Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1072

_____________________________ _____________________________

Evaluation of Alginate Microcapsules for

Use in Transplantation Of Islets of Langerhans

BY

AILEEN KING

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2001

ABSTRACT

King, A. 2001. Evaluation of Alginate Microcapsules for Use in Transplantation ofIslets of Langerhans. Acta Universitatis Upsaliensis. Comprehensive Summaries ofUppsala Dissertations from the Faculty of Medicine 1072. 43 pp. Uppsala. ISBN 91-554-5113-6

Transplantation of islets of Langerhans is a potential treatment of type 1 diabetes thataims to restore normal glucose homeostasis. Microencapsulation of islets could enabletransplantation in the absence of immunosuppression, which would be beneficial as theside effects associated with immunosuppression outweigh the potential benefits of islettransplantation. Alginate is a polysaccharide that can be harvested from brown algaeand is often used for microencapsulation of cells.The aim of this study was to evaluate alginate/poly-L-lysine/alginate capsules withregard to their biocompatibility and permeability to cytokines. Moreover, the functionof microencapsulated islets was studied in vitro as well as their ability to reversehyperglycaemia in diabetic mice.Microencapsulated rodent islets functioned well in vitro, with similar insulin releaserates and glucose oxidation rates as naked islets. However, when cultured withinterleukin-1β and tumour necrosis factor-α, microencapsulated islets werefunctionally suppressed, showing that the capsules are permeable to these cytokines.The biocompatibility of capsules varied depending on their composition. The presenceof poly-L-lysine in the capsule decreased the biocompatibility. However, thebiocompatibility of the capsules was improved when the coating alginate had beenepimerised, i.e. enyzmatically tailored. Transplantation of microencapsulated allogeneicislets to immune competent mice lowered blood glucose concentrations up to 1 monthafter implantation. The success of the microencapsulated islet graft depended on thecomposition of the alginate/poly-L-lysine/alginate capsule used, as capsules that hadpoor biocompatibility failed to reverse hyperglycaemia more than transiently in athymicnude mice.In conclusion, alginate/poly-L-lysine/alginate capsules can protect islets of Langerhansfrom allogeneic rejection in mice. However, the composition of the capsule is of criticalimportance in the success of transplantation. Epimerised alginates may provide a novelcapsule with ideal properties for microencapsulation of islets of Langerhans.

Key words: Alginate, biocompatibility, cytokines, epimerases, islet transplantation,microencapsulation.

Aileen King, Department of Medical Cell Biology, Biomedical Centre, UppsalaUniversity, Box 571, SE-751 23 Uppsala, Sweden

© Aileen King 2001

ISSN 0282-7476ISBN 91-554-5113-6Printed in Sweden by Uppsala University, Tryck & Medier, Uppsala 2001

The glory of life is not in

never falling but in getting

up every time you fall

Advice given to me by my mother

before starting my Ph D studies

REPORTS CONSITUTING THE THESIS

This thesis is based on the following papers, which will be referred to in the text by

their Roman numerals:

I King A, Sandler S, Andersson A, Hellerström C, Kulseng B, Skjåk-Bræk G:

Glucose metabolism in vitro of cultured and transplanted mouse pancreatic islets

microencapsulated by means of a high-voltage electrostatic field. Diabetes Care

22: B121-B126, 1999.

II King A, Andersson A, Sandler S: Cytokine-induced functional suppression of

microencapsulated rat pancreatic islets in vitro. Transplantation 70: 380-383,

2000.

III King A, Sandler S, Andersson A: The effect of host factors and capsule

composition on the cellular overgrowth on implanted alginate capsules. Journal

of Biomedical Materials Research 57:374-383, 2001.

IV King A, Lau J, Nordin A, Andersson A: The effect of capsule composition in the

reversal of hyperglycaemia in diabetic mice transplanted with microencapsulated

allogeneic islets. Manuscript.

V King A, Strand B, Rokstad A-M, Kulseng B, Andersson A, Skjåk-Bræk G,

Sandler S: Improvement of the biocompatibility of alginate/poly-L-

lysine/alginate microcapsules by the use of epimerised alginate as a coating.

Manuscript.

TABLE OF CONTENTS

REPORTS CONSTITUTING THE THESIS 5

ABBREVIATIONS 8

1 INTRODUCTION 9

1.1 Background 9

1.1.1 Type 1 diabetes 9

1.1.2 Pancreatic islet transplantation 9

1.1.3 Encapsulation of cells 10

1.1.4 Microencapsulation 11

1.2 Characteristics of capsules 11

1.2.1 Capsule composition 11

1.2.1.1 Materials used for cell microencapsulation 11

1.2.1.2 Alginate/poly-L-lysine/alginate capsules 12

1.2.2 Alginate capsule properties 13

1.2.2.1 Mechanical strength 13

1.2.2.2 Permeability 14

1.2.2.3 Biocompatibility 14

1.2.2.4 Size 14

1.2.3 Enzymatic tailoring of alginates 15

1.3 Biological responses affecting microencapsulated islets of Langerhans 16

1.3.1 Islet function 16

1.3.1.1 Peritoneal cavity as an implantation site 17

1.3.2 Host response to capsules 17

1.3.2.1 Cellular overgrowth as a barrier to nutrient diffusion 18

1.3.2.2 Cytokine-induced suppression of islets of Langerhans 19

1.3.3 Host response to encapsulated islets 19

1.4 Interaction of host responses and capsules 20

2 AIMS 21

6

3 OVERVIEW OF THE METHODS USED 22

3.1 Microencapsulation and transplantation of islets 22

3.1.1 Islet isolation 22

3.1.2 Microencapsulation of islets 22

3.1.3 Implantation of islets and capsules 24

3.1.4 Retrieval of implanted capsules 25

3.1.5 Induction of diabetes 25

3.2 Functional tests 25

3.2.1 Glucose oxidation rates 25

3.2.2 Insulin release rates 26

3.2.3 DNA measurements 26

3.2.4 Nitrite measurements 26

3.2.5 Real time PCR 26

4 RESULTS AND DISCUSSION 27

4.1 In vitro function of rodent islets after microencapsulation 27

4.2 Permeability of microencapsulated islets to cytokines in vitro 28

4.3 Biocompatibility of the capsules 29

4.3.1 Effect of capsule composition 29

4.3.2 Biological responses affecting cellular overgrowth of capsules 30

4.4 In vivo function of microencapsulated islets 31

4.4.1 Transplantation to nude C57BL/6 mice 31

4.4.2 Transplantation to immune competent C57BL/6 mice 32

4.5 Capsule properties required for optimal function of microencapsulated islets 33

5 CONCLUSIONS 34

ACKNOWLEDGEMENTS 35

REFERENCES 37

7

ABBREVIATIONS

G guluronic acid

IL-1 interleukin 1

iNOS inducible nitric oxide synthase

M mannuronic acid

MG alternating mannuronic and guluronic acid

NO nitric oxide

PCR polymerase chain reaction

PLL poly-L-lysine

TNF tumour necrosis factor

INTRODUCTION

9

1 INTRODUCTION

1.1 Background

1.1.1 Type 1 diabetes

The β-cells within the islets of Langerhans produce and secrete insulin, a hormone

essential for normal glucose homeostasis. Type 1 diabetes is characterised by a

deficiency in insulin, caused by the destruction of the β-cells. The aetiology of type 1

diabetes is not known, although there is evidence that most cases are due to an

autoimmune disorder (1). The current treatment of choice for type 1 diabetic patients is

insulin therapy. However, many patients develop secondary complications such as

nephropathy, neuropathy and cardiovascular problems. Intense insulin therapy decreases

the risk for such complications but increases the risk of hypoglycaemic episodes (2).

