Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. ·...
Transcript of Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. ·...
![Page 1: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/1.jpg)
Characterization of
Decellularized Extracellular
Matrix for Clinical Use
Nikhil Gheewala, PhD
Principal Scientist, ACell
CASSS - Thursday, April 5th, 2018
![Page 2: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/2.jpg)
Extracellular Matrix
Cells
Collagens
Elastin
Proteoglycans
Secreted and Bound Components
![Page 3: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/3.jpg)
Decellularization (Ideal)
Collagens
Elastin
Proteoglycans
Secreted and Bound Components
Goal: Maximize ECM retention and cell removal while minimizing ECM damage
![Page 4: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/4.jpg)
Decellularization Modifies the
Immune Response
Without decellularization, implanted xenogeneic and most
allogeneic tissues prompt a classic inflammatory (M1 macrophage)
response, often resulting in encapsulation or scar formation
More effective decellularization can result in:
Increased “anti-inflammatory” (M2 macrophage) response
Reduced encapsulation and scar formation
Constructive remodeling toward site-appropriate tissue
Brown 2009, Biomaterials;
Keane 2012, Biomaterials;
![Page 5: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/5.jpg)
Current Clinical Uses of ACell’s
decellularized Urinary Bladder Matrix
(UBM) – “MatriStem UBM”
Regulated as Medical Devices
Wounds
Chronic Wounds
Burns
Pressure Ulcers
Diabetic Ulcers
Surgical
Surgical reinforcement
Plastic and reconstructive
surgery
![Page 6: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/6.jpg)
*Preclinical data
• Host remodeling response includes
• Angiogenesis1 and innervation2
• Modulation of the inflammatory response1
• Participation of autologous progenitor cells3,6
• Resistance to infection5
• Host deposition of functional, site-appropriate tissue 4,6,7
• Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling by native cells
1. Brown et al., Acta Biomater 2012
2. Agrawal et al., J Tissue Eng
Regen Med 2009
3. Agrawal et al., PNAS 2010
4. Sicari et al., Sci Transl Med
2014
5. Medberry et al., J Surg Res
2012
6. Gilbert et al., J Surg Res 2008
7. Remlinger, et al.,
Organogenesis, 2012
Urinary Bladder Matrix (UBM)
![Page 7: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/7.jpg)
Testing and Characterization
With tissue-derived medical devices, ACell can use a
combination of physical and analytical tests
Physical Testing
• Tensile Strength
• Suture Retention
Strength
• Calorimetry (collagen
thermal stability)
• Ease of Handling
Analytical Quantification
• Moisture Content
• DNA
• Phospholipids
• Collagens
• Glycosaminoglycans
• bFGF
![Page 8: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/8.jpg)
EXTRACTION METHODS
Some material components can be extracted without requiring enzymatic
digestion of the tissue macrostructure, using appropriate buffers and
micronizing/homogenization methods
![Page 9: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/9.jpg)
Phospholipid Quantification
• Phospholipid content is indicative of the level of remaining cellular and
intracellular membrane components
• Measurable in solution using BioAssay Systems’ EnzyChrom™ Phospholipid
Assay Kit
• Need to be able to extract into solution a repeatably high yield of phospholipids
• Initial suggestion from BioAssay Systems was to use Triton X-100, also used
as the provided “assay buffer”
![Page 10: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/10.jpg)
Phospholipid Buffers
Buffers tested:
Triton X-100, baseline
Triton X-100 + 50mM Tris buffer
Methanol
• Dried after extraction, then reconstituted in Triton X-100 +
50mM Tris
Test Articles:
Micronized (milled) UBM samples, non-sterile
![Page 11: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/11.jpg)
Phospholipid Extraction Buffers
100.0%
99.6%
110.1%
1.3% 1.8% -0.9%
-20%
0%
20%
40%
60%
80%
100%
120%
140%
Triton X-100 Triton X-100 +50mM Tris
Methanol
Yie
ld
(% o
f Tri
ton
X-1
00 E
xtr
ac
tio
n)
Extraction Methods
Normalized Extraction Yields
1st extraction
2nd extraction
![Page 12: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/12.jpg)
MultiPlex ELISA
Informational multiplex assays are occasionally used for a
variety of analytes
Each analyte is not tested enough to justify full
development
We’d like to have a “one-size fits all” approach to allow
gross evaluation of a number of analytes
Do we digest the material or homogenize in MPER
(mammalian protein extraction reagent)?
