Table 1.

Methods used in decellularization processes.

Methods	Mechanism	Side Effects on the ECM	References
Chemical
Acid; Base	Solubilizes cytoplasmic components, disrupts nucleic acids	Damages collagen and GAG	[32]
Triton X-100	Breaks lipid–lipid and lipid–protein unions, while leaving the protein interactions untouched	Not recommended for ECM where the lipids and GAG are important components	[9,23,26,44,45]
SDS	Liquefies the internal and external cell membranes	Tends to denaturalize proteins and may induce nuclear and cytoplasmic waste in the remaining matrix	[24,25,26,46,47,48]
Triton X-200	Similar to its X-100 counterpart. Very effective in some tissues	Needs to be combined with a zwitterionic detergent to be effective. Damages the matrix in a similar way that SDS does.	[28]
CHAPS	Properties of ionic and nonionic detergents	Similar damage level compared to Triton X-100	[27]
TBP	Disrupts protein–protein interactions	Variable results, collagen degradation but keeping the mechanical properties	[25,49]
Hypertonic and hypotonic solutions	Osmotic pressure to make the membrane explode	High amount of cell waste in the remaining matrix	[11,33,50]
Enzymes
EDTA, EGTA	Breaks cell adhesion to matrix. It is usually combined with trypsin	Does not actually kill the cells	[29,30,46,50,51]
Trypsin	Digestion of membrane proteins leading to cell dead	Can damage the proteins in the ECM, in particular laminin and GAG	[29,30,43,52]
Pepsin	It targets peptide bounds	Causes high damage in the ECM proteins if left for too long	[31]
Endonucleases and Exonucleases	Degradation of the nuclear material inside and outside of the nucleus	Further cleaning and enzyme removal is required, as they may promote immune response	[6,29,53,54,55,56,57]
Physical
Freezing	Crystals created in the freezing process destroy the cell membrane	The overall protein structure of the ECM may be compromised	[24,35,36,37,38,58]
Force	Mechanical pressure can be enough to induce the lysis in some tissues	Limited to tissues with hard structures, as it can greatly damage the ECM structure	[39]
Agitation	Commonly used to facilitate chemical agent infiltration and to induce cell lysis	Aggressive processes like sonication can greatly damage the ECM	[40,41]
Vacuum-assisted decellularization (VAD)	Enables chemical agents to reach the more inner parts of the tissue	It is not a decellularization method but a facilitator	[42]
Hydrostatic pressure	Applies high pressure to the tissue and induces cell lysis	Excessive pressure can damage the structure	[43,59]