Transplantation of islets of Langerhans is a potential treatment of type 1 diabetes that

aims to normalise glucose homeostasis, and thereby reduce the risk of complications

and improve the quality of life of the diabetic patient.

1.1.2 Pancreatic islet transplantation

Whole pancreas transplantation can normalise blood glucose homeostasis in diabetic

patients (3). Moreover, complications are reduced and the quality of life of the patient is

improved (4). However, this process involves an extensive surgical procedure and it is

therefore more desirable to transplant only the islets of Langerhans.

The success rate of islet transplantation was low (5) until Shapiro et al in Edmonton

succeeded in restoring insulin independence in 100% of patients transplanted (6). There

were several improvements in the Edmonton protocol that probably contributed to the

high success rate. The patients received a large number of islets (on average 11,500/kg

bw) compared to previous studies, the cold ischaemia time was limited, the islets were

not cultured and perhaps most importantly, cyclosporin and steroids were excluded

from the immunosuppression regimen. Moreover, the patients transplanted were

generally free from severe secondary complications and the relatively good health of

these patients may also have contributed to the success. This study proved that islet

transplantation could consistently reverse hyperglycaemia in diabetic patients, although

the long-term success remains to be shown. However, despite the success of the

Edmonton protocol, islet transplantation is still only available to patients where the

INTRODUCTION

10

benefits of restored glucose metabolism outweigh the risks of immunosuppression.

Immunosuppression has been linked to increased susceptibility to infections, increased

risk of malignancy and general toxicity (7-9). Moreover, some immunosuppressive

drugs have been found to induce insulin resistance and/or are toxic to islets of

Langerhans (10,11). Indeed, a follow-up study from Edmonton indicated that the

patients suffered from various side effects from the immunosuppression (12). The risks

associated with such side effects presently exclude patients with stable diabetes from

treatment with islet transplantation. It is therefore evident that it would be beneficial to

perform islet transplantation without the need of immunosuppressive drugs.

1.1.3 Encapsulation of cells

Encapsulation of cells provides the means of transplanting cells in the absence of

immunosuppressive drugs. The principle of encapsulation is that the transplanted cells

are contained within an artificial compartment separated from the immune system.

Thus, the capsule should protect the cells from potential damage caused by antibodies,

complement and immune cells. However, small molecules such as nutrients should be

able to freely enter the capsules and waste products and hormones such as insulin

should be easily released. There are three main types of encapsulation (13-16), namely a

vascular shunt system, macroencapsulation and microencapsulation. In a vascular shunt

system, the islet graft surrounds a shunt so that the graft is in close vicinity to the blood.

This allows the graft to be well nourished, although the risk of thrombosis together with

the risks associated with the surgical procedure have prevented this type of encapsulated

graft being investigated in humans (17,18).

Macroencapsulation involves the encapsulation of the whole islet graft within a hollow

fibre or diffusion chamber. These types of capsules have been particularly prone to

inducing a fibrotic response (17,19,20). Moreover, the diffusion to the islets in the

centre of such devices is limited and islets tend to become necrotic due to lack of

oxygen and nutrients (17,21,22).

In microencapsulation, each islet is individually encapsulated. This offers advantages

over other types of encapsulation. An important point is that risk of breakage is spread

over a large number of capsules, and thus if one capsule breaks the whole graft is not

lost. Another advantage in comparison with macroencapsulation is that the distance of

INTRODUCTION

11

diffusion is decreased. Moreover, the spherical shape of the microcapsules is optimal in

reducing a foreign body reaction, due to the absence of corners (23).

1.1.4 Microencapsulation

Chang first described microencapsulation in 1964 (24) and this technique was used by

Lim and Sun in 1980 to encapsulate islets of Langerhans (25). Microencapsulation has

been used with a variety of cell types including PC12 cells (26) for the treatment of

Parkinsons disease, hepatocytes (27) for the treatment of liver disease and parathyroid

tissue (28) for hypoparathyroidism. Moreover, this technique has been used for the

encapsulation of genetically modified cells producing, for example, factor IX for the

treatment of haemophilia B (29) and growth hormone for the treatment of dwarfism

(30). However, the most common application is probably in the microencapsulation of

islets of Langerhans, which has been widely studied (31-33).

There are two main aspects to consider with regard to transplantation of

microencapsulated pancreatic islets. First, the characteristics of the capsule should be

considered and how its composition can affect the desired properties of the capsule.

Second, biological responses affecting the capsule and the function of

microencapsulated islets need to be understood.

1.2 Characteristics of capsules

1.2.1 Capsule composition

1.2.1.1 Materials used for cell microencapsulation

A variety of materials has been investigated for use in microencapsulation, which can be

classified into two main groups: thermoplastic polymers and hydrogel polymers.

Thermoplastic polymers are water insoluble and include polyacrylates such as

hydroxyethyl methacrylate methyl methacrylate (HEMA-MMA). These capsules are

stable, but the diffusion properties of water-soluble nutrients may be limited and long-

term cell viability in these non-aqueous capsules has to be studied in more detail (34).

Interest has also focussed on the use of polyethylene glycol (PEG). This polymer can be

used as a conformal coating, which minimises the size of the capsule as the barrier is

INTRODUCTION

12

formed directly on the islet. Although in vitro studies of PEG as a conformal coating on

islets have been promising (35), the in vivo performance still has to be proven.

Hydrogels have been favoured for microencapsulation of cells due to the mild

encapsulation conditions they provide and their good diffusion properties. One such

hydrogel is agarose, but there is little information about the physical properties of

agarose capsules and a drawback is the lack of long-term immunoisolatory properties

(34).

The most studied polymer for use in microencapsulation is alginate. Alginate is an

intercellular matrix polysaccharide in brown algae and is produced as an extracellular

coating by some types of bacteria (36). The polysaccharide consists of regions of

mannuronic acid (M-blocks), regions of guluronic acid (G blocks) and regions of mixed

sequence (MG-blocks). The ratio and sequence of these uronic acids differ depending

on the source of the alginate and can determine the properties of the alginate (37).

Alginate is non-toxic and its gelling properties make it ideal for the immobilisation of

cells.

1.2.1.2 Alginate/ poly-L-lysine (PLL)/alginate microcapsules

Alginate forms the core and a matrix for the encapsulated cells (Figure 1). PLL binds to

the alginate core, forming a polyanion-polycation complex membrane, which reduces

the porosity of the gel, and thus forms the immunoprotective barrier (38). However, the

positive charge of PLL allows cellular adhesion on the surface of the capsule.