![Page 13: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/13.jpg)
MultiPlex ELISA Digestion or
Extraction
• Results excluded when non-detectable
• Homogenate extract, with MPER, is generally more effective
0.1
1
10
100
1000
10000
100000
IL-1
raIL
-4IL
-7IL
-18
FG
F-1
FG
F-2
FG
F-2
EG
FV
EG
FV
EG
F-a
PD
GF
PLG
FM
CP
-1M
CP
-3M
DC
FIT
-3L
Fra
cta
lkin
eR
AN
TE
S
Mass c
on
ten
t (n
orm
alized
to
IL
-1ra
)
UBM MultiPlex ELISA
Pepsin tissue digest
Homogenate extract
![Page 14: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/14.jpg)
DIALYSIS AND/OR DILUTION
After extraction or sample digestion, do we need to remove potential
contaminants?
![Page 15: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/15.jpg)
bFGF Quantification
bFGF: basic Fibroblast Growth Factor (FGF-2)
Can be measured as a representative of a large number of
heparin-binding proteins
Not known to affect product performance, but we often receive
inquiries from physicians about GF content
Measurable in solution using a number of available human bFGF
ELISA kits
We need a sample solution that won’t interfere with specific
antibody binding
![Page 16: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/16.jpg)
bFGF Extraction Buffer and
Dialysis
Buffers tested:
Medium 106, with protease inhibitors
Urea, with heparin, tris, and protease inhibitors
Dialysis
Each buffer tested with or without 2x 24hr dialysis
Test Articles:
Micronized (milled) UBM samples, non-sterile
![Page 17: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/17.jpg)
bFGF Extraction Buffer and
Dialysis
0
0.5
1
1.5
2
2.5
3
Medium 106 Medium 106 w/Dialysis
Urea Urea w/ Dialysis
bF
GF
Co
nte
nt
(No
rma
lize
d t
o M
ed
ium
10
6)
Treatment Group
![Page 18: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/18.jpg)
Urea Extraction Yield Confirmation
• Spike recovery experiments performed by spiking bFGF either into
the powder, followed by drying, or into the extract
0
0.5
1
1.5
2
2.5
3
Normal Powder Spike Extract Spike
Measu
red
bF
GF
Co
nte
nt
(no
rma
lize
d t
o N
orm
al)
Spike Recovery of bFGF in UBM
Calculated Spike
Normal Pre-spike Content
Measured Values
![Page 19: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/19.jpg)
dsDNA Quantification
• DNA content is indicative of the level of remaining nuclear (or
mitochondrial) components
In addition, DNA specifically can negatively influence an immune response
• Measurable in solution using Quant-IT Picogreen
• Tissue digestion is required to release DNA into solution, creating high
levels of turbidity
• Dilution is commonly used, but to what extent for our samples?
![Page 20: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/20.jpg)
Dilutions of DNA assay digest
Test Articles:
Micronized (milled) UBM samples, non-sterile
Digested by Proteinase-K enzyme in lysis buffer
Dilutions
Single-step dilutions 1:10 to 1:100
Two-step dilutions 1:200 to 1:1000
![Page 21: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/21.jpg)
Dilutions of DNA assay digest
0
0.2
0.4
0.6
0.8
1
1.2
0 200 400 600 800 1000 1200
Ca
lcu
late
d D
NA
Co
nte
nt
(no
rma
lize
d t
o 1
:10
0)
Dilution Factor
1:100 Dilution – No change to procedure
![Page 22: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/22.jpg)
In Conclusion
• Testing of dECM products largely focuses on materials
testing and “representative” compositional assays
• With soft tissue materials, our unique challenges generally
are related to the sample preparation – bringing our
analytes into a stable solution that won’t interfere with
commercially-available assay
• Like most testing, different types of analytes are suited to
different methods, and the simplest option is often the
most effective
![Page 23: Characterization of Decellularized Extracellular Matrix for Clinical … · 2018. 4. 28. · •Extracellular Matrix materials such as UBM are resorbable/degradable and allow remodeling](https://reader035.fdocuments.in/reader035/viewer/2022071502/6121c9825c14d44ec62e3fe9/html5/thumbnails/23.jpg)
Acknowledgements
Alphaba Sacko (Phospholipid)
Kevin Joye (DNA)
Jaci Miller (bFGF)
Penelope Morel, U Pitt (Multiplex ELISA)