6.ECM

Question 1

Decellularization

Answer 1

“The processing of tissues or organs to remove cells and cellular
debris, with the aim of preserving biological activity and/or structural
integrity of the extracellular matrix”
Avoid adverse allogeneic or xenogeneic
immunologic responses;
Retain most of the ECM components,
sequestered growth factors, glycoproteins, and
other soluble and insoluble bioactive molecules.

Question 2

Steps of decellularization

Answer 2

1. Cell membrane lysis à physical treatments or ionic solutions;
2. Separation of cellular component from the ECM à enzymatic treatments;
3. Solubilization of cytoplasmic and nuclear cellular components à detergent
   treatment;
4. Removal of cellular remnants from the ECM.

Question 3

Metrics of decellularization:

Answer 3

• < 50 ng of double-stranded DNA/mg of dry weight ECM
• No visible nuclei in H&E stained histological sections
• Residual DNA < 200 base pairs in length

Question 4

physical methods of decellularization

Answer 4

-snap freezing
-mechanical force
-mechanical agitation

Question 5

enzymatic methods of decellularization

Answer 5

trypsin
endonucleases
exonuclease

Question 6

Key characterization aspects of decellularized ECMs (dECMs)

Answer 6

Histological evaluation
Assess the efficiency of decellularization à removal of cellular constituents
Evaluate the ECM components that were retained upon decellularization
Immunohistochemical evaluation
Biochemical evaluation
ECM Ultrastructure evaluation
Biomechanical evaluation
Biofunctionality Microbiological evaluation evaluation

Question 7

Decellularization – challenges

Answer 7

• Require optimization for each tissue (tissue/organ-specificity of the ECM)
• Incomplete cellular removal ( immune reactions )
• Tissue variability (e.g., thickness, architecture) => efficacy of decellularization
• Impaired bioactivity and structural integrity of the ECM (?)

Question 8

after decellularization

Answer 8

processing>sterilization>recellularization

Question 9

Recellularization

Answer 9

“The repopulation of decellularized tissues or organs by cells with the objective of ultimately reintroducing functional activity”.

Question 10

Soluble signaling molecules

Answer 10

growth factors
cytokines

Question 11

insoluble signaling molecules

Answer 11

cell-adhesion ligands
proteolytic sites

Question 12

Action mechanisms of ECM

Answer 12

1) cell recruitment and infiltration
ECM is able to attract and/or facilitate differentiation of endogenous stem or progenitor cells
2) immunomodulation
ECM has shown to be able to shift the innate immune response away from inflammation and toward remodelling