Therefore, a final layer of alginate is added to shield the PLL to improve the

biocompatibility (39). The composition of the alginate and interaction of alginate with

PLL can determine the properties of the capsules produced, such as mechanical

strength, porosity and biocompatibility (37).

INTRODUCTION

13

Figure 1. Structure of an alginate microcapsule. Immune cells are too large to penetrate thebarrier whereas nutrients and insulin can easily diffuse in and out of the capsule.

1.2.2 Alginate capsule properties

1.2.2.1 Mechanical Strength

It is important that the capsules can withstand a certain amount of physical and osmotic

stress, as breakage of the capsules will lead to immune rejection of the exposed

encapsulated cells in an allogeneic or xenogeneic transplantation. Osmosis causes the

capsules to swell, due to the exchange of Na+ with Ca2+ in the alginate. PLL strengthens

the capsule and therefore prevents excessive swelling of the capsule (40). This is

achieved by the elasticity of the polyanion-polycation interaction stabilising the capsule.

Moreover, the interaction of alginate with PLL leads to a discharge of the alginate and

therefore the amount of osmotically active counterions decreases. The strength of the

capsules also depends on the cross linking of the G-blocks with Ca2+. Thus, alginates

with a high percentage of G have a greater mechanical strength. However, Ba2+ is

capable of binding both G and M and therefore the use of this ion strengthens the

capsule (37). If the alginate gel is formed in the presence of a non-charged osmolyte

such as mannitol, inhomogeneous beads are formed, which have a higher percentage of

alginate at the surface than in the centre of the bead. These capsules are stronger than

homogenous capsules and capsules with dissolved centres (41).

Immune cells

AlginatePLLAlginate coating

Nutrients

Insulin

Islet of Langerhans

INTRODUCTION

14

1.2.2.2 Permeability

The optimal capsule should allow free diffusion of glucose and insulin but prevent cell-

mediated and humoral immunity. Indeed, it has been shown that alginate/PLL/alginate

capsules prevent cell-mediated cytotoxicity (42) and are impermeable to antibodies

(43). Several variables can influence the porosity of an alginate capsule. One important

factor is the interaction of alginate and PLL (38). Indeed, porosity can be decreased by

increasing the number of alginate-PLL ionic interactions. This can be achieved by

increasing the alginate-PLL reaction time, increasing the concentration of the PLL

solution or decreasing the PLL molecular weight. However, the composition of the

alginate also plays a role in the porosity. Not only does it determine the efficiency of the

binding of PLL to the alginate bead, but it also determines how open the structure of the

gel is. For example, alginates with a higher content of the long and inflexible G blocks

form a more open Ca-alginate network than alginates with a higher proportion of M

(44). Moreover, G (in comparison with M and MG sequences) binds less efficiently to

PLL (40)(B.Strand, personal communication) and this together with the more open gel

network forms a capsule with a higher porosity.

1.2.2.3 Biocompatibility

The composition of capsules can affect their biocompatibility. The biocompatibility of

capsules could depend on the alginate per se (45), impurities in the alginate (33,46,47)

or the interaction between alginate and PLL (48). PLL is positively charged and

therefore favours the adherence of cells. Moreover, it has been suggested that PLL is

toxic to cells and the necrosis that is induced increases the inflammatory response (49).

As described above, the composition of the alginate influences its interaction with PLL,

with alginates with high ratios of M or MG sequences binding PLL more efficiently. A

strong interaction of the alginate and PLL leads to neutralisation of the PLL, preventing

its interaction with cells and thus increasing biocompatibility of the capsule.

1.2.2.4 Size

Capsules should be small to allow rapid diffusion of nutrients. Moreover, the graft

volume should be kept to a minimum. Indeed, it should be noted that the volume of a

capsule is a function the radius to the power of three, and therefore if the diameter of the

capsule is halved, the volume will be decreased to an eighth. The size of the capsule can

INTRODUCTION

15

affect other properties of the capsule. Indeed, it has been suggested that the

biocompatibility of capsules can depend on their size (50). Smaller capsules are more

prone to osmotic stress and subsequent swelling than larger capsules, due to their

increased surface to volume ratio. Such a swelling of the capsule reduces the porosity of

the alginate (38). Moreover, excessive binding of PLL to small beads can cause them to

collapse. Thus, to avoid this, the protocol for producing small capsules requires quicker

washes with saline before PLL administration. This leads to less PLL being bound and a

consequent increase in porosity.

The size of the capsule can be controlled using a high voltage electrostatic bead

generator, by changing the voltage, needle diameter, distance between the needle and

gelling solution or pump flow rate. The composition of the alginate can also affect the

size of the capsule depending on the interaction between polymers within the alginate

and the binding of alginate to PLL. Thus, capsules with a higher proportion of MG

sequences tend to be smaller (B. Strand, personal communication), as the alginate

polymers are brought closer together by these flexible sequences. However, it should be

noted that small alginate beads containing a high proportion of MG sequences are more

resistant to swelling and thus their porosity is not affected due to this.

1.2.3 Enzymatic tailoring of alginate

With the choice of natural alginates presently available, it is difficult to create a capsule

that is strong, small, biocompatible and has the desired porosity. Often the choice of a

particular alginate for a specific property is at the expense of another.

C5-epimerases are enzymes that convert M into G in the polymer chain of alginate and

can be used to create novel alginates with improved properties (51,52). The alginate

producing bacterium Azotobacter vinelandii encodes seven different epimerases, all of

which differ in their epimerisation pattern. One of these isoenzymes, AlgE4, converts

the relatively stiff M blocks into more flexible MG-blocks in the alginate polymer (51)

(Figure 2).

The flexibility of the MG-blocks interspacing the G rich junction zone in the polymer

network of AlgE4-epimerised alginate leads to closer interaction of alginate polymers in

the gel. Furthermore, alginates epimerised in this manner bind PLL more effectively (B.

Strand, personal communication). Thus, the use of epimerised alginates can affect the

strength, porosity, size and biocompatibility of capsules.

INTRODUCTION

16

This may enable alginates to be engineered so alginate capsules can be produced that

have all the desirable properties for the successful transplantation of encapsulated

pancreatic islets.

Figure 2. The conversion of M blocks in alginate into MG sequences by the epimerase AlgE4.Kindly provided by Berit Strand, Norwegian University of Science and Technology.

1.3 Biological responses affecting microencapsulatedislets of Langerhans

1.3.1 Islet function

For successful transplantation of encapsulated islets, it is essential that the islets

function properly. Thus, islets should not be detrimentally affected by the

microencapsulation process. Moreover, the presence of the capsule should not affect the

function of the islet, which is dependent on the supply of nutrients and protection from

adverse components of the host’s immune system. The supply of nutrients is unlikely to

be a problem during culture, unless the porosity of the capsule is too low. However, the

supply of nutrients in vivo could be affected by a variety of factors including

implantation site and the host response to the capsules.

