Decellularized adipose matrix provides an inductive microenvironment for stem cells in tissue regeneration

Table 1 Different studies of decellularization and sterilization methods for preparation of DAM

Decellularization methods			Sterilization methods	Refs.
Physical treatments	Chemical treatments	Biological treatments
Freeze–thaw, 3 cycles (-80 °C to 37 °C)	99.9% isopropanol	0.25% trypsin/0.1% EDTA 15000 U DNase, 12.5 mg RNase, 2000 U lipase	70% ethanol/1% penicillin and streptomycin/UV light/1% antibiotic/antimycotic	[26,30,74,75,83,84,96,101,102]
		0.05% trypsin-EDTA 100 U/mL benzonase	70% ethanol/1% penicillin and streptomycin	[63,66]
		0.05% trypsin 500 U/mL benzonase	0.1% peracetic acid in 4% ethanol	[61]
	99.9% isopropanol 1 mol/L NaCl	1 mmol/L EDTA + Lysis buffer (1% tergitol type NP-40, 0.1% SDS, 5 mmol/L EDTA, 0.4 mol/L NaCl, 50 mmol/L Tris-HCl pH 8, 1 mmol/L PMSF)	70% ethanol/1% penicillin and streptomycin	[65]
	100% isopropanol	0.25% trypsin-EDTA; 1 mL DNase + 1 mL RNase + 2 mL lipase	70% ethanol/1% penicillin and streptomycin	[135]
	0.5 mol/L NaCl/1 mol/L NaCl/isopropanol/Triton X-100	0.25% trypsinEDTA	1% penicillin and streptomycin	[69,71,93]
Freeze-thaw, 3 cycles (-80 °C to 37 °C) + ultrasonic	0.5% SDS + 100% ethanol	—	100% ethanol	[25]
Freeze-thaw, 35 cycles (-80 °C to 37 °C) + homogenization, 5 min (12000 r/min)	1% Triton X100 + 100% isopropanol + 1 mol/L NaCl	100 U/mL DNase 100 μg/mL RNase	—	[68]
Freezethaw, 4 cycles (-80 °C to 37 °C) + ultrasonic	96% ethanol 0.5% SDS	0.05% trypsin/0.05 mmol/L EDTA + DNase	—	[87]
Freezethaw, 5 cycles (-80 °C to 37 °C)	Isopropanol	0.25% trypsin/0.1% EDTA DNase I + RNase A	Ethylene oxide	[136]
Freeze-thaw, 5 cycles (liquid nitrogen to 37 °C)	99.9% isopropanol	0.05% trypsin-EDTA 20 ng/mL DNase I + 20 ng/mL RNase	1% penicillin and streptomycin	[67]
Freezethaw, 4 or 5 cycles (liquid nitrogen to Room temperature)	0.5 mol/L acetic acid	—	—	[88]
Freezethaw, 35 cycles (liquid nitrogen to Room temperature)	0.1% SDS	0.05% trypsin + 0.05% EDTA + 20 ng/mL DNase I + 20 ng/mL RNase	1% penicillin and streptomycin	[89]
Freezethaw, 618 cycles (liquid nitrogen to room temperature)	0.1% sodium azide + 1 mol/L NaCl + 4% sodium deoxycholate	2000 K units DNase	—	[78]
	1% Triton X-100	2000 K units DNase
Homogenization, 5 min (12000 rpm)	1 mol/L NaCl/0.5% SDS	0.2% DNase + 200 μg/mL RNase	—	[27,28,81]
	—	—	—	[104,137]
	0.5% SDS + 100% isopropanol	—	—	[138]
Homogenization, 5 min + ultrasonic	—	0.25% Pancreatin	Ethylene oxide	[79]
Homogenization, 3 min (12000 r/min)	SDS	—	—	[90]
	4 mol/L urea	4 mol/L Gu	Ethylene oxide	[100,139]
Homogenization (twice)	2 mol/L urea+70% ethanol	2 U/mL dispase II + 4 mol/L GuHCl	Dialysis against chloroform	[23,24]
Homogenization	2 mol/L urea buffer	—	70% ethanol/1% antibiotic/antimycotic solution	[65]
Constant stirring	1% SDS or 2.5 mmol/L sodium deoxycholate	2.5 mmol/L sodium deoxycholate + 500 U porcine lipase + 500 U porcine colipase	365 nm UV light	[62,85]
Constant stirring	1% SDS	2.5 mmol/L sodium deoxycholate + 100 μg/mL lipase + 50 ng/mL colipase; 50 μg/mL DNase + 50 μg/mL RNase	Ethylene oxide	[86]
Mechanical processing	0.1%, 1%, 3%, or 5% Peracetic acid + 1% Triton X-100	600 U DNase	—	[31,94]
	3% Triton X100 + 4% sodium deoxycholate + 4% ethanol/0.1% peracetic acid + 100% n-propanol	0.02% trypsin + 0.05% EDTA	4% ethanol + 0.1% peracetic acid	[22]
SCCO2 (180 bar)	Ethanol	—	SC-CO2	[91]
—	1% sodium dodecylsulfate + 100% isopropanol	2.5 mmol/L sodium deoxycholate + 500 U lipase + 500 U colipase	5000 IU penicillin and 5 mg/mL streptomycin	[32]
	0.5% SDS + isopropanol + 0.1% peracetic acid + 4% ethanol	—	0.1% peracetic acid+4% ethanol	[92,103]
	1% Triton X-100	10, 20 and 100 IU/mL DNase I	—	[82]
	1-propanol	Sodium deoxycholate	Peracetic acid	[33]
	Organic solvent + surfactant/ethanol-based solution	—	Peracetic acid	[34,64]

