightly higher than 150mM hydroxyectoin (66.7 ± 4.2%).

However, the difference between the adipose tissues cryopreserved using ectoin and

hydroxyectoin is statistically insignificant (p<0.05). Similar to oil ratio, XTT assay

depicted the superiority of the conventional but harmful 10% Me2SO over the organic

osmolytes.

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Figure 5.6: Evaluation of organic osmolytes in various concentrations for

cryopreservation of adipose tissue by XTT assay. 10% Me2SO was used as a control.

G3PDH assay

The assessment of cell membrane integrity via the measurement of G3PDH level (Fig 5.7)

shows a slightly lower enzyme activity thereby a higher tissue viability was obtained with

ectoin than hydroxyectoin. Thus, it indicates that the cryopreservation of adipose tissue

was superior using ectoin than hydroxyectoin, though a substantial tissue preservation was

also shown by later. In comparison, the viability results are slightly lower than those

obtained with conventional 10% Me2SO. The corresponding G3PDH values are 2.68 ±

0.15 U/ml (150mM ectoin), 2.96 ± 0.12 U/ml (150mM hydroxyectoin) and 2.28 ± 0.07

U/ml (10% Me2SO). Furthermore, it was also analyzed that the protective efficacy

increased with increase in the concentration of organic osmolytes and overall, 150mM

ectoin gave significantly (p<0.05) higher viability than 150mM hydroxyectoin.

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Figure 5.7: Effect of ectoin and hydroxyectoin as intracellular CPAs on the

cryopreservation of adipose tissue by the assessment of G3PDH enzyme activity.

Ectoin than hydroxyectoin showed a slightly higher viability. Both CPAs show lower

efficiency 10% Me2SO.

TBARS assay

Fig 5.8 depicts the TBARS analysis data, which reveals that that 150mM ectoin (361 ± 11

nM/g wet wt) gave significantly lower lipid peroxidation products than 150mM

hydroxyectoin (397 ± 5 nM/g wet wt) indicating the higher viability achieved with ectoin

than hydroxyectoin. However, the lipid peroxidation with ectoin and hydroxyectoin were

significantly higher than Me2SO (285 ± 19 nM/g wet wt). This represents the lower

efficacy of the organic osmolytes than 10% Me2SO.

The results can be explained by the fact that ectoin possess less number of hydroxyl

groups and thus is lesser water-soluble than hydroxyectoin; hence, the distribution of

ectoin within the lipid-filled adipocytes may be much higher than hydroxyectoin [144].

Thus, 150mM ectoin and 10% Me2SO were selected as intracellular CPAs for formulating

the freezing solution cocktail for the freezing solution cocktail.

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Figure 5.8: Viability assessment of post-thaw adipose tissues using ectoin and

hydroxyectoin as CPAs by TBARS assay. The viability obtained with ectoin is

slightly higher than hydroxyectoin and 150mM is the most favorable concentration

at which ectoin performed better. However, unlike extracellular CPAs, the

conventional 10% Me2SO (control) is superior to organic osmolytes.

5.1.3 Screening of ionic compounds

Not enough studies were conducted to understand the role of carrier medium in

cryopreservation of cells and tissues. However, the significance of ionic carrier medium is

evident from the literature related to cryopreservation of sperms where extenders

comprised of various ions such NaCl, KCl, NaHCO3,MgCl2 etc. are usually used to

preserve the functionality of cryopreserved sperm [145] . Evidence proving the

significance of ionic solutes in preservation of organs and tissues is apparent from the

literature of low temperature storage of organs for organ transplantation [146, 147].

Earlier developed Euro-Collins and University of Wisconsin preservation solutions are

intracellular type solution having higher potassium ions than sodium ions. Both these

solutions were observed to offer better transplantation outcome when used for

preservation of kidney and liver. However, relatively newer developed solutions like

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Celsor, IGL-1 (Institute Georges Lopez) and ET-K (Kyoto University solution) that have

higher sodium concentration than potassium were observed to give better protection to

most of the organs including hearts, vessels and pancreas. Therefore, the effect of various

types of ionic compounds such as NaCl, Na2HPO4, KCl, KH2PO4, MgSO4 and MgCl2

towards the cryopreservation of adipose tissue in varying concentration was investigated

in the range of 80mM-240mM concentration. PBS was used as a control. The evaluation

results are described here.

Oil ratio

As described earlier, the damaged adipocytes in adipose tissue cryopreserved in different

ionic components measured by oil ratio is depicted in Fig 5.9. The extent of oil release

thereby representing the level of damaged tissue was different with different ionic

compounds and the trend was observed as NaCl<Na2HPO4<KCl<KH2PO4<MgCl2≈

MgSO4, though there is not much change in viability between NaCl and Na2HPO4. The oil

release was also found to change with a change in concentration of carrier media. The oil

release was increased with increase in concentration from 80mM-120mM and then

decreased with further increase in the concentration of both NaCl and Na2HPO4. However,

the minimum oil release means the maximum viability of adipose tissue was obtained at

160mM. Whereas in the case of KCl, a continual decrease in oil release with an increase

in concentration was observed. Overall, among the ionic compounds used, 160mM NaCl

(0.038 ± 0.002) showed the highest viability of cryopreserved tissue whereas MgSO4 and

MgCl2 showed the lowest tissue viability. Except at 240mM concentration of ionic

compounds, the trend of viability follows NaCl>Na2HPO4>KCl in all concentrations.

Thus, it has been demonstrated that the type of ionic compounds and their concentration

significantly influence the adipose tissue viability. 160mM NaCl and 160mM Na2HPO4

showed superior viability than other ionic solution and hence these compounds were

selected for further study.

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Figure 5.9: Screening of ionic compounds for cryopreservation of adipose tissue by

oil ratio analysis. The trend of oil release was observed as

NaCl<Na2HPO4<KCl<KH2PO4<MgCl2≈ MgSO4, indicating the superiority of NaCl

over other carrier media.

XTT assay

The trends of XTT assay (Fig 5.10) showed that the maximum metabolic activity was

observed with 160mM NaCl (84.1 ± 5.2 %) followed by 160mM Na2HPO4 (78.8 ± 3.7%).

Moreover, the metabolic activity of adipose tissue cryopreserved in 160mM NaCl and

160mM Na2HPO4 is significantly higher than the PBS (65.4 ± 4.1%) used as a control.

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Figure 5.10: Evaluation of selected ionic compounds for cryopreservation of adipose

tissue by XTT assay. The maximum metabolic activity was observed with NaCl

followed by Na2HPO4 at 160mM concentration. Moreover, the metabolic activity of

adipose tissue cryopreserved in NaCl and Na2HPO4 at 160mM concentration is

significantly higher than the PBS used as a control.

G3PDH assay

The results of extracellular G3PDH activity assay (Fig 5.11) of adipose tissue viability

revealed similar trend as assessed by oil ratio and XTT assay. A lower level of enzyme

activity representing a better cellular function of adipose tissue was revealed when

cryopreserved in 160mM NaCl (1.85 ± 0.06 U/ml) than that obtained using 160mM

Na2HPO4 showing the G3PDH activity of 2.01 ± 0.17 U/ml. Although, the difference in

extracellular G3PDH level between NaCl and Na2HPO4 is insignificant (p<0.5), the

difference between the individual ionic compounds and PBS (3.07 ± 0.15 U/ml) is very

much significant (p<0.05) indicating higher protective ability of individual ionic

compounds than PBS.

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Figure 5.11: Evaluation of selected ionic compounds for cryopreservation of adipose

tissue by the extracellular G3PDH activity assay. A lower level of enzyme activity

representing a better cellular function of frozen adipose tissue was depicted when

cryopreserved in 160mM NaCl than the viability obtained using 160mM Na2HPO4

and 240mM KCl. Furthermore, the individual ionic compounds have shown higher

protective ability than PBS.

TBARS assay

The lipid peroxidation products released from adipose tissue by free radical mediated

cryoinjury was measured by TBARS assay (Fig 5.12). The presence of 160mM NaCl and

160mM Na2HPO4 lead to significantly (p<0.05) lesser malondialdehyde production in

cryopreserved adipose tissue than PBS (414 ± 13 nM/g wet wt) indicating its higher

protective efficacy for cryopreserved adipose tissue than PBS. The trend suggests that in

the presence of 160mM NaCl (271 ± 7 nM/g wet wt), the lipid peroxidation is least

followed by 160mM Na2HPO4 (320 ± 48 nM/g wet wt). Although, the variation between

the quantities of lipid peroxidation products of cryopreserved adipose tissue with ionic

compounds is insignificant (p<0.05), there is a significant difference between 160mM

NaCl and PBS.

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Figure 5.12: Viability of cryopreserved adipose tissue in the presence of different

ionic compounds in freezing solution as carrier media was measured by TBARS

assay. The results indicate 160mM NaCl as the superior carrier medium.

Considering all the assay results taken together, NaCl at 160mM concentration gave

higher viability of cryopreserved adipose tissue than other ionic components. A

comparable but slightly lower viability was also achieved when adipose tissue

cryopreserved in Na2HPO4 solution. It is interesting to note that at near isotonic

concentration, NaCl performed better whereas Na2HPO4 and KCl performed well at

hyperosmotic concentration. This result may be due to the altered distribution of ions in

adipocytes during cryopreservation of adipose tissue when NaKATPase pumps cease to

function and solute concentration effect becomes prominent [148, 149]. At this time,

sodium and chloride ions might have a predominant role in the viability of cryopreserved

adipose tissue than potassium and phosphate ions. Moreover, sodium and chloride, the

most common extracellular cation and anion, may provide better protection to cells at

isotonic concentration whereas potassium and phosphate, which are the most common

intracellular cation and anion, offer better protection at hyperosmolar concentration.

However, KH2PO4, which constitutes of only intracellular type ions, showed poor results

in all concentrations indicating the presence of either sodium or chloride as extracellular

ions are necessary to have satisfactory viability even at hyperosmolar concentration. The

prominence of ionic carrier media in maintaining the viability of cryopreserved adipose

tissue may be because of the unique features of adipocytes compared to other cells of our

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body. During cryopreservation, ice-mediated and osmotic imbalance mediated injuries are

the two major factors, which govern the fate of cryopreserved cells and tissues. While

though ice-mediated injury plays dominant mode of cryoinjury for cells filled with mainly

colloidal water, adipocytes, which mainly contain lipids, may be influenced by the

osmotic factor more dominantly than ice factor during cryopreservation [150]. However,

further investigation on the detailed mechanism is necessary to understand much complex

cryopreservation phenomenon.

Therefore, to formulate ultimate freezing solution cocktail, 160mM NaCl was selected as

the potential carrier medium.

5.1.4 Formulation and evaluation of freezing solution for adipose tissue

cryopreservation

The selected extracellular and intracellular CPAs in 160mM NaCl carrier medium was

evaluated individually as well as in combination to formulate an optimized freezing

solution for adipose tissue cryopreservation. To this end, different batches of freezing

solutions such as 160mM NaCl/90mM trehalose, 160mM NaCl/150mM ectoin, 160mM

NaCl/90mM trehalose/150mM ectoin and 160mM NaCl/10% Me2SO were formulated to

evaluate their efficiency towards adipose tissue cryopreservation.

Oil ratio

The results of oil analysis (Fig 5.13) indicate that among the prepared freezing solutions,

160mM NaCl/10% Me2SO has shown the most effective providing maximum viability of

0.032±0.004 followed by 160mM NaCl/90mM trehalose (0.035 ± 0.002) indicating both

Me2SO and trehalose containing freezing solution are efficient for the preservation of

adipose tissue. Though a slightly higher tissue viability was obtained with 10% Me2SO in

NaCl carrier media than viability achieved with 160mM NaCl/90mM trehalose, the

difference in viability is statistically insignificant. Nevertheless, both these freezing

solutions showed higher viability when compared with viability obtained with 160mM

NaCl, 160mM NaCl /150mM ectoin and 160mM NaCl/90mM trehalose/150mM ectoin.

Interestingly, the presence of ectoin in the formulated freezing solution did not offer any

additional advantage in cryopreservation of adipose tissue.

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Figure 5.13: Post-thaw viability of cryopreserved adipose tissue in freezing solutions

formulated from trehalose and ectoin in NaCl as carrier media by oil ratio analysis

(D, Me2SO; T, Trehalose; E, Ectoin). The freezing solution comprising of 160mM

NaCl/10% Me2SO has shown the most effective providing maximum viability

followed by 160mM NaCl/90mM trehalose indicating freezing solution containing

either Me2SO or trehalose are efficient for the preservation of adipose tissue.

XTT assay

Unlike oil ratio results, all the formulated freezing solutions have shown more or less

similar XTT values (Fig 5.14) representing the comparable level of adipose tissue

viability. Overall, 160mM NaCl/90mM trehalose achieved a slightly higher viability (84.8

± 4.3%) than other freezing solutions prepared from ectoin and thus proven to be the most

potential. When compared the viability was also comparable to the viability obtained

using 10% Me2SO in NaCl (85.3 ± 3.9%). Thus, 160mM NaCl/90mM trehalose is proven

to be a potential non-toxic freezing solution for adipose tissue.

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Figure 5.14: Cellular viability of cryopreserved adipose tissue in freezing solutions

prepared from trehalose and ectoin in NaCl carrier media by XTT assay (D, Me2SO;

T, Trehalose; E, Ectoin). Overall, the formulated 160mMNaCl/90mM trehalose

achieved a slightly higher viability (84.8 ± 4.9%) than other freezing solutions and

thus proven to be the most efficient freezing solution. The viability was also

comparable to the viability obtained using 10% Me2SO.

G3PDH assay

The cell membrane integrity of adipose tissue was assessed by G3PDH activity and the

experimental data is shown in Fig 5.15. The enzyme activity was shown by freezing

solution containing ectoin (2.008 ± 0.147 U/ml) as CPA is higher that represents higher

adipocyte damage than the trehalose-containing freezing solution (1.751 ±0.121 U/ml).

The freezing solution containing trehalose has also shown similar viability outcome with

cryopreserved adipose tissue.

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Figure 5.15: Viability of cryopreserved adipose tissue in freezing solutions prepared

from trehalose and ectoin in NaCl carrier media assessed by G3PDH activity (D,

Me2SO; T, Trehalose; E, Ectoin). A higher adipocyte damage in cryopreserved

adipose tissue was shown by freezing solution containing ectoin as CPA than

trehalose, representing the higher tissue viability achieved with trehalose.

TBARS assay

Fig 5.16 shows the TBARS assay results obtained with adipose tissue cryopreservation in

the formulated freezing solution as 160mM NaCl/10% Me2SO ≈ 160mM NaCl/90mM

trehalose > 160mM NaCl/150 ectoin and 160mM NaCl/90mM trehalose/150M ectoin. The

results follow the trend of viability is comparable to the values obtained with other assays.