INTRODUCTION

17

1.3.1.1 Peritoneal cavity as an implantation site

Due to the large volume of microencapsulated islet grafts, the peritoneal cavity has to be

used as a transplantation site. Interestingly, this was the first site used for experimental

islet transplantation (53). It is easily accessible and thus the surgical risk is minimised

for implantation of capsules. Although the peritoneal cavity is not the physiological

route for insulin delivery, studies using insulin pumps have shown that blood glucose

concentrations are lowered by intraperitoneal delivery of insulin (54). The peritoneal

cavity is, however, a rather harsh environment compared to the other more commonly

used sites for islet transplantation, such as the intraportal site or beneath the kidney

capsule. These sites are richly vascularised, as are endogenous islets (55). However,

there is a low oxygen tension in the peritoneal cavity (56,57). Moreover, the peritoneal

cavity is rich in macrophages (58), which is the most active cell type in the host

response to biomaterials.

1.3.2 Host response to capsules

The host inflammatory reaction is a normal response to the implantation of a biomedical

device. However, it is important that this reaction does not interfere with the function of

the microencapsulated islets. The reaction usually starts with adsorption of proteins on

to the surface of the biomedical polymer, the most important being fibrinogen (59).

Macrophages may then adhere to the surface and produce cytokines such as IL-

1β, TNF−α and TGF-β, which further activate macrophages and fibroblasts (59-63).

The grafted tissue can also affect the host response to the capsule. Insulin released from

islets can affect, for example, the metabolism and function of macrophages (64).

Moreover, it has been suggested that cells within islets may produce cytokines such as

IL-1 (65), which could increase the inflammatory response. A foreign body reaction and

the resulting cellular overgrowth on the capsule may be detrimental for encapsulated

islets for two reasons. First, it may cause a physical and metabolic barrier to effective

nutrient diffusion into the capsule. Second, the islets may be exposed to macrophage-

derived cytokines, for example interleukin-1β (IL-1β) and tumour necrosis factor (TNF-

α), which are known to be detrimental to the function of the islet.

INTRODUCTION

18

1.3.2.1 Cellular overgrowth as a barrier to nutrient diffusion

Macrophages and fibroblasts account for the majority of cells found in the cellular

overgrowth on capsules (48,66). Both of these cell types have a high metabolic rate with

high rates of glucose utilisation (67,68). These cells attach to the surface of the capsule

(Fig 3) and it can be assumed that the nutrient diffusion to the islets within the capsules

is decreased due to the presence of these cells. Moreover, fibroblasts produce different

types of fibres, which could act as a physical barrier to nutrient diffusion (Fig 4).

Fig 3. Cells on the surface of a capsule can act as a metabolic barrier to nutrient diffusion andproduce cytokines. Scanning Electron Micrograph of an empty capsule retrieved 1 week afterintraperitoneal implantation to C57BL/6 mice. The bar is 10µm.

Fig 4. The fibre network produced by fibroblasts may build a physical barrier to nutrientdiffusion. Scanning Electron Micrograph of an empty capsule retrieved 1 week afterintraperitoneal implantation to C57BL/6 mice. The bar is 10 µm.

INTRODUCTION

19

1.3.2.2 Cytokine-induced suppression of islets of Langerhans

The cytokines IL-1β and TNF-α are produced in activated macrophages. The

expression of these cytokines has been shown to occur in macrophages in contact with

biomaterials (69,70). Moreover, these cytokines have been implicated in the

pathogenesis of type 1 diabetes and have been shown to inhibit the function of

pancreatic islets (71-73). Experimental evidence has also suggested a role for these

cytokines in rejection of allografts (74,75). One effect of these cytokines on β-cells is

the induction of inducible nitric oxide synthase (iNOS) and the subsequent production

of nitric oxide (NO) (76). NO has several detrimental effects on the islets including

inhibition of the mitochondrial enzyme aconitase and induction of DNA damage (77),

although some of the suppressive effects of IL-1β on islets of Langerhans are

independent of NO (78,79). Nevertheless, NO has been shown to suppress encapsulated

islets co-cultured with macrophages (80) and it is therefore evident that cytokine and/or

NO production in the vicinity of the islets is highly undesirable.

1.3.3 Host response to encapsulated islets

There are two specific immunological processes that may affect encapsulated islets. One

is immune rejection and the other is recurrence of the autoimmune disease. For immune

destruction of islets to occur, foreign antigen must be presented to T-cells. There are

two types of antigen presentation: direct and indirect presentation (81-83). Direct

presentation requires cell-to-cell interaction and is therefore avoided by encapsulation of

cells. The indirect pathway is initiated by soluble antigens, which may be shed from the

encapsulated islet. The subsequent activation of T-cells leads to an inflammatory

response, with the activation of macrophages and the production of cytokines in the

vicinity of the capsules. The direct presentation of antigen is probably the most

important pathway in allogeneic transplantation. However, the indirect pathway of

antigen presentation is important in xenogeneic transplantation and may be of

importance in autoimmune diabetes (82). Thus, the transplantation of encapsulated

islets in such circumstances may be more problematic.

INTRODUCTION

20

1.4 Interaction of host responses and capsules

The function of microencapsulated islets is dependent on both capsule composition and

biological factors. The capsule composition can affect the strength, size, porosity and

biocompatibility of the capsule, all of which can affect the function of the islet.

Biological parameters such as the implantation site and the host response to the capsule

can also affect the function of microencapsulated islets. Whereas biological factors are

difficult to control, capsule characteristics may be engineered to provide a capsule that

induces a minimal host response and allows good function of the microencapsulated

islet. In order to create a capsule that is ideal for the optimal function of

microencapsulated islets, a careful evaluation of microencapsulated islets has to be

carried out to understand the specific problems relating to this procedure.

AIMS

21

AIMS

The overall aim of these studies was to evaluate microcapsules for the transplantation of

islets of Langerhans.

The specific aims were:

In vitro:

1. To study the function of islets directly after microencapsulation and after further

culture for two weeks.

2. To determine the ability of the cytokines IL-1β and TNF-α to penetrate the

capsule and affect the function of islets.

In vivo:

1. To investigate the biocompatibility of different types of capsules, with regard to

capsule composition and host factors.

2. To study the ability of microencapsulated allogeneic islets to reverse

hyperglycaemia in diabetic mice.

OVERVIEW OF THE METHODS USED

22

3. OVERVIEW OF THE METHODS USED

An overview of the methods used is described below. All methods as well as

manufacturers of the equipment and chemicals are described in more detail in the

individual papers.

3.1 Microencapsulation and transplantation of islets

3.1.1 Islet isolation (Papers I-V)

Islets were isolated from rats (Sprague-Dawley) or mice (C57BL/6 or BALB/c). The

pancreas was removed and shaken in a solution of collagenase at 37 °C. This enzyme

preferentially digests the exocrine acinar tissue. After washing, the islets were

handpicked from the exocrine digest using a braking pipette. Islets were cultured in

RPMI + 10% foetal calf serum in a glucose concentration of 11.1 mM. It has been

previously been shown in our laboratory that these are ideal conditions for the culture of

mouse islets (84).

