characterization of decellularized extracellular matrix for clinical … · 2018. 4. 28. ·...

Characterization of

Decellularized Extracellular

Matrix for Clinical Use

Nikhil Gheewala, PhD

Principal Scientist, ACell

CASSS - Thursday, April 5th, 2018

Extracellular Matrix

Cells

Collagens

Elastin

Proteoglycans

Secreted and Bound Components

Decellularization (Ideal)

Collagens

Elastin

Proteoglycans

Secreted and Bound Components

Goal: Maximize ECM retention and cell removal while minimizing ECM damage

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;

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

*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)

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

EXTRACTION METHODS

Some material components can be extracted without requiring enzymatic

digestion of the tissue macrostructure, using appropriate buffers and

micronizing/homogenization methods

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”

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

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

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)?

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

DIALYSIS AND/OR DILUTION

After extraction or sample digestion, do we need to remove potential

contaminants?

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

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

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

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

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?

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

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

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

Acknowledgements

Alphaba Sacko (Phospholipid)

Kevin Joye (DNA)

Jaci Miller (bFGF)

Penelope Morel, U Pitt (Multiplex ELISA)