Thus, 160mM NaCl/10% Me2SO showing TBARS value of 253 ± 15 nM/g wet wt and

160mM NaCl/90mM trehalose showing TBARS value of 256 ± 16 nM/g wet wt) are

comparable and the solutions provided superior efficacy than other freezing solution for

adipose tissue cryopreservation. Interestingly, similar to other assays, ectoin did not show

any impact as an intracellular cryoprotectant on cryopreservation outcome.

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Figure 5.16: Viability of cryopreserved adipose tissue in freezing solutions prepared

from trehalose and ectoin in NaCl carrier media measured by TBARS assay. The

results indicate 160mM NaCl/10%Me2SO and 160mM NaCl/90mM trehalose

achieved superior viability than other freezing solutions. (D, Me2SO; T, Trehalose; E,

Ectoin)

Overall, the present study demonstrated that 160mM NaCl/90mM trehalose and 160mM

NaCl/ 10% Me2SO offer better viability of cryopreserved adipose tissue compared to

160mM NaCl alone. However, 160mM NaCl alone offers significantly higher viability

compared to other ionic compounds and cryoprotectants representing the prominent role

of osmolarity and carrier media in the cryopreservation of adipose tissue. The higher

viability obtained with ectoin in PBS than PBS alone was not reflected in the viability

outcome using ectoin in NaCl, indicating the limited role of ectoin in the cryopreservation

of adipose tissue. Further study on enhancement of the protective role of the developed

freezing solution in adipose tissue cryopreservation is important to ensure its higher

viability on long-term storage.

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5.2 Improvement of adipose tissue viability by the addition of anti-oxidants in

freezing solution

The oxidative stress mediated damage has been reported to be more pronounced in

cryopreservation of adipose tissue than other tissue types as adipose tissue is usually

harvested from obese patient and adipocytes in obese are known to have a higher

concentration of free radicals compared to other body tissues [151, 152]. Furthermore,

mechanical manipulation of adipose tissue during liposuction and further downstream

processes, increase the susceptibility of the tissue towards free radical generation. Besides

ice related injury, the role of free radical mediated cryoinjury due to ROS generation is

also a critical factor that causes cell death during cryopreservation.

The addition of antioxidant as a supplement in freezing solution is reported to be a

promising strategy to overcome cryoinjury during cryopreservation of cells and tissues.

Curcumin, a phytochemical having antioxidative property increased the various cells and

tissue viability as reported earlier [153]. Besides curcumin, other antioxidants such as

carnitine and Vit E decreased cytoplasmic lipid content and increased the cytotolerance of

oocytes during vitrification [154]. L-carnitine also significantly improved epididymal

sperm motility after cryopreservation as reported elsewhere [155].

Keeping the above in view, we hypothesized that supplementation of antioxidants in

160mM NaCl/90mM trehalose freezing solution may improve the cryopreservation

outcome of adipose tissue. Therefore, the present study focusses on exploring potential

antioxidants for achieving improved viability of cryopreserved adipose tissue and

investigates their influence on the cryopreservation efficiency of the previously formulated

freezing solution for adipose tissue preservation. In this study, curcumin, carnitine, lipoic

acid, Vit E, gallic acid, ellagic acid and quercetin were chosen as antioxidants based on the

literature. Similar to the earlier section, oil ratio, XTT, G3PDH and TBARS have been

measured to assess the viability of post-thawed adipose tissue.

5.2.1 Screening of antioxidants

Oil ratio

As in the case of CPAs, the screening of potential antioxidants was done by measuring oil

release during cryopreservation (Fig 5.17). As indicated, a varying oil ratio representing

the varying level of tissue damage was found with different types of antioxidants. Among

the various antioxidants, curcumin and carnitine achieved higher tissue viability than other

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antioxidants. The tissue viability was also observed to be concentration dependent though

curcumin and carnitine showed the superior antioxidant property providing less oil release.

The viability of cryopreserved adipose tissue decreased with increase in the concentration

of antioxidants except Vit E, which showed a reversed trend of oil release. As compared,

1mg/ml curcumin (0.040 ± 0.004) achieved least oil ratio followed by 3mg/ml of carnitine

(0.041 ± 0.002). The lower oil ratio shown by 1mg/ml curcumin represents its superiority

in achieving higher tissue viability than carnitine and other antioxidants used under study.

A comparable oil ratio was also achieved with curcumin and carnitine. Both curcumin and

carnitine were studied further to compare and confirm their superiority over each other.

Figure 5.17: Screening of antioxidants for cryopreservation of adipose tissue

measured by oil ratio. Measured oil ratio was varied representing the varying level of

tissue damage with varying types of antioxidants. Among the various antioxidants,

curcumin and carnitine achieved higher tissue viability than others. Overall, 1mg/ml

curcumin (0.040 ± 0.004) achieved least oil ratio followed by 3mg/ml of carnitine

(0.041 ± 0.002)

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XTT assay

XTT assay data as shown in Fig 5.18 reveals the higher metabolic activity (84.9 ± 2.9%)

of adipose tissue was shown with curcumin and 1mg/ml is the most favorable

concentration of curcumin. The increase in concentration beyond 1mg/ml did not show

any benefit in the preservation of adipose tissue and no change in viability between

3mg/ml and 5mg/ml of curcumin. Whereas a typical trend of viability was observed when

cryopreservation of adipose tissue was performed using carnitine. The viability of adipose

tissue increased with increase in the concentration of carnitine from 1mg/ml to 3mg/ml

(82.78 ± 3.65%) and then decreased with further increase in concentration. In comparison

between curcumin and carnitine, an equivalent level of adipose tissue viability was

achieved at 1mg/ml and 3mg/ml respectively. Thus, the study has demonstrated the

superior efficiency of curcumin using comparatively lesser amount than carnitine.

Figure 5.18: Evaluation of curcumin and carnitine for cryopreservation of adipose

tissue measured by XTT assay. An equivalent level of adipose tissue viability was

achieved at 1mg/ml and 3mg/ml respectively.

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G3PDH assay

The measurement of the extracellular G3PDH enzymes released by lysed adipocytes

indicates the lower viability of cryopreserved adipose tissues with a higher concentration

of curcumin (Fig 5.19). The results show that quantity of extracellular G3PDH was lowest

with 1mg/ml curcumin (1.91 ± 0.10 U/ml) representing its superior antioxidant efficiency

in reducing ROS generated tissue damage. Furthermore, similar to XTT assay, a non-

linear tissue damage and hence tissue viability of cryopreserved adipose tissue was

observed when carnitine was used as an antioxidant. The viability of cryopreserved tissues

in 3mg/ml carnitine was the maximum, which was also comparable to the viability

achieved with 3mg/ml curcumin. Furthermore, the viability difference between carnitine

and curcumin at 5mg/ml is not statistically significant.

Figure 5.19: Performance evaluation of curcumin and carnitine for cryopreservation

of adipose tissue measured by G3PDH assay and confirm the superior antioxidant

activity shown by curcumin providing higher tissue viability at comparatively lower

concentration than carnitine.

TBARS assay

The quantity of malondialdehyde production during cryopreservation of adipose tissue

measured by TBARS assay corroborated with the findings of both XTT and G3PDH assay

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(Fig 5.20). The adipose tissue cryopreserved with 1mg/ml curcumin (270 ± 24 nM/g wet

wt) showed lesser lipid peroxidation than tissues cryopreserved with other concentrations

of curcumin and carnitine. However, the difference in the level of protection achieved

with 3mg/ml carnitine (302 ± 15 nM/g wet wt) and 1mg/ml curcumin is statistically

insignificant (p<0.05) that signifies the concentration level at which individual

antioxidants functions well in preventing tissue damage. Therefore, all the assays taken

together curcumin was proven to be the best antioxidants and 1mg/ml is the most effective

concentration.

Figure 5.20: Evaluation of curcumin and carnitine for cryopreservation of adipose

tissue measured by TBARS assay. The assay result corroborated with the findings of

both XTT and G3PDH assay revealing the higher efficiency of curcumin.

The results revealed that curcumin and carnitine were effective and ellagic acid, gallic acid

and quercetin were found to be ineffective in maintaining the viability of cryopreserved

adipose tissue. The result may be explained by the fact that antioxidants when used in

inappropriate concentration may also act as free radical or ROS generator imparting

additional damage to the tissues [156-158]. Furthermore, compared to only water (polar)

soluble antioxidants such as ellagic acid, gallic acid and quercetin, antioxidants that are

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soluble in only non-polar solvents (curcumin and Vit E) as well as both polar and non-

polar solvents (carnitine and lipoic acid) performed better for cryopreservation of adipose

tissue [159]. The result may be explained by the fact that adipocytes are filled mostly with

triglycerides, a nonpolar molecule and thus, permeation and distributions of non-polar

antioxidants within the cells performed better compared to polar antioxidants [160].

Therefore, 1mg/ml curcumin and 3mg/ml carnitine were selected for further study to

investigate their effectiveness in improving the cryopreservation of adipose tissue by

supplementing with the most efficient 160mM NaCl/90mM trehalose freezing solution.

5.2.2 Improvement of 160mM NaCl/90mM trehalose freezing solution supplemented

with antioxidants

As it has already been mentioned that antioxidants have an important role in overcoming

free radical mediated cryoinjury during cryopreservation of cells and tissues. In our

previous work, carnitine and curcumin were found to be efficient antioxidants performing

well towards cryopreservation of adipose tissue in PBS solution. Therefore, it was

hypothesized that the addition of these antioxidants may improve the cryopreservation

outcome of the 160mM NaCl and the formulated 160mM NaCl/90mM trehalose freezing

solution for adipose tissue cryopreservation. Therefore, in this phase of dissertation work,

cryopreservation experiments were carried out with these freezing solutions with the

addition of carnitine and curcumin to investigate their effect on the cryopreserved tissue

viability.

Oil ratio

Oil ratio data (Fig 5.21) indicates that the supplementation of antioxidants with 160mM

NaCl and 160mM NaCl/90mM trehalose freezing solutions significantly improved the

viability of cryopreserved adipose tissue. Although there was no significant difference

(p<0.05) among oil ratios of 160mM NaCl/1mg/ml curcumin (0.032 ± 0.004), 160mM

NaCl/3mg/ml carnitine (0.033 ± 0.002) and 160mM NaCl/90mM trehalose (0.035 ±

0.002), there was significant difference between the oil ratios obtained with 160mM

NaCl/90mM trehalose/1mg/ml curcumin (0.02 ± 0) and 160mM NaCl/90mM

trehalose/3mg/ml carnitine (0.025 ± 0.002). Overall, freezing solutions containing

curcumin achieved higher viability than carnitine supplemented freezing solutions and the

highest viability was shown by 160mM NaCl/90mM trehalose/1mg/ml curcumin

representing its superior performance than other freezing solution.

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Figure 5.21: Oil ratio analyses indicate the viability of cryopreserved adipose tissue

using curcumin and carnitine in freezing solution. 160mM NaCl was used as carrier

media. The viability obtained with 160mM NaCl/90mM trehalose/1mg/ml

curcumin(0.02 ± 0.00) and 160mM NaCl/90mM trehalose/3mg/ml carnitine (0.025 ±

0.002) freezing solutions was higher than the viability obtained with 160mM

NaCl/10% Me2SO (0.032 ± 0.004).

XTT assay

The XTT results (Fig 5.22) shows that the difference in metabolic activity achieved with

the cryopreserved adipose tissues in different freezing solutions was not found to be

statistically significant. However, adipose tissue cryopreserved in 160mM NaCl/90mM

trehalose/1mg/ml curcumin freezing solution achieved the highest metabolic activity

(89.7± 4.4%).

Interestingly, as also observed in oil ratio analysis, 160mM NaCl/1mg/ml curcumin (86.2

± 3.4%) freezing solution gave better viability than 160mM NaCl/90mM trehalose (84.8 ±

4.3%) indicating a dominant role of redox state in adipocytes in determining the viability

of cryopreserved adipose tissue. However, combined effect of trehalose and 160mM

NaCl/1mg/ml curcumin improved the viability revealing the independent and additive

cryoprotective mechanisms of curcumin and trehalose. Freezing solutions supplemented

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with carnitine provided comparable results to that obtained with 160mM NaCl/90mM

trehalose freezing solution as assessed by both oil ratio and XTT assay indicating a similar

level of efficacy of carnitine and trehalose. However, similar to curcumin-supplemented

solutions, the addition of trehalose to carnitine supplemented freezing solution further

improved the viability of cryopreserved adipose tissue.

Figure 5.22: The viability assessment of cryopreserved adipose tissue using curcumin

and carnitine as antioxidants in freezing solution. 160mM NaCl was used as carrier

media. adipose tissue cryopreserved in 160mM NaCl/90mM trehalose/1mg/ml

curcumin freezing solution achieved the highest metabolic activity (89.7 ± 4.4%).

G3PDH assay

As indicated in Fig 5.23 the extracellular G3PDH concentration was the least with the

cryopreserved adipose tissue when cryopreserved in 160mM NaCl/90mM

trehalose/1mg/ml curcumin (1.62 ± 0.07 U/ml) freezing solution. However, the result was

not statistically significantly different (p<0.05) from other freezing solution except when

the adipose tissue cryopreserved in 160mM NaCl/3mg/ml carnitine.

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Figure 5.23: G3PDH assay indicate the viability of cryopreserved adipose tissue

using curcumin and carnitine in freezing solution. 160mM NaCl was used as carrier

media. The extracellular G3PDH concentration was the least and hence the

maximum viability of post-thawed adipose tissue when cryopreserved in 160mM

NaCl/90mM trehalose/1mg/ml curcumin freezing solution.

TBARS assay

The trend in malondialdehyde production and thus the adipocyte viability in cryopreserved

adipose tissue (Fig 5.24) were similar to the viability results obtained with oil ratio, XTT

and G3PDH assay results proving the addition of antioxidants improved the viability of

cryopreserved adipose tissue. The tissue preserved in 160mM NaCl/90mM

trehalose/1mg/ml curcumin freezing solution undergoes lipid peroxidation (204 ± 11 nM/g

wet wt), which is significantly lower than the conventional 10% Me2SO freezing solution

(253 ± 15 nM/g wet wt). Although, freezing solution comprising of 160mM NaCl/90mM

trehalose/3mg/ml carnitine (219 ± 15 nM/g wet wt) performed significantly higher than

160mM NaCl/10% Me2SO, the adipose tissue viability obtained with 160mM

NaCl/3mg/ml carnitine were comparable to the viability obtained with 160mM NaCl/10%

Me2SO freezing solution. The difference between the concentration of lipid peroxidation

products with 160mM NaCl/90mM trehalose/3mg/ml carnitine and 160mM NaCl/90mM

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trehalose/1mg/ml curcumin freezing solutions was not statistically significant (p<0.05) as

also revealed by XTT and G3PDH assay.