3.1.2 Microencapsulation of islets (Papers I-V)

In the present studies, islets were microencapsulated using a high-voltage electrostatic

technique (Figure 5). An electrostatic potential is created between the needle of the

droplet generator and the CaCl2 solution, thus pulling down the beads rapidly before

large droplets form. This allows the production of uniformly sized capsules, the size of

which can be regulated by the voltage. As well as acting as an electrode, a metal rod

submerged in the CaCl2 solution controls the level of the liquid by use of a vacuum.

This is achieved through a hole in the hollow rod that is at the solution surface. Thus,

when the volume of the solution increases as beads accumulate, the excess liquid is

removed. This ensures that the distance between the needle and the solution surface is

constant, both within and between experiments.

The liquid alginate (1.8% (w/v)) is mixed with islets. Droplets are formed through a

needle and fall into a solution containing divalent cations such as calcium, which gels

the alginate beads. The CaCl2 solution contains 0.3 M mannitol as an osmolyte. As

mannitol is uncharged, an inhomogeneous alginate bead is formed, with a higher

concentration of alginate at the surface of the bead than in the centre (41). The beads are

OVERVIEW OF THE METHODS USED

23

Islet of Langerhans

1.8% Alginate mixedwith islets

Improvement ofbiocompatibility

Formation ofimmunoprotectivebarrier

Poly-L-Lysine

0.1% Alginate

Gelling ofalginate toform bead

Syringe pump

Vacuum

Electrode

Ca Cl2

Figure 5. Microencapsulation of islets of Langerhans using an electrostatic field.

Each step of the process is shown. Capsules are washed in saline between stages.The text to the left of the figure indicates the function of each process.

OVERVIEW OF THE METHODS USED

24

then washed in saline before being shaken in PLL, in the present studies either at a

concentration of 0.05% for 5 min or 0.1% for 10 min. A final layer of alginate at a

concentration of 0.1% is added as an outer coating, which shields the positively charged

PLL. Empty capsules can be produced using the same procedure, but omitting the islets.

Capsules used in Paper I-IV are described in Table 1. The capsules used in Paper V

were produced in Trondheim, Norway and are described in detail in Paper V. All

solutions in the microencapsulation procedure contained 1 mM HEPES and were

adjusted to pH 7.4. The equipment used was autoclaved and solutions were sterile

filtered. Microencapsulated islets were cultured as islets (see above), although

microencapsulated islets to be transplanted were cultured in serum-free medium before

transferral to saline for the transplantation.

Alginate Composition PLL ExposureDesignation

Guluronicacid

Mannuronicacid

Concentration Time

Size

(µm)

70%G-0.1% PLL 70% 30% 0.1 % 10 min 550

42%G-0.05% PLL 42% 58% 0.05 % 5 min 650

63%G-0.05% PLL 63% 37% 0.05% 5 min n.d

Table 1. Capsules used in Papers I-IV. Coating alginates were the same alginate as used

for the core (0.1%, 10 min).

3.1.3 Implantation of islets and capsules (Papers I, III-V)

Mice were anaesthetised by either a subcutaneous injection of xylazine hydrochloride

(16 mg/kg) and ketamine (150 mg/kg) (Paper I) or an intraperitoneal injection of 2 ml/g

Avertin [2.5% (vol/vol) solution of 10 g 97% 2.2.2-tribromoethanol in 10 ml 2-methyl-

2-butanol] (Paper III-V). A small incision was made in the skin and then in the linea

alba in the peritoneal membrane. The linea alba was used as a point of incision to

decrease the inflammatory reaction. Naked or microencapsulated islets in RPMI 1640

medium or saline, respectively, were gathered in a pipette and injected into the

peritoneal cavity in a volume of approximately 0.5 ml. Empty capsules were

transplanted in the same manner as microencapsulated islets. One to two sutures were

OVERVIEW OF THE METHODS USED

25

used to close the peritoneal membrane and 3-4 sutures were used to close the skin. Mice

were then left under a warm lamp to recover.

3.1.4 Retrieval of implanted capsules (Papers I, III-V)

After killing the mice by cervical dislocation, capsules were retrieved by the injection of

4-5 ml of Hanks’ solution or saline into the peritoneal cavity. The peritoneal cavity was

then opened and capsules were washed out. A low retrieval rate of capsules is generally

associated with low biocompatibility, with the assumption that the capsules with the

most overgrowth adhere within the peritoneal cavity. This is probably the case,

presuming the capsules are stable. In Paper III, retrieval was determined by counting the

capsules, whereas in Paper V retrieval was calculated by the volume and size of the

capsules retrieved.

3.1.5 Induction of diabetes (Paper V)

Alloxan (90 mg/kg in nude mice and 75 mg/kg in C57BL/6 mice) was injected

intravenously to induce diabetes in mice. The mice became hyperglycaemic 3 days after

the injection, with blood glucose concentrations exceeding 20 mM as measured by

sampling from the tail tip using a glucometer. The diabetes induced is chemical, and in

this respect, probably does not resemble the autoimmunity of type 1 diabetes.

3.2 Functional tests

3.2.1 Glucose oxidation rates (Papers I-III,V)

Glucose oxidation rates were used to measure the aerobic metabolism of glucose in

islets and the cells attached to the capsules after implantation. In Krebs’ cycle, carbon-

14 labelled glucose is converted to 14CO2, which is released and entrapped in hyamine

and can then be measured by liquid scintillation (85). In islets, the glucose oxidation

rate should increase with increasing glucose concentrations (86), which implies good β-

cell viability, as this provides the energy necessary to allow the release of insulin. In the

cells on the overgrown capsules, high oxidation rates probably reflect a high number of

cells consuming nutrients. A high glucose oxidation rate of empty capsules thus

indicates a low degree of biocompatibility.

OVERVIEW OF THE METHODS USED

26

3.2.2 Insulin release rates (Papers I,II)

The main biological function of β-cells is to secrete insulin in response to glucose. Insulin

secretion was therefore measured at low (1.7 mM) and high (16.7mM) glucose concentrations

during 1-hour incubations. In this way, the ability of islets to increase their insulin release in

response to glucose can be studied. Moreover, the measurements at low glucose

concentrations can establish whether the insulin release is controlled or whether insulin is

being leaked. Insulin accumulation to the medium during culture was also measured, to detect

the longer-term insulin release. Moreover, insulin content of the islets was measured, which

can be used together with the release rates to suggest whether synthesis or secretion is

affected. Insulin was measured by use of a radioimmunoassay (87).

3.2.3 DNA measurements (Papers I-III,V)

DNA was measured by fluorometry, where the ribose moieties in the DNA are quantified by

their reaction with 3,5-diaminobenzoic acid (88,89). It should be noted that even degraded

DNA is measured and thus this assay should be regarded as an approximate quantification of

the number of cells and not their viability.

3.2.4 Nitrite measurements (Paper II)

Nitrite is a stable product of the reaction between NO and oxygen. Thus, measuring the

concentration of nitrite provides a reliable indication of the amount of NO produced by cells.

The nitrite reacts with Greiss reagent (90) and can then be measured by use of a

spectrophotometer at 546 nm. A standard curve of known concentrations of nitrite is used to

calculate the amount of nitrite in the samples.

