Table 3.

Methodology of decellularized tissue or cell-derived ECM

Agents/techniques	Mode of action	Effects on ECM
Physical treatments
Freeze and dry	Xenogeneic cellular compounds can be washed away after microscopic ice crystals disrupt cell membrane	Disrupt or fracture ECM fibers [92–94]
Mechanical-shaking force	Shaking action promotes cell debris removal from matrix	Disrupt ECM structure and clean up the cellular fragments [95–97]
NTIRE	Electrical pulse disrupts cellular membranes	Can disrupt ECM [98,99]
scCO2	Deeply penetrates into tissues and solubilizes non-polar molecules	Can disrupt ECM when the system is rapidly depressurized [81]
Chemical treatments
Acids and bases	Disrupts both intracellular organelles and cell membranes	Break down collagen and GAGs and denature proteins or growth factors [95,100]
Ionic detergents	Solubilizes plasma membranes and nuclear membranes	Denature proteins via damaging bonds between proteins [82,101,102]
Non-ionic detergents	Disrupts bonds between lipids and between lipids and proteins	Beneficial to keep the ECM intact, may disrupt ultrastructure and GAGs [83,101,102]
Enzymatic treatments
Trypsin	Cleaves cell adhesion from ECM	Extended exposure can destroy the structure of ECM, remove fibronectin, laminin, elastin, GAG [103–105]
Dispase	Cleaves collagen IV and fibronectin	Extended exposure can destroy the ultrastructure of ECM [95,106]
Nuclease (DNase and RNase)	Degrades nucleic acids	Hard to remove, may induce immune reaction [107–109]
FBS (serum containing DNase and RNase)	Retains bioactive proteins, degrades remaining DNA/RNA	Can minimize the loss of major bioactive proteins, decrease xenogeneic immune response [86–88]
Combined methodologies
Shaking action + FBS	Optimizes approaches to remove xenogeneic cellular compounds by maintaining bioactive proteins and ECM structure

ECM, extracellular matrix; GAGs, glycosaminoglycans; NTIRE, non-thermal irreversible electroporation; scCO₂, supercritical carbon dioxide; FBS, fetal bovine serum.