Figure 5.24: TBARS assay indicate the viability of cryopreserved adipose tissue using

curcumin and carnitine in freezing solution. Among the freezing solutions, 160mM

NaCl/90mM trehalose/1mg/ml curcumin underwent significantly lower lipid

peroxidation (204 ± 11 nM/g wet wt) than the freezing solution containing

conventional 10% Me2SO. A comparable tissue viability using 160mM NaCl/90mM

trehalose containing carnitine at a comparatively higher concentration (3mg/ml) was

obtained.

Overall, considering the results of all assays taken into account, it was observed that

160mM NaCl/90mM trehalose/1mg/ml curcumin freezing solution performed better than

other freezing solution including Me2SO containing freezing solution. Thus, the study

provides a Me2SO free, non-toxic freezing solution that has the ability to cryopreserve

adipose tissue with maintained tissue viability. Furthermore, the presence of curcumin in

the post-thawed adipose tissue may offer the additional advantage of better wound healing

and aesthetic outcome after lipofilling operation. Besides antioxidant property, curcumin

is known to exhibit anti-inflammatory and anti-infective properties, which make it a

unique molecule for wound healing applications [161]. Curcumin treatment resulted in

increased formation of granulation tissue, neovascularization and enhanced biosynthesis of

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extracellular matrix proteins such as collagen [162]. Curcumin treated cutaneous wound

showed decreased levels of lipid peroxides (LPs) and cross-linking of collagen. Therefore,

all these factors may provide additional benefit in achieving better wound healing in the

lipofilling operation and tissue regeneration by the use of cryopreserved adipose tissue

through tissue engineering technique. Moreover, in the case of autologous fat grafting

needs to be performed at the tumor resected site, curcumin may kill the residual tumor

cells because of its anticancer property [163, 164]. Thus, the presence of curcumin at graft

site may lead to less resorption of grafts, maintaining the contour of the surgical site and

improve aesthetic outcome of the surgery. The hypothesis is supported by the fact that

antioxidants N-acetylcysteine and Coenzyme Q10, treatment resulted in improved graft

retention [165, 166].

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5.3 Effect of signaling pathway inhibitors

Cryoinjury during cryopreservation accentuates the apoptosis mediated death of

adipocytes in adipose tissue [167]. The initiation of apoptosis is possible by many

mechanisms and pathways such as intrinsic and extrinsic pathways. The intrinsic pathway

contains proteins such as second mitochondria-derived activator of caspase (SMAC) or

cytochrome c, while the extrinsic pathway starts with special TNF-receptors, also called

‘death receptors.' All pathways result in the activation of substrate specific cysteine

proteases, also called caspases that are the effectors of apoptosis [168]. Molecules that

inhibit caspases are known to inhibit apoptosis-mediated cell death [169]. Z-VAD-FMK is

a cell permeable, pan-caspase inhibitor, which prevents apoptosis-mediated cell death

during cryopreservation of cells and tissues thereby, increases the post-thaw viability

[170].

Similar to caspases, Rho/ROC kinase also plays a negative role in maintaining the

viability of adipose tissue [171]. Increased Rho/ROC kinase activity was observed in

adipose tissue of obese and has linkage to adipose hypertrophy, metabolic dysregulation

and adipose tissue inflammation. Furthermore, Rho/ROCK inhibits the cytoskeletal

reorganization and interferes with insulin pathway necessary for adipogenesis of hADSCs.

Rho/ROCK inhibitor such as Y-27632 reverses the negative effects thereby maintains a

healthy state of adipose tissue [172]. The ROCK inhibitor Y-27632 increased the viability

of various cryopreserved cells and tissue [173].

Although the role of caspase and ROCK inhibitors in increasing the viability of

cryopreserved cells and tissues are widely recognized, the molecules have not been

evaluated so far for their efficacy in cryopreservation of adipose tissue. Therefore, it was

hypothesized that the addition of Z-VAD-FMK and Y-27632 in the developed freezing

solution might improve the viability of post-thawed adipose tissue. Thus, an effort was

given to investigate the impact of Z-VAD-FMK and Y-27632 after supplementing them

with the developed freezing solution. Similar to the previous methodology, the viability of

post-thaw adipose tissue was evaluated by oil ratio, XTT, G3PDH and TBARS assays.

Oil ratio

The oil release due to damage of adipocyte present in adipose tissue was assessed by

performing cryopreservation experiment using the most effective freezing solution

160mM NaCl/90mM trehalose/1mg/ml curcumin formulated in the previous chapter

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supplementing with 10nM Z-VAD-FMK and 100mM Y-27632. The concentrations of

inhibitors were based on literature [174, 175]. The results in Fig 5.25 reveal that there is

no significant difference in oil released from post-thawed adipose tissue cryopreserved

with the most efficient 160mM NaCl/90mM trehalose/1mg/ml curcumin freezing solution

(0.0213 ± 0.0023) with and without inhibitors, though a slightly lower viability was shown

by Y-27632 (0.0227 ± 0.0023). This represents that the addition of inhibitors has

negligible influence on the tissue viability.

Figure 5.25: Effect of Rho kinase and caspases inhibitors supplemented with the

most effective 160mM NaCl/90mM trehalose/1mg/ml curcumin freezing solution on

the viability of cryopreserved adipose tissue by oil ratio. It is indicated that the

addition of inhibitors has no significant influence on the tissue viability.

XTT assay

XTT results (Fig 5.26) shows a marginal increment of adipose tissue viability by adding

inhibitors and the viability showed by both inhibitors is comparable. The corresponding

viability values were found to be 89.6 ± 3.6% and 89.4 ± 3.4% with Z-VAD-FMK and Y-

27632. Though a slightly higher adipose tissue viability obtained with the freezing

solution supplemented with inhibitors but the difference between the viability results with

and without inhibitors are statistically insignificant (p<0.05) thereby demonstrating that

the inhibitors do not play any significant role in maintaining the viability of cryopreserved

adipose tissue.

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Figure 5.26: XTT assay indicates the viability of cryopreserved adipose tissue using

Rho kinase and caspase inhibitors supplemented with the formulated freezing

solution. Results indicate a slightly higher adipose tissue viability obtained by adding

inhibitors, but the difference between the viability results with and without inhibitors

are statistically insignificant (p<0.05) which signifies that the inhibitors do not play

any significant role in maintaining the viability of cryopreserved adipose tissue.

G3PDH assay

Similar to the findings of oil ratio and XTT assays, the results of G3PDH assay (Fig 5.27)

also indicate that there is no significant difference in enzyme activity during

cryopreservation of adipose tissue using formulated 160mM NaCl/90mM

trehalose/1mg/ml curcumin with and without inhibitors. The enzyme activities of 1.67 ±

0.10 U/ml and 1.61 ± 0.07 U/ml were shown with the addition of Z-VAD-FMK and Y-

27632 to the freezing solution (1.62±0.07 U/ml) respectively. Thus, the results represent

the limited role of Z-VAD-FMK and Y-27632 compounds in the protection of

cryopreserved adipose tissue. However, similar to oil ratio analysis results, the freezing

solution containing Y-27632 gave slightly better performance than Z-VAD-FMK.

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Figure 5.27: G3PDH assay indicates the viability of cryopreserved adipose tissue

using Rho kinase and caspases inhibitors supplemented with the freezing solution.

TBARS assay

TBARS assay (Fig 5.28) also showed that there was no beneficial effect of Z-VAD-FMK

and Y-27632 when added to 160mM NaCl/90mM trehalose/1mg/ml curcumin freezing

solution. However, 160mM NaCl/90mM trehalose/1mg/ml curcumin/10nM Z-VAD-FMK

(215 ±15 nM/g wet wt) performed slightly higher than 160mM NaCl/90mM

trehalose/1mg/ml curcumin /100mM Y-27632 (217 ±13 nM/g wet wt).

Figure 5.28: TBARS assay indicates the effect of Rho kinase and caspases inhibitors

on the viability of cryopreserved adipose tissue using

160mMNaCl/90mMtrehalose/1mg/ml curcumin freezing solution. Though there is no

significant improvement in viability was observed using inhibitor, Z-VAD-FMK

inhibitor shows slightly better performance than Y-27632.

Overall, the results proved that addition of Z-VAD-FMK and Y-27632 did not improve

the viability of post-thaw adipose tissue.

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5.4 Control rate freezing of adipose tissue using formulated freezing solution

Cooling rate during cryopreservation has a profound effect on the viability of

cryopreserved cells and tissues. Slow cooling leads to extracellular ice formation and

hence the dominant mechanism of cryoinjury is solute concentration effect [176].

Whereas, rapid cooling leads to intracellular ice formation and thus ice-mediated injury

becomes dominant cause of cell death [177]. Therefore, to achieve maximum cell

viability, the optimal cooling rate is essential.

In the present research, initially the adipose tissue was cryopreserved in -80°C freezer in

isopropanol containing bath so as to maintain 1-2°C/min cooling rate [178]. Keeping this

in view, adipose tissue was subjected to control rate freezing in a controlled rate freezer

(CRF) to achieve more accurate and precise temperature control using the most effective

160mM NaCl/90mM trehalose/1mg/ml curcumin freezing solution [38].

5.4.1 Effect of cooling rate

The effect of cooling rate on the cryopreservation of adipose tissue was studied at varying

cooling rate such as 0.5°C/min, 1°C/min, 2.5°C/min, 5°C/min in CRF from 4°C to -120°C

followed by preservation in LN2 After 48h, the cryopreserved tissues were thawed rapidly

in a 37°C water bath. Rapid thawing method was selected based on the reported literature

that suggests that rapid thawing is superior to slow thawing method. The viability of post-

thawed adipose tissue was assessed as usual by oil ratio, XTT, G3PDH and TBARS

assays.

Oil ratio

The experimental results on oil release from cryopreserved adipose tissue at varying

cooling rate are shown in Fig 5.29. The viability results indicate that the adipose tissue

cryopreserved at -1°C/min cooling rate released less oil from the damaged adipose tissue

than either below or above -1°C/min and thus retained more viability. The trend of oil

release at different cooling rate is as follows- -1°C/min (0.019 ± 0.002) <0.5°C/min (0.021

± 0.002) <2.5°C/min (0.023 ±0.002) < 5°C/min (0.029 ± 0.002). Although, mean oil ratio

at -1°C/min cooling rate is higher than 0.5°C/min, the difference between them is

insignificant (p<0.05). Thus, with increasing cooling rate, the viability of the post-thawed

adipose tissues decreased rapidly. In comparison, oil ratios between adipose tissue

preserved using isopropanol filled container in -80°C freezer (0.021 ± 0.002) and

controlled rate freezer at -1°C/min cooling rate and storage at LN2, is comparable though a

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slightly lower oil ratio representing superior tissue viability was achieved with the later

condition.

Figure 5.29: Oil ratio analyses indicate the effect of cooling rate on the

cryopreservation of adipose tissue using 160mM NaCl/90mM trehalose/1mg/ml

curcumin freezing solution. The trend of oil release at different cooling rate is as

follows- -1°C/min (0.019 ± 0.002) <0.5°C/min (0.021 ± 0.002) <2.5°C/min (0.023 ±

0.002) <5°C/min (0.029 ± 0.002) indicating -1°C/min is the best cooling rate

providing maximum adipose tissue viability.

XTT assay

As indicated in Fig 5.30, -1°C/min cooling rate showed the maximum metabolic activity

(93.1± 2.2%) followed by adipose tissues cryopreserved at -0.5°C/min (89.7 ± 2.9%), -

2.5°C/min (84.1 ± 3.5%) and -5°C/min (83.9 ± 3.8%). As also observed with oil ratio

measurement, the difference in metabolic activity between adipose tissue preserved in

isopropanol filled container and -1°C/min controlled rate freezing is statistically

insignificant (p<0.05). However, the mean metabolic activity of controlled rate frozen

samples was higher than samples frozen using Mr. Frosty. This indicates the superiority of

the control rate freezing in the maintenance of adipose tissue viability.

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Figure 5.30: XTT assay showing the effect of cooling rate on the viability pattern of

cryopreserved adipose tissue using 160mM NaCl/90mM trehalose/1mg/ml curcumin

freezing solution. The maximum viability was obtained at -1°C/min.

G3PDH assay

The viability in terms of cell membrane integrity evaluated by G3PDH assay (Fig 5.31)

indicated the similar trend of tissue damage and reconfirmed -1°C/min cooling rate is the

most effective cooling rate providing maximum tissue viability. The tissue damage in

terms of enzyme activity level observed at different cooling rate follows the following

trends -1°C/min (1.57 ± 0.10 U/ml) < -0.5°C/min (1.72 ± 0.07 U/ml) < -2.5°C/min

(1.77±0.03 U/ml) < -5°C/min (1.86 ± 0.12 U/ml) representing the decrease in viability

with rapid cooling rate above -1°C/min as well as below -1°C/min as slow cooling.

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Figure 5.31: G3PDH assay indicates the effect of cooling rate on the cryopreservation

of adipose tissue using 160mM NaCl/90mM trehalose/1mg/ml curcumin freezing

solution. -1°C/min is observed to provide the maximum post-thaw viability by

showing least extracellular G3PDH enzyme activity.

TBARS assay

The results of TBARS assay (Fig 5.32) corroborate with the findings of other assays,

which suggested slow cooling particularly the cooling rate of -1°C/min is superior to other

cooling rates for cryopreservation of adipose tissue. The lipid peroxidation products in

post-thawed adipose tissue cryopreserved with -1°C/min cooling rate (200 ± 13 nM/g wet

wt) is significantly lower than the adipose tissue cryopreserved with other cooling rates.

Furthermore, the slow cooling rate below -1°C/min did not favor the adipose tissue

viability cryopreserved in 160mM NaCl/90mM trehalose/1mg/ml curcumin freezing

solution.

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Figure 5.32: Effect of cooling rates on the post-thaw viability adipose tissue

cryopreserved in 160mM NaCl/90mM trehalose/1mg/ml curcumin freezing solution

assessed by TBARS assay. The viability shows a similar trend that evaluated by XTT

and G3PDH assays.

The study on the cooling rate established that the formulated 160mM NaCl/90mM

trehalose/1mg/ml curcumin freezing solution is the most effective at -1°C/min cooling rate

achieving most tissue viability than other cooling rate and -80°C freezer. Therefore, -

1°C/min cooling rate was selected for the subsequent controlled rate freezing of adipose

tissue.

5.4.2 Effect of Seeding

Nucleation of ice is one of the most significant uncontrolled variables in conventional

cryopreservation leading to sample-to-sample variation in cell recovery, viability and

function [179]. Predictable nucleation of ice allows a way of increasing viability of cells

and reducing variability between samples. Therefore, nucleation of ice should be

controlled to allow standardization of cryopreservation protocols of cells and tissues for

biobanking [180].