3.2.5 Real Time PCR (Paper III)

Real time PCR was used to examine the mRNA expression of IL-1β and TNF-α in peritoneal

macrophages after implantation of capsules. RNA was isolated from the macrophages using

an RNeasy mini kit. The PCR reactions were carried out using a TaqMan assay. This assay

measures each cycle of the PCR reaction by measuring fluorescence in the sample generated

by a fluorophore, which is cleaved from the probe.

RESULTS AND DISCUSSION

27

4. RESULTS AND DISCUSSION

4.1 In vitro function of rodent islets after microencapsulationGlucose oxidation and insulin release rates in mouse islets one day after microencapsulation

in 70% G-0.1% PLL capsules, were comparable to naked islets, thus showing that the

function of mouse islets was not disturbed by the microencapsulation procedure (I). After two

weeks in culture, the function of the microencapsulated islets was unchanged compared with

one day after microencapsulation, which indicates that the presence of the capsule does not

detrimentally affect the function of the islets. However, naked islets had increased glucose

oxidation and insulin rates at this time point (3 weeks after isolation) compared with islets 1

week after isolation, the reason for which is unknown.

In rat islets, the glucose oxidation rates and the glucose-induced insulin secretion of

microencapsulated islets 3 days after microencapsulation tended to be lower than what was

observed for naked islets, although this did not reach statistical significance (II). Moreover,

the insulin accumulation to the medium during 48 h was slightly reduced and the insulin

content slightly increased in the microencapsulated rat islets, suggesting that insulin secretion

but not insulin synthesis was vulnerable to suppression. This interpretation of the data is based

on the assumption that insulin degradation is not decreased by the encapsulation process per

se. In a previous study, an initial suppression of insulin release of microencapsulated rat islets

reverted to the level of control islets after 6 days in culture (91). Thus, it is possible that the

mild effects seen in rat islets are a reversible result of the microencapsulation procedure rather

than the presence of the capsule. However, it should be noted that in a study by Tatarkiewicz

et al, in vitro insulin release by rat islets 4 days after microencapsulation was significantly

lower than insulin release by control islets (92).

In a perifusion system, microencapsulated rat islets have been shown to respond to high

glucose challenges as quickly as naked islets (93). However, the absolute amount of insulin

released was decreased compared with naked islets. This may have been due to the use of

citrate to liquefy the centre of the capsules. Indeed, it has since been shown that the use of

citrate severely inhibits insulin secretion from islets (94). The centres of the capsules in the

present studies were not liquefied and thus the islets were not exposed to citrate.

It has been previously shown that the alginate composition does not affect the viability or

function of cultured microencapsulated islets (93). However, it has been suggested that PLL

RESULTS AND DISCUSSION

28

in capsules may be toxic to encapsulated Jurkat cells (49), although no such studies have been

carried out with islets. In summary, our own results and other studies show that

microencapsulated mouse islets have adequate function in vitro, whereas rat islets may be

slightly impaired by the microencapsulation process.

4.2 Permeability of microencapsulated islets to cytokines in vitroRat islets microencapsulated in 70 % G-0.1 % PLL capsules were functionally suppressed

after exposure to the macrophage derived cytokines IL-1β and TNF-α, indicating the

permeability of this capsule to these cytokines (II). At higher concentrations of IL-1β (10

U/ml and 25 U/ml), microencapsulated islets were more suppressed than naked islets exposed

to the same activities of the cytokines. This was not associated with an increased nitrite

concentration in the medium compared to naked islets, suggesting that the increased

sensitivity of microencapsulated islets was not dependent on increased NO levels. After a 48 h

exposure to 25 U/ml IL-1β, neither microencapsulated islets nor naked islets were able to

recover their function after 6 days. However, after a 48 h exposure to 2.5 U/ml IL-1β, both

microencapsulated and naked islets were able to regain approximately 90 % of their function

after a 6-day recovery period. These results show that the alginate/PLL/alginate capsules

used in the present study do not confer protection against the effects of cytokines. However,

microencapsulated islets can recover after a short-term exposure to low concentrations of IL-

1β.

Other studies have shown various results with regard to the permeability of capsules to

cytokines. This is probably due to different capsules used in the studies. Cole et al also found

that IL-1β and TNF-α were permeable to alginate/PLL/alginate capsules (95). Tai et al

claimed that alginate/PLL/alginate capsules protected islets against functional suppression

induced by IL-1 and TNF (96). However, a high concentration of PLL (0.5%) was used in that

study, which may explain their data.

In a study by Kulseng et al, IL-1β but not TNF-α penetrated the capsule (97). The capsules

used in that study were approximately 700 µm, compared to 550 µm in our study. This size

difference may explain the difference in porosities, as discussed in section 1.2.2.4. In

Kulseng’s study, reduction of the PLL concentration from 0.1% to 0.05% led to TNF

penetration into the capsule. This further emphasises the importance of the PLL concentration

in determining the porosity of capsules. Indeed, in the studies of 42% G capsules in Papers

III-V, the low concentration and exposure time of PLL used (0.05%, 5 min) most probably

allowed the penetration of IL-1β and TNF-α, although this was not studied in this capsule

RESULTS AND DISCUSSION

29

type. Interestingly, the use of an epimerised alginate as a core in alginate/PLL/alginate

capsules reduces the permeability of the capsule to TNF by 26% (B. Strand; personal

communication), which demonstrates how the alginate can be engineered to change specific

properties of the capsule.

With regard to the permeability of cytokines, the three dimensional structure of the compound

should be considered. TNF-α forms conical shaped trimers with a molecular weight of

approximately 55 kD (98,99). The three dimensional structure of this protein may therefore

impede its penetration into the capsule. It could therefore be feasible to exclude TNF-α from

capsules, whereas preventing the penetration of IL-1β may be more problematic. IL-1β is a

small protein with a molecular weight of 17.5 kD. It is likely that if the porosity of the capsule

were reduced to prevent the penetration of IL-1β, insulin diffusion would probably be

hindered. Thus, it would be more desirable to prevent cellular overgrowth on capsules and

consequently inhibit the production of cytokines in the vicinity of the microencapsulated

islets.

4.3 Biocompatibility of the capsules4.3.1 Effect of capsule composition

Empty microcapsules induced a foreign body reaction with a cellular overgrowth, which was

metabolically active as indicated by the glucose oxidation rates (I, III and V). Indeed, the

oxidation rates of the cellular overgrowth on 10 empty capsules were often in the range of

glucose oxidation rates for 10 naked islets. It can thus be speculated that the cellular

overgrowth could act as a metabolic barrier as well as a physical barrier to nutrient diffusion.

Indeed, in vivo experiments showed that capsules with the best biocompatibility functioned

best (IV). When lower concentrations of PLL (0.05%) were used in capsules, it was evident

that improvement of binding of PLL to the alginate, either by using an alginate with a high M

content (III) or an epimerised alginate coating (V), increased the biocompatibility of the

capsules. Moreover, the cellular overgrowth on capsules was markedly reduced when PLL

was omitted from the capsule (III and V). In capsules with no PLL, there were no differences

in biocompatibility in capsules produced from epimerised alginate compared with capsules

produced from non-epimerised alginate (V). It is therefore likely that this small change in

composition of the alginate does not affect the biocompatibility of PLL-free capsules.