Seeding or introduction of small ice crystals into the undercooled sample is a way of

controlled nucleation of ice [181, 182]. The seeding is performed manually by generating

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a cold spot on the outside of a closed cryovials using a stainless steel electrode. To find

out the effect of seeding temperature on the viability of cryopreserved adipose tissue, the

adipose tissue was seeded at -5°C, -7°C, and -9°C while cooling from 4°C to -120°C at -

1°C/min. The freezing solution used was 160mM NaCl/90mM trehalose/1mg/ml

curcumin. The samples were stored at LN2 for 48 h before being thawed rapidly and

assessed for viability.

Oil ratio

The oil release from cryopreserved adipose tissue was found to depend on the seeding

temperature. The viability (Fig 5.33) was initially increased with decreased in seeding

temperature from -5°C to -7°C and the viability remained constant afterward. Therefore, -

7°C is the most effective seeding temperature that provided maximum tissue viability with

minimum oil release. The corresponding oil ratios achieved were 0.021 ± 0.002 (-7°C),

0.024 ± 0.004 (-5°C), 0.021 ± 0.002 (9°C). However, the difference in results between

them is insignificant (p<0.05). Interestingly, when compared to samples frozen with -

1°C/min cooling rate without seeding, seeding decreased the viability of cryopreserved

adipose tissue, albeit insignificantly (p<0.05).

Figure 5.33: Measured oil ratio showing the effect of seeding temperature on the

cryopreservation of adipose tissue using 160mM NaCl/90mM trehalose/1mg/ml

curcumin freezing solution. As observed, -7°C is the most effective seeding

temperature that provided maximum tissue viability with minimum oil release from

adipose tissue.

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XTT assay

As in the previous assay, the metabolic activity (Fig 5.34) of cryopreserved adipose tissue

indicates that there is no significant change in post-thaw viability (92.1 ± 2.4%).

Furthermore, seeding also decreased the metabolic activity of post-thawed adipose tissue

indicating lesser viability than non-seeded samples.

Figure 5.34: Effect of seeding temperature on the cryopreservation of adipose tissue

using 160mM NaCl/90mM trehalose/1mg/ml curcumin freezing solution evaluated by

XTT measurement. The trend shows the similar viability pattern observed with all

the seeding temperature.

G3PDH assay

The results in Fig 5.35 indicate that the G3PDH activity at different seeding temperature

follows a similar trend as that of the oil ratio. Seeding at -7°C showed the least

extracellular concentration of G3PDH (1.61 ± 0.07 U/ml) in the cryopreserved samples

than other seeded and non-seeded samples. However, the difference is not statistically

significant (p<0.05) between them.

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Figure 5.35: G3PDH assay showing the effect of seeding temperature on the post-

thaw viability of adipose tissue cryopreserved in 160mMNaCl/90mM

trehalose/1mg/ml curcumin freezing solution. The assay result confirms that seeding

at -7°C is favorable for the maintenance of adipose tissue by offering minimum oil

release.

TBARS assay

The results of TBARS assay (Fig 5.36) also indicate similar finding to oil ratio, XTT and

TBARS assay. Samples preserved at -7°C showed least generation of lipid peroxidation

products (202 ± 10 nM/g wet wt) than others. However, similar to other assays, TBARS

assay also proved the deteriorating effect of seeding on the cryopreservation of adipose

tissue.

Figure 5.36: The viability pattern for post-thaw adipose tissue evaluated by TBARS

assay indicating -7°C as the seeding temperature that provided maximum tissue

viability with producing minimum lipid peroxidation products.

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Overall, the results show that the seeding temperature does not have any significant

influence on the cryopreservation of adipose tissue. As also observed in the previous

results, maintaining osmotic balance and preventing oxidative stress is more significant in

the case of adipose tissue cryopreservation than preventing ice related cryoinjury.

A comparison of the viability of adipose tissue cryopreserved in different freezing solution

is shown in Table 5.1. As indicated, the maximum tissue viability was achieved using

160mM NaCl /90mM trehalose/1mg/ml curcumin freezing solution with control rate

freezing at -1°C/min cooling rate followed by storage in LN2

Table 5.1 Comparison of viability using different freezing solution

Freezing solution Oil ratio XTT assay

(%)

G3PDH

assay (U/ml)

TBARS assay

(nM/g wet wt)

PBS 0.083±0.005 65.39±4.13 3.069±0.147 414±13

90mM trehalose/PBS 0.047±0.002 82.55±2.97 2.249±0.100 321±7

150mM ectoin/PBS 0.077±0.002 69.79±3.45 2.683±0.154 361±11

160mM NaCl 0.039±0.002 84.14±5.22 1.848±0.056 271±7

90mM trehalose/160mM

NaCl

0.035±0.002 84.76±4.28 1.751±0.121 256±16

10% Me2SO/PBS 0.043±0.006 80.20±3.21 2.281±0.074 285±19

10% Me2SO/160mM NaCl 0.032±0.004 85.35±3.88 1.719±0.169 253±15

160mM NaCl /90mM

trehalose

0.035±0.002 84.76±4.28 1.751±0.121 256±16

160mM NaCl /90mM

trehalose/1mg/ml curcumin

(-80°C)

0.02±0 89.72±4.41 1.622±0.074 204±10

160mM NaCl /90mM

trehalose/1mg/ml curcumin

(-1°C /min cooling rate

followed by storage in LN2)

0.019±0.002 93.13±2.21 1.574±0.100 200±13

160mM NaCl /90mM

trehalose/1mg/ml curcumin

(-1°C/min cooling rate,

seeding at -7°C followed by

storage in LN2)

0.021±0.002 92.09±2.38 1.607±0.074 202±10

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5.5 Long-term viability of adipose tissue by controlled rate freezing using formulated

freezing solution

The maintenance of viability with long shelve-life is of prime importance. Long-term

preservation is required to hold the potential therapeutic modality of adipose tissue in

reserve for use at a future date. Given increasing application of adipose tissue in a

cosmetic and aesthetic surgery, the significance of long-term preservation of adipose

tissue is increasing. Although the viability of cryopreserved cells and tissues is known to

decrease over time because of free radicle formation and other events, the incidence of

adverse events on the long-term viability of cryopreserved adipose tissue is unclear, often

with conflicting conclusion [183]. Therefore, after achieving a successful result on a short-

term basis, the developed freezing solution having composition 160mM NaCl/90mM

trehalose/1mg/ml curcumin was evaluated for its efficacy towards the maintenance of

tissue viability on a long-term perspective. The adipose tissue was cryopreserved by

control rate freezing using the developed freezing solution and the tissue viability was

assessed in an interval of 7, 15, 30, 60 and 90 days of storage in liquid nitrogen. The

controlled rate freezing was performed at -1°C/min cooling rate from 4°C to -120°C after

equilibrating with the freezing solution for 1h at 4°C.

Oil ratio

The results depicted in Fig 5.37 revealed that the oil release and hence adipocyte damage

of cryopreserved adipose tissue was increased gradually over storage time that represents

the gradual loss in tissue viability with time. However, the viability of adipose tissue after

90 days of storage (0.027 ± 0.002) was not significantly different from viability even after

7 days (0.024 ± 0.004) in terms of oil ratio indicating that the developed freezing solution

is efficient in maintaining adipose tissue viability for a longer period of storage.

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Figure 5.37:Measured oil ratio showing the effect of 160mMNaCl/90mM trehalose/

1mg/ml curcumin freezing solution on the long-term viability of cryopreserved

adipose tissue, which demonstrates that the developed freezing solution is efficient in

maintaining adipose tissue viability during 90 days period of storage.

XTT assay

The results of XTT assay, as shown in Fig 5.38, corroborate with the finding of oil ratio

analysis. The metabolic activity and hence the viability was gradually decreased with

storage period. However, the difference in viability loss was not statistically significant.

As for example, the viability of 89.7 ± 3.1% after 7 days of storage was decreased only to

88.00 ± 3.6% after 90 days indicating that the maximum viability loss was only 2% during

the 90 days of storage of adipose tissue.

Figure 5.38: Assessment of the long-term viability of cryopreserved adipose tissue

measured by XTT assay. The result indicates the efficacy of the developed 160mM

NaCl/90mM trehalose/1mg/ml curcumin freezing solution towards preserving

adipose tissue during the 90 days storage.

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G3PDH assay

Fig 5.39 depicted an increase in the concentration of extracellular G3PDH over time. The

activity of the enzyme was shown to be 1.65 ± 0.1 U/ml after 7 days and the activity was

increased to 1.80±0.07 U/ml after 90 days. The nominal change in G3PDH concentration

indicates less damage of cryopreserved adipose tissue even after 90 days of storage.

Therefore, the developed freezing solution is proven to be efficient in preserving adipose

tissue with long shelve life.

Figure 5.39: G3PDH assay shows the efficiency of 160mM NaCl/90mM

trehalose/1mg/ml curcumin freezing solution on the long-term viability of

cryopreserved adipose tissue. The cryopreserved adipose tissue was maintained well

during the 3months period of storage.

TBARS assay

The assay measures the formation of lipid peroxidation products after 7 days, 15 days, 30

days, 60 days and 90 days. As indicated from Fig 5.40, the malondialdehyde concentration

increases as the cryopreserved adipose tissues undergo lipid peroxidation over time. The

malondialdehyde level 209 ± 6 on first week changed to 234 ± 15 nM/g wet wt. of adipose

tissue at the end of 3rd month. The results reveal an increase in the concentration of

malondialdehyde over time further indicating a decrease in viability or continuation of

malondialdehyde formation. However, the reduction in viability is not statistically

significant representing the efficiency of freezing solution for cryopreservation of adipose

tissue.

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Figure 5.40: TBARS assay shows the viability pattern of adipose tissue

cryopreserved in the developed 160mM NaCl/90mM trehalose/1mg/ml curcumin

freezing solution during the 90 days period of storage.

5.5.1 Morphological assessment of cryopreserved adipose tissue using CLSM

To assess the structural and functional integrity of adipose tissue cryopreserved for 90

days, phase contrast microscopy, fluorescent imaging with PI were performed.

Furthermore, inherent fluorescence of the developed freezing solution, because of the

presence of curcumin was also evaluated and used to assess the morphology of

cryopreserved adipose tissue. The results were also compared with fresh adipose tissue.

Phase contrast images (Fig 5.41) has revealed the distinct morphology of adipocytes in

fresh and cryopreserved adipose tissue.

Figure 5.41: CLSM images of fresh and post-thaw cryopreserved (90 days) adipose

tissue. The images reveal the distinct morphology of adipocytes in fresh and

cryopreserved adipose tissue.

Viability of adipocytes in cryopreserved adipose tissue by PI

The structural integrity of cryopreserved adipose tissue was based mostly on H&E

staining. However, judging adipocyte health by shape or nuclear appearance can be

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misleading, and histologic sections are too thin to show most nuclei of healthy adipocytes.

Therefore, fluorescent staining with PI was used to assess the status of both fresh and

cryopreserved tissues. PI is impermeable to cell membrane until there is a breach in the

cell membrane. Once inside the cells, PI bind with nucleic acids and then fluoresce

revealing the dead cells.

Figure 5.42: CLSM images are showing the structural integrity of fresh (a) and

cryopreserved adipose tissue of 90 days storage (b) using PI as fluorescence dye. The

discrete distribution of red stains mainly represents stained nuclei of dead adipocytes

and the arrays of red stains cells mainly represent dead endothelial cells of the

vessels.

Fig 5.42 has revealed the structural integrity of fresh and cryopreserved adipose tissue

after staining with PI. As revealed, there was not much difference in viability between

fresh and cryopreserved tissues. Both fresh and cryopreserved adipose tissue mainly

constituted of non-stained viable adipocytes.

Vessels made up of endothelial cells provide sufficient nutrient and oxygen support for

active metabolism and survival of the tissue. As can also be seen in Fig 5.43, the

adipocytes are mostly viable; however, the most cells of vasculatures are dead in both

fresh and cryopreserved adipose tissue supporting the recently reported literature on the

importance of diffusion and neovascularization on long-term fat grafts survival [184]. The

finding also supports the existing theory of fat grafts, which states that long-term graft

volume consists primarily of grafted adipocytes that have survived the entire procedure

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and grafted fats initially lack vascular support and the oxygen; nutrient supply to grafts

occurs primarily through diffusion and neovascularization from surrounding tissue [185].

Figure 5.43: CLSM of fresh (a) and cryopreserved (90 days) adipose tissue (b) using

PI indicating dead endothelial cells.

Assessment of structural integrity of adipocytes using the developed freezing solution

Curcumin is a well-known fluorescence molecule with an excitation wavelength in the

range of 420-480nm and emission band of 490-520nm [186-189]. As the developed

freezing solution contains curcumin, the inherent fluorescence property of the freezing

solution was used to assess the status of the cryopreserved adipose tissue. Fresh adipose

after staining with curcumin acted as a control. As seen in the Fig 5.44a, curcumin was

insoluble in PBS and appeared as heterogeneously distributed fluorescent particulates over

the adipose tissue. As revealed in Fig 5.44b, the curcumin forms homogeneous solution

after incubation with 2.5% Me2SO for 30min and the depth of penetration of curcumin

increased. Furthermore, the non-permeation of homogenously distributed curcumin inside

the cells helped in developing the contrast between adipocytes. The less permeability of

curcumin inside the cell is also resulted in less bioavailability of it because of less

permeation of curcumin inside cells [190].

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Figure 5.44:CLSM images of adipose tissue using curcumin in PBS (a) and curcumin

in 2.5% Me2SO. The heterogeneous distribution of changed to homogenous

distribution with more depth of penetration after solubilizing curcumin in Me2SO.

To evaluate the potential of curcumin to be able to differentiate between live and dead

cells, adipose tissue was incubated with 0.25% SDS solution in the presence of 0.1%

curcumin/2.5%% Me2SO solution for 30min. SDS, a surfactant molecule is supposed to

compromise the plasma membrane and let the dye permeate inside the cells easily as seen

in Fig 5.45a. Furthermore, because of fat solubility of curcumin, it stains the lipids of

adipocytes, differentiating the dead cells from live cells. Therefore, curcumin may be used

as live/dead differential stains for adipocytes. To evaluate the status of the cryopreserved

adipose tissue frozen with 160mM NaCl/90mM trehalose/1mg/ml curcumin, the post-

thawed tissue was incubated with 2.5% Me2SO for 30min in the presence of the freezing

solution containing curcumin. Fig 5.45b, c reveals that most of the adipocytes of

cryopreserved adipose tissue retained the viability like fresh adipose tissue.

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Figure 5.45: shows the structural and functional integrity of SDs treated (a), fresh (b)

and cryopreserved (90 days) adipose tissue (c) using 0.1% curcumin/ 2.5% Me2SO.

Arrow indicate dead adipocytes

5.5.3 Survival of stem cells in cryopreserved adipose tissue

The retention of mesenchymal stem cells in the adipose tissue has several advantages. It

has been reported that supplementation of hADSCs along with adipose tissue for

reconstructive and aesthetic surgery improved the outcome of the surgery. Furthermore,

successful isolation of hADSCs from cryopreserved adipose tissue provides an alternative

cheaper source of hADSCs, supplementing the benefits of hADSCs cryobanks. Thus, an

attempt was made to isolate hADSCs from cryopreserved adipose tissue (90 days) and the

isolated hADSCs were compared with hADSCs isolated from fresh adipose tissue.

Morphology of isolated hADSCs

The morphological characteristic of the cultured hADSCs isolated from fresh and

cryopreserved adipose tissue (90 days) was assessed by observation under phase contrast

microscope.

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Figure 5.46: Phase contrast microscopic images shows gradual change of round to

fibroblastic cell morphology of hADSCs isolated from fresh (a) and cryopreserved

(90 days) adipose tissue (b) observed upon culture in DMEM containing 10% FBS.

The cells reached confluency on the 12th day of culture. (Scale bar 100µm).

Fig 5.46b depicts that the initial round shape like morphology of isolated hADSCs from

cryopreserved adipose tissue was gradually changed to fibroblast-like appearance over 12

day culture period. Moreover, the cells were observed to adhere to the culture dish after 8h

of incubation and fibroblast-like morphology was noticed after 18h. The cultured cells

reached confluence after 12 days of culture. Similar morphology was also observed with

fresh hADSCs (Fig 5.46a).

Proliferation kinetics

As indicated from Fig 5.47, the hADSCs isolated from cryopreserved cells were

proliferated normally as the hADSCs isolated from fresh adipose tissue indicating the

freezing solution has no adverse influence on the proliferation of hADSCs representing its

ability to maintain cryopreserve adipose tissue. Upon seeding at a concentration of 5x103

cells/well, the hADSCs from fresh adipose tissue proliferated to 67.33±2.08 x103

cells/well, whereas hADSCs from cryopreserved adipose tissue proliferated to 68.33±3.05

x103 cells/well on the 9th day.

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Figure 5.47: Proliferation kinetics of hADSCs isolated from fresh and cryopreserved

adipose tissue of 3months storage. Cryopreserved hADSCs were proliferated

normally as the hADSCs isolated from fresh adipose tissue indicating the freezing

solution has no adverse effect on the proliferation of hADSCs of adipose tissue and

thus able to maintain the adipose tissue viability.

Differentiation potential of hADSCs

Osteogenesis (Fig 5.48) of hADSCs was indicated by staining of deposited calcium with

alizarin red whereas staining of lipid droplet by Oil Red O indicates adipogenesis of

hADSCs. Thus, the results showed that the isolated hADSCs from cryopreserved adipose

tissue differentiated successfully to osteogenic and adipogenic lineages paving its

application in tissue engineering and regenerative medicine.

Figure 5.48: Assessment of differentiation potential of hADSCs isolated from fresh

(a, b) and cryopreserved (90 days storage ) adipose tissue(c, d). (a, c) Osteogenic

(Alizarin red staining) and (b, d) adipogenic lineages (Oil Red O staining). As

indicated the isolated hADSCs differentiated successfully to osteogenic and

adipogenic lineages.

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Overall, though a slightly lower viability of adipose tissue frozen with the developed

160mM NaCl/90mM trehalose/1mg/ml curcumin freezing solution was observed in

comparison to fresh tissue, but the developed freezing solution was able to maintain high

viability even after 3 months of storage in LN2 as evident from Table 5.2. shows the

comparison of viability between fresh and cryopreserved adipose tissue after 3 months of

storage. Furthermore, hADSCs were successfully isolated from the cryopreserved adipose

tissue (90 days) frozen with the developed freezing solution. The isolated hADSCs

showed adherancy with plastics, regular morphology and proliferation and also retained

the differential ability to osteogenic and adipogenic lineages. Thus, the developed freezing

solution has no adverse influence on the mesenchymal stem cells present in the adipose

tissue. Therefore, adipose tissue frozen with 160mM NaCl/90mM trehalose/1mg/ml

curcumin freezing solution is not only suitable for reconstructive and aesthetic surgery but

also a good source of hADSCs when need arises.

Table 5.2 Comparison of viability between fresh and cryopreserved (3months)

adipose tissue

Tests Fresh AT Cryopreserved AT

Oil ratio 0 0.027±0.002

XTT (%) 100% 88 ±3.6

G3PDH assay (U/ml) 1.09±0.05 1.80±0.07

TBARS assay (nM/g wet wt.) 142.47±11.54 233.62±15.13

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Chapter 6

Cryopreservation of Adipose tissue-

derived stem cells

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6.1Screening of potential cryoprotectants and carrier media towards the formulation

of freezing solution for cryopreservation of adipose tissue-derived stem cells

Mesenchymal stem cells (MSCs) are potential for tissue regeneration because they are

multipotent and differentiate into different cell types such as osteoblasts, adipocytes,

chondrocytes, myoblasts and the like [191]. Adipose tissue is an attractive source of MSCs

because of its easy availability and accessibility, thereby offers its application in

producing therapeutic and tissue engineering products [192]. In this context, an effective

cryopreservation strategy for preservation of hADSCs with long shelve-life is important

for their clinical application.

Cryopreservation using conventional freezing solution consisting of Me2SO and FBS has

several detrimental effects including, the acute and chronic toxicity of patients and

genotoxicity of preserved cells. Furthermore, the most commonly used DMEM carrier

media having multicomponent constituent creates more complex and unpredictable cell

environment at sub-zero temperature because of ice crystallization. Hence, development of

a Me2SO and serum-free freezing solution in a suitable carrier media for preserving

hADSCs is inevitable.

Therefore, efforts have been made in this phase of the dissertation to investigate the

potentiality of various hydrocolloids and organic osmolytes as natural CPAs and

phosphate buffered saline (PBS) and its individual components as carrier media to

formulate a freezing solution for cryopreservation of hADSCs. The performance of the

formulated freezing solution was evaluated by a battery of routine as well as advanced

tests such as immunophenotype (flow cytometric analysis), cell viability and cell

proliferation (Trypan blue; MTT assay), cytoskeletal analysis (CLSM study) and

differential potential (alizarin red; Oil Red O assays). The experimental results obtained in

the above research are discussed in this chapter.

6.1.1 Morphological and Immunophenotypic characterization of hADSCs

The morphology of the isolated and cultured hADSCs from fresh adipose tissue was

assessed microscopically (Fig 6.46a). The initial round cells gradually changed to

fibroblast-like appearance. The cultured cells have expressed 99% CD90, 89% CD73,

98% CD105 as positive and 1.2% CD34,2% CD45, and 0.5% HLA-DR as negative

surface markers indicating the cells are mesenchymal stem cells type (Fig 6.1).

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Figure 6.1: Flowcytometric analysis showing the expression of positive CD90 (99%),

CD73 (89%), and CD105 (98%) markers and negative HLA-DR (0.5%), CD34

(1.2%) and CD45 (2%) markers representing the cells are of mesenchymal stem cells

in characteristics.

6.1.2 Evaluation of hydrocolloids

Hydrocolloids are polysaccharides that prevent ice crystallization, thereby act as

extracellular cryoprotectants. The importance of hydrocolloids as cryoprotectants (CPA)

was reported in the literature [193, 194]. Polyvinylpyrrolidone (PVP) and methylcellulose

(MC) hydrocolloids have shown promise in cryopreservation of hADSCs [92, 93].

Furthermore, the hydrocolloids namely gums, CMC, MC and inulin were selected based

on their use as food additives and various medicinal products without any cytotoxicity

[195, 196].

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Figure 6.2: Effect of types of hydrocolloids and their concentrations on the viability

of cryopreserved hADSCs assessed by Trypan blue assay. Among the hydrocolloids,

has performed best achieving the maximum viability of 55.7 ± 3.2%. The viability

trend observed as 10% PVP > 0.3% tragacanth gum (43 ± 4.6%) > 1% (w/v)

methylcellulose (37% ± 2.1%) > others

Trypan blue assay results (Fig 6.2) shows that 10% (w/v) PVP/ PBS is the most efficient

cryoprotectant achieving maximum viability (55.7 ± 3.2 %) followed by 0.3% (w/v)

tragacanth gum (43 ± 4.6%) and 1% (w/v) methylcellulose (37% ± 2.1%). The viability

obtained with 10% PVP is 12% and 18% higher than tragacanth gum (TG) and

methylcellulose (MC) respectively. Among natural hydrocolloids, tragacanth gum showed

higher viability than other polysaccharides, which may be due to its molecular weight and

neutral characteristics. The viability obtained with 10%PVP/PBS was also higher than

10%Me2SO/PBS (control) and viability achieved with 10%Me2SO/PBS supplemented

with 20%(v/v) FBS was comparable. Inulin, pectin and carrageenan showed the lowest

viability. The viability trend observed by MTT assay was similar to Trypan blue assay

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(Fig 6.3). The results confirm PVP as the superior cryoprotectant to the other

hydrocolloids used in this study and the 10% Me2SO as the conventional CPA for

hADSCs.

Figure 6.3: Effect of selected hydrocolloids on the viability of cryopreserved hADSCs

assessed by MTT assay. The viability data obtained by MTT assay followed similar

trends as Trypan blue assay. The results confirm PVP as the superior cryoprotectant

to the other hydrocolloids used in this study as well as 10% Me2SO as the

conventional CPA for hADSCs preservation.

6.1.3 Evaluation of PVP in combination with PBS components as carrier media

The most commonly used PBS as carrier media consists of NaCl, Na2HPO4, KCl and

KH2PO4. It was hypothesized that these components may have a specific role in

cryopreservation of stem cells, which has not been explored. Hence, these compounds

were evaluated individually at a buffered isotonic concentration in combination with

10%PVP. As indicated in Fig 6.4a, 10%PVP/ 0.9% NaCl shows the highest viability

(70.3 ± 1.5%) with the lowest viability shown by 10%PVP/ Na2HPO4 (34.3 ±3.1%). The

viability obtained with 10% PVP in isotonic NaCl is 15% more than the viability obtained

using other carrier media. The viability (60.7 ± 2.1%) of hADSCs with 10%PVP/KCl is

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comparable to the viability (55.7 ± 3.2%) obtained using 10%/PVP/PBS. The results also

revealed that phosphate containing ionic compounds offer lower viability than chloride-

containing compounds. The viability data measured by MTT assay (Fig 6.4b) followed

the similar trend as viability assessed by Trypan blue method thereby confirming

10%PVP/ 0.9% NaCl as a superior freezing solution.

Interestingly, the viability observed with NaCl containing freezing solution in our

experiment is in good agreement with viability achieved with DMEM containing freezing

solution. Thus, the results prove that the effectiveness of DMEM and normal saline on

cryoprocess is comparable, and normal saline may replace DMEM due to its lower price

and easy availability. Furthermore, chloride based ionic compounds performed better than

phosphate based compounds indicating the freezing solution containing extracellular

anions and cations provide better viability compared to the respective intracellular

counterpart. The results may be contrary to many other reports stating that the intracellular

type freezing medium offers better viability [5]. The concept of intracellular preservation

media is mainly for the hypothermic preservation of organs and tissues, and the same

concept may not be appropriate for the cryopreservation of mesenchymal stem cells.

Figure 6.4: Effect of PBS and its individual ionic components as carrier media on the

viability of cryopreserved hADSCs in 10% PVP assessed by Trypan blue assay (a)

and MTT assay (b). 0.9% NaCl is found to be the best among the carrier media. The

results also revealed that phosphate containing ionic compounds performed better

than chloride-containing compounds thus signifies that the freezing solution

containing extracellular anions and cations provide better viability compared to the

respective intracellular counterpart.

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6.1.4 Improvement of the developed freezing solution by the addition of organic

osmolytes

Natural osmolytes are small organic molecules that maintain cell volume and fluid balance

of cells under stress and act as intracellular cryoprotectants. Organic osmolytes are non-

cytotoxic and used in skin ointments, nasal sprays and eye drop. Although organic

osmolytes namely ectoin and hydroxyectoin as intracellular CPAs have shown promise in

cryopreservation of human endothelial cells and murine embryos, have not so far been

explored for cryopreservation of hADSCs.

The effect of natural organic osmolytes in combination with the best freezing solution

(10%PVP /0.9%NaCl was investigated with the aim of achieving improved viability of

cryopreserved hADSCs.

Fig 6.5a shows that the supplement of ectoin and hydroxyectoin increased the viability of

cryopreserved hADSCs although a slightly higher viability was achieved with ectoin. The

viability was also increased with increase in the concentration of osmolytes in freezing

solution and the maximum cell viability of 81.7 ± 2.1 %) was obtained using ectoin at

60mM concentration. Thus, an increase of 11% viability of 10%PVP/0.9%NaCl was

obtained with the addition of osmolytes. The cell recovery assessed by MTT also

followed the similar trends. (Fig 6.5b). Thus, it is demonstrated that

10%PVP/0.9%NaCl/60mM ectoin was established as the most efficient freezing solution

that provided the highest cell viability than other freezing solutions.

Usually, the supplementation of intracellular CPA with extracellular CPA complements

each other to increase the viability of cryopreserved cells. Therefore, PVP, an extracellular

CPA was supplemented with intracellular CPA such as ectoin and hydroxyectoin. Ectoin

was found to be more efficient than hydroxyectoin, as the superior cryopreservation

outcome was obtained using ectoin. Furthermore, ectoin was also reported to have the

potential to replace Me2SO as a cryoprotectant in a serum-free cryomedium for MSCs

[139]. Thus, the viability obtained with developed freezing solution

10%PVP/0.9%NaCl/60mM ectoin is also comparable to the conventional

10%Me2SO/DMEM supplemented with FBS (80%).

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Figure 6.5: Effect of organic osmolytes on the viability of cryopreserved hADSCs.

The Trypan blue assay (a) and MTT assay (b) results revealed that

10%PVP/0.9%NaCl freezing supplemented with organic osmolytes improve cell

viability. Furthermo0re, ectoin is found to be more efficient in maintaining cell

viability than hydroxyectoin. Overall, 10%PVP/0.9%NaCl/60mM ectoin was

established as the most efficient freezing solution that provided the highest cell

viability than other freezing solutions.

6.1.5 Cytoskeletal analysis

F-actin, one of the major components of the cytoskeletal system has a significant role in

maintaining cellular integrity and function. At extremely low temperature, the ultra-

structure of F-actin becomes distorted that causes cell damage, which was assessed by

cytoskeletal analysis [197]. Fig 6.6a (Priyanka Goyal M.Tech thesis, 2015) and 6.6b

showed the normal cytoskeletal distribution pattern of F-actin, indicating hADSCs

cryopreserved in 10%PVP/0.9%NaCl/60mM ectoin retain their normal cell morphology

like fresh hADSCs proving the efficiency of the formulated freezing solution towards their

maintenance.