However, with regard to the composition of the alginate, it has been claimed that M is more

effective in eliciting an immune response (45,48), whereas others have claimed that alginate

with a high M content is less immunogeneic (100). It has been suggested that these

discrepancies may depend on the purity of the alginate (33). The integrity of the capsules is

RESULTS AND DISCUSSION

30

also important, as any exposure of PLL in the capsule increases the cellular reaction against

the capsule (48,101). Differences in comparing alginate per se (45) or intact capsules (100)

may also account for the differences in these studies.

4.3.2 Biological responses affecting the cellular overgrowth of capsules

The presence of syngeneic islets within the capsules increased the overgrowth compared with

what was observed on empty capsules (I). Indeed, other studies have shown that the presence

of cells within capsules increases the cellular overgrowth on the capsules (66,102-105).

It has been shown in several studies that macrophages are the main cell type involved in the

response against capsules (66,106). Indeed, even when capsules are transplanted to nude mice

and rats, the response is intensive (91,103,III), indicating that T-cells are not involved in this

process.

In Paper III, the role of NO in the host response to capsules was investigated by studying the

cellular overgrowth on capsules implanted to knockout mice lacking iNOS. NO is an effector

molecule in macrophages and has previously been detected in macrophages in foreign body

inflammation (107). Interestingly, there was a tendency for an increased foreign body reaction

to capsules transplanted in the mice lacking iNOS. Furthermore, iNOS-deficient mice have

shown more severe trinitrobenzene-induced colitis, indicating that NO can play a protective

role in this model of inflammation (108). Indeed, NO can act as either a proinflammatory or

anti-inflammatory molecule (109) and in this model of cellular overgrowth on implanted

capsules it seems to act as an anti-inflammatory molecule. This may be due to the anti-

adhesive properties of NO (110,111). However, NO has been shown to be detrimental to

microencapsulated islets co-cultured with macrophages (80) and from this viewpoint, NO

should be avoided. It has been previously reported that cellular overgrowth differs between

species and is increased in animal models of diabetes such as the BB rat (112). It has thus

been suggested that cytokines may be involved in this process. Moreover, it has been shown

that microcapsules can induce IL-1 production from macrophages in vitro (113). Indeed, we

were able to show that there were differences in the host response to capsules depending on

the mouse strain used. C57BL/6 mice had a more severe host reaction to capsules than

BALB/C mice. C57BL/6 mice also had an increased mRNA expression of IL-1β and TNF-α

in their peritoneal macrophages after capsule implantation compared with BALB/c mice,

suggesting a role of these cytokines in the reaction against the capsules.

In subsequent studies to Paper III, we used the C57BL/6 mouse model, as this represents a

“tougher” model than the BALB/c mouse and thereby may be more beneficial in investigating

the problems associated with cellular overgrowth on microcapsules.

RESULTS AND DISCUSSION

31

4.4 In vivo function of microencapsulated mouse islets4.4.1 Transplantation to nude (nu/nu) C57BL/6 mice

Transplantation of empty capsules did not affect blood glucose concentrations. Alloxan-

induced diabetes in nude mice was reversed transiently after transplantation of 1000 Balb/c

islets microencapsulated in 70% G-0.1 % PLL capsules. However, normoglycaemia (<11.1

mmol/L) could not be maintained for more than a few days (IV). When BALB/c islets

microencapsulated in 42% G-0.05 % PLL capsules were transplanted to nude mice, blood

glucose concentrations were significantly reduced as long as one month after transplantation,

although these mice were moderately hyperglycaemic. Indeed, function of the graft at 1

month was evidenced by an intravenous glucose tolerance test, although it should be noted

that the basal glucose concentrations were high. However, when 837±39 of these capsules

were transplanted (n=5), normoglycaemia was not maintained for more than a few days (Fig

6), indicating that 1000 microencapsulated islets is a borderline size of the transplant to

achieve normoglycaemia.

Day

0 1 2 3 4 5 6 7

Bloo

d G

luco

se (m

M)

0

5

10

15

20

25

30

35

Figure 6. Blood glucose concentrations of nude C57BL/6 mice after transplantation of 1000±0 (opendiamonds, data from Paper IV) or 837±39 (closed diamonds) islets microencapsulated in 42% G-0.05% PLL capsules. Mice transplanted with 1000 microencapsulated islets had lower blood glucoseconcentrations on day 4, 6 and 7 after transplantation in comparison with mice transplanted with837±39 capsules (p<0.05 ANOVA with Student-Newman-Keuls post hoc test, n=4-5)

RESULTS AND DISCUSSION

32

The number of naked islets required to reverse hyperglycaemia after transplantation in other

sites is considerably lower (114). Thus, it is evident that the microencapsulation of islets

and/or transplantation to the peritoneal cavity increases the number of islets required. Indeed,

it has been shown previously that transplantation to the peritoneal cavity requires an increased

mass (115).

The present results show the importance of capsule composition in the function of

microencapsulated islet grafts. The failure of the 70% G-0.1% PLL capsules was most

probably due to the poor biocompatibility of these capsules, as shown in Paper III. The 42%

G-0.05% PLL capsules were further studied to assess whether these capsules could protect

islets in immune competent mice.

4.4.2 Transplantation to immune competent C57BL/6 mice

Alloxan-induced diabetes in immune competent C57BL/6 mice was reversed after allogeneic

transplantation of naked or microencapsulated Balb/c islets. However, the blood glucose

concentrations of mice transplanted with naked islets rose to an average of over 20 mmol/L by

day 10, indicating immune rejection of the islet grafts. The microencapsulated grafts

continued to function until the end of the study at day 28, as shown by an intravenous glucose

tolerance test. However, as in the nude mice, these mice were moderately hyperglycaemic at

the end of the study. Again, the importance of good biocompatibility for islet function was

emphasised, as the only completely normoglycaemic mouse at day 28 had no overgrowth on

the capsules, as determined by histology. Grafts that had partially failed had overgrowth on

the capsules.

The average blood glucose concentrations of the immune competent mice 1 month after

implantation of microencapsulated islets were slightly higher than in the nude mice (18.6±5.5

mM vs 13.7±3.5 mM). It cannot be ruled out that some capsules broke, which in the immune

competent mice would lead to rejection of the islet. This was not studied in detail, although it

should be noted that these small capsules with a relatively low content of PLL are weaker

than 70% G-0.1% PLL capsules. Indeed, after histology, some of the 42% G-0.05% PLL

capsules were seen to have broken, although it is not known whether this occurred in vivo or

during the histological procedure.

In a recent study by Duvivier-Kali et al, (116), a simple barium-alginate bead protected islets

in an allograft and allowed function for over 1 year. It had previously been assumed that PLL

was required to reduce porosity to an appropriate level to prevent allograft rejection.