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Figure 6.6: CLSM images of frozen hADSCs in 10%PVP/0.9%NaCl/60mM ectoin

freezing solution. Nuclear stain (blue) used was DAPI and the cytoskeletal stain was

TRITC-Phalloidin. The distribution of F-actin is predominantly beneath the cell

membrane, indicating its normal distribution in cryopreserved hADSCs.(Scale bar

25µm)

6.1.6 Differentiation Potential

Freezing solutions have a significant influence on the differentiation ability of

cryopreserved cells. The impact of the formulated 10%PVP/0.9%NaCl/60mM ectoin on

the osteogenic and adipogenic differentiation ability of frozen hADSCs was investigated.

When the post-thaw hADSCs were exposed to the adipogenic medium, the cell

morphology underwent a transition from elongated fibroblastic to a round or polygonal

structure. After 14 days of induction, a consistent cell vacuolation was evident in the

induced cells. On 0.36% Oil Red O staining, the differentiated cells showed the deposition

of red lipid droplets throughout the cytoplasm (Fig 6.7b) that indicates the adipogenic

induction of cryopreserved hADSCs.

The osteogenic differentiation potential of cryopreserved hADSCs was evident from the

deposition of calcium as an osteogenic differentiation marker during 21 days of culture

(Fig 6.7a). Thus, the freezing solution can maintain the osteogenic and adipogenic

differentiation potential of the cryopreserved hADSCs

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Figure 6.7: Osteogenic and adipogenic differentiation potentiality of hADSCs

cryopreserved in 10%PVP/0.9%NaCl/60mM ectoin freezing solution (a) and (b).

Osteogenic potential of hADSCs is represented by the deposition of red calcium on 21

days cell culture (a) with Alizarin red assay. Oil Red O staining shows the deposition

of red lipid droplets throughout the cytoplasm on 14 days (b) indicating the

adipogenesis. (Scale bar 200µm).

Overall, Me2SO-free, serum free and efficient freezing solution 10%PVP/ 0.9%NaCl/

60mM ectoin was formulated, which will be used in subsequent sections for further

improvement in its efficacy in cryopreservation of hADSCs.

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6.2 Optimization of freezing solution composition to improve cryopreservation

outcome of adipose tissue-derived mesenchymal stem cells

In the previous work, we have screened and evaluated cryoprotectant efficacy of a number

of hydrocolloids and organic osmolytes for preserving hADSCs. The study has shown

polyvinylpyrrolidone (PVP) and ectoin as the potential CPA achieving superior hADSCs

viability upon cryopreservation than other osmolytes as well as conventional Me2SO.

Besides cryoprotectants, the normal saline solution was found to be an effective carrier

media that can replace the complex and much expensive DMEM media used traditionally

in cryopreserved hADSCs. Based on this study, a novel freezing solution was formulated

from the combination of PVP as extracellular CPA, ectoin as intracellular CPA, and NaCl

as the carrier media. The freezing solution (0.9% NaCl/10% PVP/60mM ectoin) was

found to be effective in terms of cell viability, and differentiation ability of cryopreserved

hADSCs. Therefore, an attempt has been given in the present study to improve further the

cryopreservation efficacy of the above-formulated freezing solution by optimizing the

concentration of its individual components following Taguchi orthogonal design method.

Taguchi’s optimization method is an attractable and widely used technique because of its

several advantages; it minimizes trials for generating useful information regarding the

response of interest, reduces the experimental time and robustness of the process [198,

199]. In addition, catalase, a natural antioxidant was incorporated in the formulation of

freezing solution because catalase is reported to mitigate free radicals mediated cell

damage during freezing, thereby, an improved viability of cryopreserved cells on long-

term storage is expected [200].

6.2.1 Formulation of Freezing solution

For the development of an optimal freezing solution, Taguchi’s orthogonal design method

was applied. To this end, four factors namely NaCl, PVP, ectoin, catalase as freezing

media components and three levels of their different concentrations were selected (Table

6.1) to prepare the freezing solutions with varying composition as shown in Table 6.2.

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Table 6.1: Four control factors and three levels of concentration

NaCl

(Carrier media)

PVP (Extracellular

CPAa)

Ectoin (Intracellular

CPAa)

Catalase

(Antioxidant)

80mM 8% 30mM 50µg/ml

120mM 10% 60mM 100µg/ml

160mM 12% 90Mm 150 µg/ml

a) CPA represents cryoprotectant

Table 6.2: Formulation of freezing solutions based on Taguchi L9 (3(4)) array

Cryopreservation

Solution

NaCl (mM) PVP (%) Ectoin(mM) Catalase (µg/ml)

Solution-1 80 8 30 50

Solution-2 80 10 60 100

Solution-3 80 12 90 150

Solution-4 120 8 60 150

Solution-5 120 10 90 50

Solution-6 120 12 30 100

Solution-7 160 8 90 100

Solution-8 160 10 30 150

Solution-9 160 12 60 50

6.2.2 hADSCs viability assessment by Trypan blue dye exclusion assay

Trypan blue permeates the compromised cell membrane of dead cells and stains them

blue, whereas the intact membrane of live cells excludes the Trypan blue. This property of

the dye distinguishes the stained dead cells from live cells easily under an optical

microscope. The viability of the post-thawed hADSCs evaluated by the Trypan blue dye

assay was also compared with the viability obtained with 10% Me2SO used as a control.

Experimental results (Table 6.3) revealed that the freezing solution consists of 160mM

NaCl/10% PVP/ 30mM ectoin/150 µg/ml catalase (solution 8) provides the highest

viability followed by the freezing solution comprising of 120mM NaCl/10% PVP/90mM

ectoin/50µg/ml catalase (solution 5). The corresponding viability values are measured to

be 81% and 80% respectively. In addition to these solutions, solution 2 (80mM

NaCl/10% PVP/60mM ectoin/100 µg/ml catalase) and solution 7 (160mM NaCl/8%

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PVP/90mM ectoin/100 µg/ml catalase) also showed good viability of 78%. The viability

of all other freezing solutions was less than 70%. Interestingly, no statistically significant

difference was observed in the viability of solution 8 and solution 5. When compared with

viability results obtained with control (10% Me2SO in PBS), all the freezing solutions

except solution 1(80mM NaCl/8% PVP/30mM ectoin/50 µg/ml catalase), showed

significantly higher viability. An increase of 30% viability was obtained with solution 8 in

comparison to control. The freezing solution 8 has also shown superior cryopreservation

outcome than 0.9% NaCl/10% PVP/60mM ectoin (81%) representing the significant role

of catalase as antioxidant.

Table 6.3: Trypan blue dye exclusion assay of post-thawed hADSCs

Sample Repetition-1 Repetition-2 Repetition-3 Mean

Viability

SDa) S/N

ratiob)

Solution-1 56 55 54 55.00 1.00 34.80

Solution-2 78 79 77 78.00 1.00 37.84

Solution-3 71 65 69 68.33 3.05 36.67

Solution-4 67 64 63 64.66 2.08 36.20

Solution-5 82 80 80 80.66 1.15 38.13

Solution-6 69 67 68 68.00 1.00 36.64

Solution-7 78 76 79 77.66 1.52 37.80

Solution-8 83 80 81 81.33 1.52 38.20

Solution-9 67 68 65 66.66 1.52 36.47

10% Me2SO 52 51 48 50.33 2.08 --

Optimised

Solutionc)

86 83 85 84.66 1.52 --

a)SD denotes Standard Deviation

b) S/N ratio represents Signal to Noise ratio

c)Optimized solution was comprised of 160mM NaCl/10% PVP/90mM ectoin/100µg/ml

catalase

6.2.3 Taguchi statistical analysis

The Trypan blue assay results were used to calculate the signal-to-noise ratio (S/N). S/N

ratio, expressed in decibels, is an important performance indicator of Taguchi statistical

analysis. The S/N ratio was calculated based on Taguchi’s –“larger-the-better” approach

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that means larger S/N value indicate superior viability results [201]. The results indicate

that S/N ratio follows similar trends as that of the Trypan blue viability assay results

(Table 6.3) obtained with cryopreserved hADSCs. Moreover, the main effects plot (data

mean) for S/N ratios revealed the influence of individual column over the Taguchi results

(Fig 6.8). The results indicate that there is a significant variation in viability with different

concentrations of individual components in freezing solution. Overall, the trend suggests

that the viability is increased with increase in the concentrations of NaCl and ectoin.

Whereas among the concentrations tested, 10% PVP and 100 µg/ml catalase showed the

maximum viability of cryopreserved hADSCs.

Figure 6.8: Illustrates Main effect plot for mean of S/N ratios. S/N ratio increases

with increase in the concentration of NaCl (A) and ectoin (C). PVP (B) and catalase

(D) showed the highest S/N ratio at a concentration of 10% (w/v) and 100µg/ml

respectively.

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The response table of the mean was tabulated with mean response across levels of a factor

to find out the influence of individual column over Taguchi results (Table 6.4). The

influence was quantified by the value of Delta, which is the difference between the

maximum and minimum mean response across levels of a factor. The higher is the value

of Delta; the higher is the influence. The response table indicates that PVP showed the

stronger influence over the viability results followed by NaCl, ectoin and catalase in that

order.

Table 6.4: Response table for mean viability of cryopreserved hADSCs

Level NaCl PVP Ectoin Catalase

1 67.11 65.78 68.11 67.44

2 71.11 80.00 69.78 74.56

3 75.22 67.67 75.56 71.44

Delta 8.11 14.22 7.44 7.11

Rank 2 1 3 4

ANOVA analysis assessed the contribution of factors to the overall cryopreservation

outcome. The highest contribution of PVP was also confirmed (55.12%) followed by

NaCl (15.20%), ectoin (14.10%) and catalase (11.75%) as indicated in Table 6.5.

However, the effect of concentration variation within a column is statistically

insignificant.

Table 6.5: ANOVA analysis of the four factors

Factors Degree of

Freedom

Sum of

squares

Mean

square

F P Contribution

(%)

NaCl 2 98.69 49.34 0.56 0.60 15.20

PVP 2 357.95 178.97 4.03 0.08 55.12

Ectoine 2 91.58 45.79 0.51 0.62 14.10

Catalase 2 76.25 38.12 0.42 0.68 11.75

Error 2 24.88 03.83

Total 10 649.35

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The result signifies that the post-thaw viability using PVP as extracellular cryoprotectant

was the highest, followed by NaCl as carrier media, ectoin as intracellular cryoprotectant

and catalase as an antioxidant. Further, a small variation in the concentration of individual

factors might have contributed towards non-significance of ANOVA results.

6.2.4 Flow cytometry

PI is excluded by viable cells because of its high molecular weight, i.e. 668.4 D. The cell

membrane of necrotic cells becomes permeable to the dye and the dye binds with nucleic

acids and becomes a fluorescent molecule inside the necrotic cells. This property is

utilized by flow cytometry to detect and quantify the non-viable cells. The flow cytometry

data is presented in Fig 6.9, which indicates a similar trend in the result as that of Trypan

blue dye exclusion assay. The results confirm that the 160mM NaCl/10% PVP/30mM

ectoine/150 µg/ml catalase is the most efficient freezing solution achieving maximum

hADSCs viability of 81 %.

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Figure 6.9: Flow cytometry analysis of cryopreserved hADSCs using PI as a

fluorescent dye. 10% Me2SO freezing solution was used as control (A). B-J

represents the viability of cells using the freezing solutions formulated by Taguchi’s

orthogonal design. The optimized solution (K) comprising of 160mM NaCl/10%

PVP/90mM ectoin/100µg/ml catalase showed the maximum viability of ~85%.

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6.2.5 MTT Assay

MTT assay was used to assess the metabolic activity of cells and it is one of the most

commonly used methods to determine cell viability. The experimental viability data is

shown in Fig 6.10.

Figure 6.10: MTT assay of cryopreserved hADSCs. 10% Me2SOwas used as control

freezing solution. The optimized solution comprised of 160mM NaCl/10%

PVP/90mM ectoin/100µg/ml catalase showed the higher metabolic activity of

cryopreserved cells than other freezing solutions after 24h post-thaw culture.

Overall, the trend in viability follows that of the flow cytometry results indicating solution

8 achieves higher metabolic activity than other freezing solution. Furthermore, all the

formulated freezing solutions superseded the viability obtained with control as 10%

Me2SO in PBS. However, the higher metabolic activity, shown by the cryopreserved cells

frozen in solution 1, was statistically insignificant to the metabolic activity shown by the

control group. Similarly, the metabolic activity of hADSCs preserved in solution 8 was

statistically insignificant to that of solution 5.

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6.2.6 Validation of Taguchi results

The experimental confirmation test is known to be the final step in verifying the results

drawn based on Taguchi’s design approach. Thus, a confirmation experiment was

conducted by utilizing the obtained optimal concentration of the control factors. The

optimal concentration of the components was selected based on the main effect plot (Fig

6.8). The result showed that the optimal concentration of the control factors is 160mM

NaCl, 10% PVP, 90mM ectoin and 100µg/ml catalase. Therefore, for validation of the

results obtained by the Taguchi’s design, a freezing solution comprised of the

concentrations mentioned above of the individual components was prepared. The solution

comprised of 160mM NaCl/ 10%PVP/90mM ectoin/100 µg/ml catalase achieved post-

thawed cell viability of 85% assessed by Trypan blue dye exclusion assay (Table 6.3).

This result was further confirmed by flow cytometry and MTT assay (Fig 6.8K and 6.10),

which showed the superior viability of 160mM NaCl/ 10%PVP/90mM ectoin/100 µg/ml

catalase solution over solution 8. The obtained viability (85%) using the optimized

freezing cocktail is the highest among the solutions used in the experiment.

Overall, the study provides an optimal composition of a novel freezing solution that

comprised of 160mM NaCl/10% PVP/90mM ectoin/100µg/ml catalase achieving

maximum viability of 85%.

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6.3 Evaluation of signaling pathway inhibitors for cryopreservation of hADSCs

Apoptosis is one of the important mechanisms of cell death during cryopreservation. Low-

temperature stress induces apoptosis, the stepwise-programmed cell death [202]. The

proteins over the cell surface receive the stress signal and send the signals downstream

inside the cells to activate caspases. The activated caspases stimulate cascades of the

proteolytic process to initiate the self-destruction process leading to DNA degradation and

cell membrane disruption. Similar to apoptosis, altered cytoskeleton of cells during

cryopreservation is another important cause of cell death [203-205]. The cytoskeleton

provides strong support to cell plasma membrane maintains the organizational distribution

of organelles and helps in executing signaling cascade properly. Thus, any alteration in the

cytoskeleton because of ice-mediated mechanical damage during cryopreservation alters

the morphology and functions of cells leading to altered rehydration and viability of post-

thawed cells.

Supplementation of molecules that prevent or inhibit the caspases activation and maintains

the stability of cytoskeleton, in freezing solution was reported to increase the viability of

post-thaw cells. Z-VAD-FMK, an irreversible inhibitor of caspases, enhanced the viability

of post-thawed human embryonic stem cells when supplemented in freezing solution and

post-thaw culture. Supplementation of Z-VAD-FMK in freezing solution also improved

the post-thaw viability of porcine hepatocytes and human amniotic fluid-derived

mesenchymal stem cells [169, 206]. Another molecule Y-27632, a Rho kinase inhibitor,

stabilizes the cytoskeleton and observed to improve the survival of post-thawed cells. Li X

et al. demonstrated that Y-27632 added to the post-thaw culture medium for 24h post-

thaw increased human embryonic stem cell survival rate [207]. Studies by Martin-Ibanez

et al. replicated this initial finding [208]. Furthermore, the effect of ROCK inhibitor Y-

27632 was shown to have similar effects on the recovery from cryopreservation of adult

stem cells and bone marrow-derived MSCs as well as human induced pluripotent stem

cells [37, 209, 210].

Given the beneficial effect of Z-VAD-FMK and Y-27632 on the recovery of post-thawed

cells and in the effort to enhance the viability of cryopreserved human adipose-derived

mesenchymal stem cells, it was hypothesized that the supplementation of the above two

molecules might enhance the viability of post-thawed hADSCs. Thus, in this phase of

work, we have supplemented 10Nm Z-VAD-FMK and 100mM Y-27632 with the most

effective 160mM NaCl/ 10%PVP/90mM ectoin/100µg/ml catalase freezing solution

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obtained in the previous chapter and the efficacy of the new freezing solutions was

evaluated towards for cryopreservation of hADSCs.

6.3.1 Trypan blue dye exclusion

The immediate viability of post-thawed hADSCs was evaluated by Trypan blue dye

exclusion assay. The results (Fig 6.11) reveal that the addition of 10nM Z-VAD-FMK and

100mM Y-27632 has no impact on the viability of post-thawed hADSCs. Although, the

mean viability obtained with Y-27632 (85.33± 3.78%) was higher than Z-VAD-FMK

(84.67± 3.21%), the difference is statistically insignificant (p<0.05). Moreover, the

difference in viability obtained with the ROCK inhibitor and the general caspases inhibitor

was insignificant to the viability shown with control (84.67±2.08).

Figure 6.11: Trypan blue assay results showing the effect of signaling pathway

inhibitors supplemented with the formulated freezing solution on the cryopreserved

hADSCs viability. The results reveal that the addition of Z-VAD-FMK and Y-27632

has no significant impact on the viability of post-thawed hADSCs.

6.3.2 MTT assay

Post-thaw recovery of the cells in culture was assessed after 24h by MTT assay (Fig 6.12).

MTT assay also observed similar findings of no significant benefit with the addition of Z-

VAD-FMK and Y-27632 in the recovery of post-thawed hADSCs.

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Figure 6.12: MTT assay showing the post-thaw viability of cryopreserved hADSCs

using signaling pathway inhibitor supplemented with the developed freezing solution.

As indicated, no significant benefit with the addition of Z-VAD-FMK and Y-27632 in

the recovery of post-thawed hADSCs was obtained.

The results corroborated with the findings of Lamas NJ et al., which stated that in contrast

to other stem cell types, Y-27632 supplementation is not a suitable strategy to enhance

hADSCs expansion in culture rather a continuous supplementation Y-27632 in culture led

to decrease metabolic activity and numbers of hADSCs [211]. Similar negative findings

on the use of Y-27632 were earlier reported for cord blood-derived hematopoietic

progenitor cells [212]. However, Qu CQ et al. reported that addition of ROCK inhibitor

along with glutathione increased the viability of cryopreserved porcine adipose-derived

stem cells [213]. The beneficial effect may be due to glutathione than ROCK inhibitor.

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6.4 Optimization of controlled rate freezing parameters for cryopreservation of

hADSCs using the developed freezing solution

The cooling rate is one of the most important factors that controls the viability of cells.

While subjected to slow cooling rate leads to extracellular ice formation and the

segregated solute concentration effect over cell membrane become an important cause of

cryoinjury, fast cooling rate induces intracellular ice formation and ice crystals mediated

injury thereby causes cell death. Thus, a trade-off between fast and slow cooling rate is

essential to achieve an optimized cooling rate providing maximum cell viability. The

problem is further complicated by the fact that the cryoprocessing steps are

interdependent, and thus cannot be optimized independently. For example, the optimal

cooling rate is dependent on the CPA types and concentration.

In this part of research work, an attempt has been given to achieve an optimized cooling

rate for the cryopreservation of hADSCs using the developed 160mM NaCl/

10%PVP/90mM ectoin/100µg/ml catalase freezing solution.

6.4.1 Effect of cooling rate

To achieve the optimized cooling rate, the cryopreservation experiment was carried out in

different prenucleation cooling rate such as -0.5°C/min,-1°C/min,-2.5°C/min,-5°C/min

from 4°C to -120°C. The samples were kept at 4°C for 1h incubation period before being

subjected to controlled rate freezers. The one-hour incubation period was necessary for

equilibrating the freezing solution with the cells. Upon completion of controlled freezing,

the cryovials were transferred immediately into LN2 to avoid recrystallization injury to

cells. The efficiency of the optimum freezing condition on the viability was assessed as

described here.

Trypan blue dye exclusion assay

The viability and recovery of the post-thawed hADSCs were assessed by Trypan blue dye

exclusion and MTT assay after 48h. The results (Fig 6.13) show that cooling at the rate of

-1°C/min achieved the highest viability of 89.3± 3.8%. The viability is 8% higher than the

viability obtained using -0.5°C/min (81.3±3.8%) and 4% better than the sample frozen at -

80°C freezer in Mr. Frosty, an isopropanol containing commercial container for

cryostoring cells. Thirumala et al. also reported similar finding that -1°C/min provided the

maximum viability of hADSCs compared to other cooling rates.

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Figure 6.13: Trypan blue assays showing the effect of cooling rate on post-thaw

viability of hADSCs cryopreserved in 160mM NaCl/ 10%PVP/90mM

ectoin/100µg/ml catalase freezing solution.

MTT assay

The metabolic activity measured by MTT assay is shown in Fig 6.14. As indicated, the

OD was increased by increasing rate from -0.5 to -1°C/min and then the OD was slowly

decreased with the higher cooling rate. Thus, -1°C/min is confirmed as the most favorable

cooling rate achieving maximum viability of frozen hADSCs.

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Figure 6.14: Effect of cooling rate on post-thaw viability of hADSCs cryopreserved in

160mM NaCl/ 10%PVP/90mM ectoin/100µg/ml catalase freezing solution assessed by

MTT assay

6.4.2 Effect of seeding temperature

As described in the previous section, at low-temperature seeding causes the quick

formation of extracellular ice that minimizes the detrimental effect of phase change from

liquid to ice in the course of cooling thereby prevents ice-mediated damage. Seeding

should be performed at the temperature above spontaneous intracellular ice formation,

which is usually in the range of −7°C and −12°C. Thirumala et al. reported that for

hADSCs, freezing usually does not start below -6°C. Therefore, to evaluate the effect of

seeding temperature on the viability of hADSCs, the cells were frozen in a controlled rate

freezer at -1°C/min cooling rate from 4°C to -120°C and the seeding was induced

mechanically using the facilities of the CRF at -5°C,-7°C and -9°C. The vials were stored

in LN2 and viability, and recovery of cells was assessed after 48h of storage.

Trypan blue dye exclusion assay

The results in Fig 6.15 indicate that among the seeding temperatures under study, the

maximum cell viability of 89.4 ± 3.6% was obtained with seeding at -7°C. A comparative

hADSCs viability was also shown at -9°C. Therefore,-7C was established as the most

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favorable seeding temperature. However, compared to non-seeded samples, the seeded

samples at -7°C showed no additional improvement in cell viability.

Figure 6.15: Trypan blue assay showing the effect of seeding temperature on post-

thaw viability of hADSCs cryopreserved in the formulated 160mM

NaCl/10%PVP/90mM ectoin/100µg/ml catalase freezing solution.

MTT assay

MTT assay (Fig 6.16) corroborates with the viability result obtained by Trypan blue assay

that the seeding at -7°C achieved higher viability than other seeding temperature. Similar

to Trypan blue assay, the viability results are not significantly different from each other

indicating the negligible influence of seeding in improving the viability of hADSCs

cryopreservation.

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Figure 6.16: Effect of seeding temperature on post-thaw viability of hADSCs

cryopreserved in 160mM NaCl/ 10%PVP/90mM ectoin/100µg/ml catalase freezing

solution assessed by MTT assay.

Overall, it has been demonstrated that -1°C/min cooling rate is effective in improving the

viability of hADSCs cryopreserved with 160mM NaCl/ 10%PVP/90mM ectoin/100µg/ml

catalase freezing solution. Furthermore, the study on the effect of seeding temperature

indicates that the influence of seeding temperature on the post-thaw viability temperature

of hADSCs is negligible.

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6.5 Effect of long-term storage on cryopreserved hADSCs using the developed

freezing solution

The effect of long-term storage on the viability of hADSCs cryopreserved in 160mM

NaCl/10% PVP/90mM ectoin/100 µg/ml catalase freezing solution. Long-term evaluation

is necessary as free radical and apoptosis-mediated cell death cause loss of viability over

time. The cells were equilibrated with the freezing solution for one hour at 4°C before

being controlled rate frozen at -1°C/min from 4°C to -120°C and plunged into LN2.The

vials were retrieved after 15 , 30 , 60 and 90 days of storage in LN2 and thawed rapidly to

assess the viability and recovery of cells.

6.5.1 Trypan blue dye exclusion assay

The results in Fig 6.17 indicate the decrease in viability over time and thus, viability

dropped from 88.6 ± 4.5% from 15 days to 85 ± 5% after 90 days of storage. The

difference in viability over time is insignificant indicating the freezing solution is able to

maintain viability for long-term.

Figure 6.17: Long-term viability study on cryopreserved hADSCs using 160mM

NaCl/10% PVP/90mM ectoin/100 µg/ml catalase freezing solution assessed by

Trypan blue assay. The viability change over 90 days storage time is insignificant

indicating the freezing solution is able to maintain hADSCs viability for long-time.

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6.5.2 MTT assay

The metabolic activity in term of OD measured during the 15-90 days of storage period is

shown in Fig 6.18. No statistically significant difference in viability was noticed during

this period thus proving the efficiency of the developed freezing solution in the

maintenance of cryopreserved hADSCs.

Figure 6.18: Long-term viability study on cryopreserved hADSCs using 160mM

NaCl/10% PVP/90mM ectoin/100µg/ml catalase freezing solution assessed by MTT

assay. Similar trend in viability as assessed by Trypan blue assay confirming the

suitability of the developed freezing solution in preserving hADSCs for a long time.

6.5.3 Morphology of cryopreserved hADSCs

The regular fibroblastic morphology and adherancy to plastics are important features for

the structural and functional integrity of hADSCs. Therefore, the morphological

characteristics of cryopreserved (90 days) hADSCs were assessed by phase contrast

microscopy.

As observed in Fig 6.19, on retrieval, the cryopreserved hADSCs adhered to the surface

and the changed to fibroblastic appearance before reaching confluency on 12th day like

fresh mesenchymal stem cells proving that the developed freezing solution has no

detrimental or adverse effect on the adherancy and morphology of cryopreserved

hADSCs.

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Figure 6.19: Morphological changes of cryopreserved (90 days storage) hADSCs in

160mM NaCl/10% PVP/90mM ectoin/100µg/ml catalase freezing solution as

observed under phase contrast microscope. 0-day (a), 6th day (b) and 12th day(c).

(Scale bar 100µm)

6.5.4 Cytoskeleton distribution of cryopreserved hADSCs

The hADSCs were stained with Phalloidin-Alexa fluor-488 and Hoechst to stain the actin

cytoskeleton and nucleus respectively and observed under CLSM. As can be observed in

Fig 6.20, there is the peripheral distribution of actin, which is a regular feature of normal

cells. Thus, the developed freezing solution and the cryopreservation strategy had no

adverse effect over the cytoskeleton of hADSCs and retained the regular cytoskeletal

distribution pattern like fresh cells.

Figure 6.20: Cytoskeleton distribution of cryopreserved (90 days) hADSCs in 160mM

NaCl/10% PVP/90mM ectoin/100 µg/ml catalase freezing solution. The peripheral F-

actin distribution represents regular arrangement of cytoskeleton.

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6.5.5 Proliferation kinetics

The proliferation kinetics of the cryopreserved (90 days) hADSCs was compared with

non-cryopreserved hADSCs (P6) using Trypan blue assay (Fig 6.21). The cryopreserved

hADSCs has been shown to retain the normal proliferation pattern indicating

cryopreservation process, and the freezing solution has no adverse effect on the functional

integrity of cryopreserved hADSCs.

Figure 6.21: Proliferation kinetics of cryopreserved (90 days) hADSCs in 160mM

NaCl/10% PVP/90mM ectoin/100 µg/ml catalase freezing solution. The hADSCs has

shown to retain the normal proliferation pattern indicating the freezing solution has

no adverse effect on the functional integrity of cryopreserved hADSCs.

6.5.6 Differentiation ability

The differentiation of cryopreserved (90 days) hADSCs into osteogenic and adipogenic

lineages was performed to evaluate the maintenance of differentiation ability of

cryopreserved hADSCs.

The histological results of the osteogenic differentiation of hADSCs are shown in Fig

6.22a. hADSCs cryopreserved in 160mM NaCl/10% PVP/90mM ectoin/100 µg/ml

catalase freezing solution for 90 days is observed to be sufficiently differentiated into

osteoblasts as shown by the deposition of calcium on 21 days of culture. Thus, the

129 | P a g e

cryopreserved hADSCs retained the differentiation ability to osteogenic lineages thereby

proven the ability of formulated freezing solution in maintaining the differentiation

potential of hADSCs. Furthermore, as can be seen in Fig 22b, lipid droplets accumulated

inside the hADSCs on 14th day culture in adipogenic differentiation media proving the

cryopreserved hADSCs also retained the differential ability to adipogenic lineages.