However, it is evident from that study that a simple barium alginate bead prevents cell-

RESULTS AND DISCUSSION

33

mediated rejection. Moreover, the improvement in biocompatibility of the capsules by

omitting PLL has most likely also played an important role in the success of this model. It

should be noted, however, that the capsules used were rather large (approximately 1mm) and

it still remains to be seen whether the same results could be achieved with smaller capsules.

4.5 Capsule properties required for optimal function of microencapsulatedislets

From the present studies, it can be concluded that good biocompatibility is an essential

property of alginate/PLL/alginate capsules for use in transplantation of islets.

The desired porosity of capsules is uncertain concerning allogeneic transplants. The exclusion

of immune cells is probably adequate to avoid immune rejection of microencapsulated islet

allografts. However, it is likely for xenogeneic transplantation that antibodies and

complement may have to be excluded from the capsules.

The ideal size of alginate-PLL-alginate capsules has been discussed. Whereas it has been

argued that smaller capsules should be used to decrease diffusion distances (117), the risk of

protruding islets increases with smaller capsules (118). However, it is likely that the total graft

volume should be decreased to enhance function and decrease the host response. Recent

studies have investigated transplanting smaller capsules into the liver (119), which may be a

more suitable site than the peritoneal cavity. However, the safety of this site has to be more

thoroughly investigated before it can be considered as an alternative that is suitable for

encapsulated islet transplantations in humans.

The strength of alginate capsules has to be sufficient to prevent breakage or excessive

swelling, as broken capsules will lead to exposure and rejection of the islet and excess

swelling will affect the porosity. However, it is presently difficult to assess the mechanical

and osmotic stress that capsules are exposed to in vivo. Nevertheless, manipulations of the

capsules that decrease the strength should be avoided.

All of the above properties can be manipulated by the use of epimerised alginates. Thus, the

use of such alginates may allow a capsule to be produced without the need for PLL, which is

small, strong, and biocompatible with an ideal porosity. Epimerised alginates for use in

microencapsulation of islets may therefore solve some of the problems presently associated

with microencapsulation technology and for this reason represent a very exciting development

in this field.

CONCLUSIONS

34

5 CONCLUSIONS

• Microencapsulated mouse islets are not functionally affected by the

microencapsulation procedure or the presence of an alginate/PLL/alginate capsule

• Alginate microcapsules (70% G-0.1% PLL) are permeable to the macrophage

cytokines IL-1β and TNF-α

• The presence of PLL in capsules aggravates the host response to capsules

• The host response to capsules varies between mouse strains, which may be a result of

different expressions of IL-1β and TNF-α in peritoneal macrophages

• Microencapsulated islets can function in vivo to reverse hyperglycaemia, although the

success depends on the biocompatibility of the capsule

• Enzymatic tailoring of alginates using epimerases may produce capsules which have

the ideal properties for encapsulation of islets of Langerhans

ACKNOWLEDGEMENTS

35

ACKNOWLEDGEMENTS

This work was carried out at the Department of Medical Cell Biology, Uppsala University.

I would like to express my sincere gratitude to

Prof. Arne Andersson, my supervisor, for welcoming me to Sweden and into his researchgroup as an exchange student and later as a Ph D student. The opportunity he provided mewith has changed the course of my life. Sharing his immense knowledge on diabetes researchand scientific writing has been essential to my scientific development.

Prof. Stellan Sandler, my co-supervisor, for always being available to discuss scientificproblems, for a great deal of support and for never failing to help me with practical problemsencountered in Sweden.

Associate Prof. Leif Jansson, my co-supervisor, for sharing his vast knowledge on manyaspects of research, for asking questions that I had never considered and teaching me newwords in English.

Astrid Nordin, Eva Törnelius, Ing-Britt Hallgren and Margareta Engkvist for superb technicalassistance. Their infinite patience in teaching me everything I know about practical laboratorywork has been admirable. I would like to thank them for teaching me Swedish and for all theirsupport, especially when I first came to Sweden.

My collaborators from Trondheim, Norway, especially:-Gudmund Skjåk-Bræk for sharing his great knowledge and enthusiasm for alginate, wine andsalmon. I never thought I would find seaweed so fascinating! -Berit Strand, for never-ending patience in answering questions about alginate and capsules,for being my congress-buddy and for making the collaboration so enjoyable.-Anne-Mari Rokstad and Bård Kulseng for “rolig” collaboration and trying to teach mestrange Norwegian dialects.

To the members of the PolyEng collaboration network, for extremely pleasant andinformative meetings.

To the present chairman of the Department, Godfried Roomans and the former chairmanBirger Petersson, for efficiently running the Department and providing a pleasant workingenvironment.

Agneta Bäfwe for excellent administrative assistance and for contributing to great lunchtimediscussions. Also thanks to Karin Öberg, Birgitta Jönzén and Gun-Britt Lind foradministrative help.

Claes Hellerström, Michael Welsh, Nils Welsh, Håkan Borg and Ulf Eriksson for sharingtheir knowledge of subjects outwith my own field.

ACKNOWLEDGEMENTS

36

Birgitta Bodin, Lisbeth Sangulin, Ing-Marie Mörsare and Kristina Mustajärvi for skillfultechnical assistance and for always being willing to help.

Göran Ståhl for his ability to efficiently fix things before I had noticed they were broken andfor adding to the atmosphere at the laboratory.

Anders Ahlander, Leif Ljung and Marianne Ljungkvist for expert help with electronmicroscopy and always making me feel welcome at “Anatomen”.

To everyone in the Animal Department for expert care of the animals.

Carina Carlsson for being an essential source of advice regarding both work-related and otherproblems. She has been a great support which I really appreciate.

To the present and former Ph Ds and students at the Department of Medical Cell Biology forbeing good friends, for learning the art of spontaneity and contributing to the fantasticatmosphere at the department. I will always remember the fun we have had together. Extrathanks to Linda, Caroline and Göran for special friendship and to Joey Lau for herenthusiastic contributions to Paper IV.

To my friends outside the Department, especially those I met in Coleraine, at Västgöta nationand at Örsundsbro Sport Klubb, for making my years in Sweden an experience that I’ll neverforget.

To my brother Alasdair and his “sambo” Emma for always letting me and my friends stay attheir house in London, no matter how many we are.

To my brother Andrew for being persuaded to drive to Sweden with all my belongings,despite his aversion for boats. Also my thanks to him and his fiancée Gillian for not gettingmarried on the same day as the defence of my thesis.

To Mum and Dad for their love and support throughout the years and for taking an interest inmy education and research. Their “suggestion” that I study as far away from Dundonald aspossible has lead to great personal development and fantastic experiences in the last fewyears.

During my Ph D studies I have received financial support from the Swedish Society forMedical Research, the Swedish Diabetes Association, the European Association for the Studyfor Diabetes, the Scandinavian Association for the Study for Diabetes and the UpsalaLäkareförening.

This work was supported by grants from the Swedish Research Council, the Swedish DiabetesAssociation, the Novo-Nordisk Insulin Fund, the Family Ernfors Fund, the Juvenile DiabetesFoundation International and the Wallenberg Foundation and BIOMed 5 of the EuropeanCommunity.

REFERENCES

37

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