Figure 6.22: Differential ability of frozen (90 days storage) hADSCs in 160mM

NaCl/10% PVP/90mM ectoin/100 µg/ml catalase freezing solution for osteogenesis (a)

and adipogenesis (b) seen under fluorescent microscope. Calcium deposition in

osteogenesis was evaluated by Alizarin red stain (a) and intracellular lipid

accumulation in adipogenesis was assessed by Oil Red O stain. (Scale bar 100µm)

Overall, among the freezing solutions, 160mM NaCl/10% PVP/90mM ectoin/100µg/ml

catalase was found to be the most effective in maintaining cryopreserved adipose tissue as

indicated in Table 6.6 providing the maximum viability of 89% by control rate freezing

at -1°C/min cooling rate followed by storage in LN2. The developed 160mM NaCl/10%

PVP/90mM ectoin/100µg/ml catalase freezing solution may be used for the

cryopreservation of hADSCs thereby paving its future clinical application.

Table 6.6 Comparison of hADSCs viability cryopreserved in various freezing

solution

Freezing solution Trypan blue

(% viability)

PBS+10% Me2SO+20% FBS 60

PBS+ 10% PVP 56

0.9% NaCl+10% PVP 70

0.9% NaCl+10% PVP+60mM ectoin 81

160mM NaCl+10% PVP+90mM ectoin+100µg/ml catalase 85

160mM NaCl+10% PVP+90mM ectoin+100µg/ml catalase:

Cooling at -1°C/min and storage in LN2 for 48h

89

160mM NaCl+10% PVP+90mM ectoin+100µg/ml catalase:

Cooling at -1°C/min and storage in LN2 for 3 months

85

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Chapter 7

Summary & Conclusion

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Lipofilling, which is an important surgical procedure, requires a lot of adipose tissue to fill

up the depressed region or scar of patient body. Besides lipofilling, there is a growing

demand of adipose tissue-derived stem cells for tissue engineering and stem cell therapy

applications. Therefore, in recent past, research interest has been generated towards the

harvesting of adipose tissue as well as production of stem cells, (mesenchymal stem cells

in particular) isolated from adipose tissue. In this context, development of an effective

cryopreservation strategy is of utmost importance for preserving adipose tissue and

hADSCs with long shelve-life for the advancement of these products for clinical

applications. Cryopreservation using conventional freezing solution consisting of dimethyl

sulfoxide and fetal bovine serum has several detrimental effects including, the acute and

chronic toxicity of patients and genotoxicity of preserved cells. Furthermore, the most

commonly used DMEM carrier media having multicomponent constituents creates more

complex and unpredictable cell environment at sub-zero temperature because of ice

crystallization.

Keeping the above issues in view, the present research aims to explore potential natural

cryoprotective agents and suitable carrier media for developing a Me2SO-free and serum-

free freezing solution for preserving adipose tissue and adipose tissue derived stem cells

with long storage life.

The most interesting results obtained from the above research is summarized as follows-

A. Cryopreservation of adipose tissue

I. In the first phase of the thesis work, the efficiency of various hydrocolloids and natural

organic osmolytes as extracellular and intracellular CPAs as well as individual PBS

components such as NaCl, Na2HPO4, KCl and KH2PO4 as possible carrier media were

evaluated by performing cryopreservation experiments with adipose tissue. Based on the

viability results measured by oil ratio as a preliminary screening criteria followed by XTT,

G3PDH and TBARS analysis, trehalose, ectoin and NaCl were selected as the most

efficient CPAs and carrier media respectively.

II. The selected extracellular and intracellular CPAs in NaCl as carrier media were

evaluated individually and in combination to formulate an freezing solution cocktail. To

this end, different batches of freezing solutions namely 160mM NaCl/90mM trehalose,

160mM NaCl/150mM ectoin and 160mM NaCl/90mM trehalose/150mM ectoin were

132 | P a g e

prepared and the conventional 10% Me2SO was used as control. Among these, freezing

solution consisting of 160mM NaCl/90mM trehalose was found to be the most efficient

achieving adipose tissue viability of 85%. The viability was comparable to the viability

shown by 160mM NaCl/10% Me2SO as a control. Interestingly, ectoin in freezing

solutions did not show any significant impact in cryopreserving adipose tissue, whereas a

substantial tissue viability (84%) was obtained using 160mM NaCl only.

III. The oxidative stress mediated damage is a critical phenomenon in cryopreservation,

which is expected to be higher in the case of adipose tissue than other body tissues.

Therefore, it was hypothesized that supplementation of antioxidants in freezing solution

might improve the cryopreservation outcome of adipose tissue. Therefore,

cryopreservation experiments were performed with adipose tissue using the formulated

160mM NaCl and 90mM trehalose/160mM NaCl freezing solutions supplemented with

different antioxidants such as curcumin, carnitine, lipoic acid,Vit E, gallic acid, ellagic

acid with varying concentration level. Among these, 1mg/ml curcumin and 3mg/ml

carnitine were found to be the potential antioxidants improving the crypreservation

outcome of both the freezing solutions and the measured viability was higher than the

viability obtained with the conventional 10% Me2SO. Overall, freezing solutions

supplemented with curcumin has shown higher viability than carnitine and 160mM

NaCl/90mM trehalose/1mg/ml curcumin achieved the maximum viability of 90%. Thus,

the study provides a Me2SO free superior freezing solution for adipose tissue

cryopreservation.

IV. An effort has been also given for further improvement of the cryopreservation

efficiency by performing the cryopreservation of adipose tissue in a control rate freezer

which resulted in 93% maximum post-thaw tissue viability at -1°C/min as an optimum

cooling rate.

V. The maintenance of tissue viability for a long time is the most vital aspect of freezing

solution to be used as cryopreservation strategy. Therefore, the efficiency of the

formulated freezing solution towards maintaining tissue viability was evaluated during 90

days of storage of cryopreserved adipose tissue in liquid nitrogen. The result revealed that

a gradual decrease in tissue viability occurred over time. However, the viability after 90

days of storage was not significantly different from viability after 7 days as evident by oil

133 | P a g e

ratio, XTT, G3PDH and TBARS assays representing the ability of the freezing solution in

maintaining long-shelve life. The structural and functional integrity of cryopreserved

adipose tissue were maintained by the freezing solution which are evident from the

morphological characteristics (Laser Scanning Confocal Microscopic image analysis) as

well as ability of frozen adipose tissue to provide hADSCs with retained metabolic

activity (MTT assay), proliferation, and differentiation (Alizarin Red assay and Oil Red O

staining) potential .

Thus, it has been concluded that the formulated freezing solution comprising of 160mM

NaCl/90mM trehalose/1mg/ml curcumin has the ability in maintaining the viability for a

long time and cryopreserved adipose tissue provides hADSCs with desired metabolic

acvitivy, proliferation and differentiation potential.

B. Cryopreservation of hADSCs

I. Besides adipose tissue cryopreservation, an effort has been given to isolate hADSCs

from adipose tissue and develop a cryopreservation strategy for their preservation by

formulating an effective freezing solution. Similar to adipose tissue, the efficiency of

various hydrocolloids and natural organic osmolytes as extracellular and intracellular

CPAs were evaluated for their efficacy in maintaining viability of adipose tissue by

performing cryopreservation experiments. Based on the viability results measured by

Trypan blue and MTT assays, among the hydrocolloids, 10% PVP in PBS as carrier media

was found to be the most effective freezing solution achieving maximum cell viability of

56% followed by tragacanth gum (43%). The viability obtained with 10% PVP/PBS was

higher than 10%Me2SO/PBS solution (control).

II. PBS as carrier media consists of NaCl, Na2HPO4, KCl and KH2PO4. The specific role

of these components as carrier media in cryopreservation of stem cells has not been

explored so far and hence these components were evaluated by supplemented with 10%

PVP. Among the various compositions, 10%PVP/0.9%NaCl showed the maximum cell

viability of 70% measured by Trypan Blus assay. The chloride based ionic compounds

was found to perform better than phosphate based compounds indicating the freezing

solution containing extracellular anions and cations provide better viability compared to

the respective intracellular counterpart. These findings were also supported by MTT assay.

134 | P a g e

III. Efforts have been given for further improvement of the efficiency of the

10%PVP/0.9%NaCl by the addition of ectoin and hydroxyectoin as the potential organic

osmolytes. The experimental study has revealed that the addition of osmolytes in freezing

solution increased the viability of cryopreserved hADSCs and the highest viability of 82%

was achieved by developing 10%PVP/0.9%NaCl/60mM ectoin freezing solution.

The cytoskeletal analysis from confocal microscopic images shows the normal

cytoskeletal distribution pattern of F-actin, that indicate that hADSCs cryopreserved in

10%PVP/0.9%NaCl/60mM ectoin was able to retain normal cell morphology like fresh

hADSCs, proving the suitability of the formulated freezing solution.

The impact of the formulated 10%PVP/0.9%NaCl/60mM ectoin on the differentiation

ability of frozen hADSCs was investigated. The study on the differentiation potential of

cryopreserved hADSCs by incubating in osteogenic and adipogenic media for a period of

21 and 14 days respectively has confirmed that the developed freezing solution is able to

maintain the osteogenic and adipogenic differentiation ability of the cryopreserved

hADSCs as assessed by Alizarin Red assay and Oil Red O staining.

IV. The cryopreservation efficacy of the formulated 10%PVP/0.9%NaCl/60mM ectoin

freezing solution was further optimized by adopting Taguchi orthogonal design

methodology. Catalase, a natural antioxidant was also incorporated into the formulation of

freezing solutions with different concentrations to mitigate free radicals mediated cell

damage during freezing. The optimal composition of the freezing solution was obtained as

160mM NaCl/10% PVP/90mM ectoin/100µg/ml catalase, providing the maximum post-

thaw viability of 85%. Furthermore, the addition of caspase and Rho-kinase inhibitors did

not show any influence on the post- thaw viability of frozen hADSCs. Thus, 160mM

NaCl/ 10% PVP/ 90mM ectoin/100µg/ml catalase was obtained as the most favorable

freezing solution.

V. To achieve better cryopreservation outcome, the cryopreservation experiment was

carried out in a controlled rate freezer at different cooling rate such as -0.5C/min,-

1°C/min,-2.5°C/min,-5°C/min from 4°C to -120°C. The measured viability was enhanced

to 89% in control rate freezing with the optimized cooling rate of -1°C/min.

135 | P a g e

The effect of long-term storage on the viability of hADSCs cryopreserved in 160mM

NaCl/10%PVP/90mM ectoin/100µg/ml catalase freezing solution indicated a slow

reduction of viability over time and the viability was dropped from 89% in 15 days to 85%

after 90 days of storage. The decrease in viability is not significant representing the ability

of the developed freezing solution in maintaining viability of adipose tissue for a long

time.

The structural and functional integrity of cryopreserved (90 days storage) hADSCs

assessed by phase contrast and confocal microscopy, indicate that the cryopreserved

hADSCs maintained the fibroblastic morphology and attained 80-85% confluence on

12thday of culture. The cytoskeletal analysis has revealed the peripheral distribution of F-

actin thereby the maintenance of the structure of hADSCs. The study on the proliferation

and differentiation to osteogenic and adipogenic lineages of 90 days storage cryopreserved

hADSCs were performed and compared with fresh hADSCs. The cryopreserved hADSCs

retained the normal proliferation and differentiation ability similar to fresh hADSCs

confirming that the freezing solution has no adverse effect on the functional integrity of

cryopreserved hADSCs.

All the above taken together, the developed Me2SO free, serum free and non-toxic 160mM

NaCl/10% PVP/90mM ectoin/100µg/ml catalase has been proven to be a potential

freezing solution in maintenaning viability, proliferation and differentiation capability of

hADSCs for long e time.

Overall, a Me2SO free, serum free and non-toxic freezing solution was developed from

natural cryoprotective agents, NaCl as carrier media and curcumin as antioxidant. The

freezing solution comprising of 160mM NaCl/90mM trehalose/1mg/ml curcumin is

effective for long-term preservation of adipose tissue with maintained tissue viability

(89%) and provide hADSCs with retained metabolic activity, proliferation, and

differentiation ability. Similarly, a novel freezing solution that is devoid of the harmful

Me2SO and serum was developed for the cryopreservation of hADSCs. The freezing

solution comprising of 160mM NaCl/10% PVP/90mM ectoin/100µg/ml catalase is

effective for cryopreservation of hADSCs with long-shelve life. It is, thus, concluded that

these developed freezing solutions might provide effective cryopreservation strategies for

the supply of these products for clinical applications in future.

136 | P a g e

Suggested further study

The present research work offers effective freezing solutions for preserving adipose tissue

and hADSCs. However, keeping in view of the limitation of the present work, the

following future work has been suggested-

I. Evaluation of the efficiency of the developed freezing solutions for longer period

of storage time than 3 months period of study undertaken in the present work.

II. Detail study on the parametric sensitivity of the control rate freezing process

III. In-vivo study must be performed using animal model, keeping in view of their

cilinical application

137 | P a g e

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List of Publications from thesis

1.Sirsendu Sekhar Ray, Krishna Pramanik, Sunil Kumar Sarangi, Nirved Jain. Serum-free

non-toxic freezing solution for cryopreservation of human adipose tissue-derived

mesenchymal stem cells. Biotechnology letters, May (2016) pp 1-8

2.Sirsendu Sekhar Ray, Krishna Pramanik, Sunil Kumar Sarangi, Nirved Jain.

Optimization of a Dimethylsulfoxide and Serum Free Freezing Solution Composition to

Enhance Viability of Adipose Tissue-Derived Stem Cells. Archives of plastic surgery

(Manuscript under review)

3.Sirsendu Sekhar Ray, Krishna Pramanik, Sunil Kumar Sarangi, Nirved Jain.Cryopreserv

ation of Adipose Tissue: Evaluation of cryoprotectants and ionic compounds. (Manuscript

under review)

4.Sirsendu Sekhar Ray, Krishna Pramanik, Sunil Kumar Sarangi, Nirved Jain.Cryopreserv

ation of Adipose Tissue: Role of antioxidants. (filing of patent under process)

5.Sirsendu Sekhar Ray, Krishna Pramanik, Sunil Kumar Sarangi, Manoj Khanna. Role of

phytochemicals in human Adipose tissue cryopreservation. Advances in low temperature

biology" London, 11th to 13th October 2012

157 | P a g e

Dr. Sirsendu Sekhar Ray

Assistant Professor

Department of Biotechnology and Medical Engineering

National Institute of Technology Rourkela

Educational Qualification

2007: MMST (Master in Medical Science and Technology)

Indian Institute of Technology Kharagpur, India

2003: MBBS (Bachelor in Medicine and Bachelors in Surgery)

Dr. Sampurnanand Medical College, Jodhpur / Rajasthan University

Research Interest: Cryobiology, Tissue Engineering, Cell -Material Interaction,

Nanobiotechnology

Publications: Journal-22, Conference-40, Book chapter-3

Teaching: Medical Science, Clinical Science, Cryo-tissue engineering, Material in

medical science, Health informatics etc.

Address

FR-40, NIT Rourkela campus

Date of birth: 06.12.1979

Nationality: Indian