SDS: Sodium dodecyl sulfate; EDTA: Ethylenediaminetetraacetic acid; SC-CO2: Supercritical carbon dioxide; Gu: guanidine.

Table 2 Comparison of each physical, chemical, and biological treatments in the adipose tissue decellularization protocols

Agent/method	Function or advantages	Impact or disadvantages
Physical
Freezing thawing	Ice crystals destroy cell membranes	Ice crystals also destroy the continuity of
	Preserve component integrity	DAM composition and microstructure
	Reduce immune response
Homogenization	Fully destroy the cell membrane structure and promote dissociation from basement membrane	Mechanical shear forces break the microstructure and component continuity
Constant stirring	Cleave the cell membrane	Stirring forces destroy microstructure
	Full exposure accelerates the effect of chemical agents	Mechanical properties are affected
Mechanical processing	Promote cell membrane rupture and release from the basement membrane	Pressure directly destroys microstructure; ultrastructure and basement membrane integrity are destroyed
SC-CO2 treatment	Supercritical inert gas penetrates tissues to remove cell residues/sterilization	Entrainer may reduce structural composition; supercritical pressure may destroy the structure
Ultrasonic	Ultrasonically break cell membrane	-
Chemical
Hypotonic/hypertonic solutions	Dissociate DNA from proteins; Osmotic pressure ruptures cell membranes	Little influence on the structure and composition of DAM
Alcohols
Isopropanol	Cell dehydration, cell membrane lysis	DAM protein components are precipitated; destruction of ultrastructure; degreasing alone has poor effect
Ethanol	Effectively remove lipid residue
Acids and bases
Acetic acid	Hydrolyze biomolecules to remove residual nucleic acids; little effect on the structure; better retention of GAGs components	Some collagen components are destroyed and removed; reduced strength of DAM; collagen, growth factors, and GAGs are damaged
Peracetic acid		Little effect on the structure and composition of DAM
Nonionic detergents
Triton X-100	Disturbing DNA–protein, lipid–lipid, and lipid–protein associations; moderate effect/stable in solution	Destruction of ultrastructure; remove GAGs
Agent/ Methods	Function or advantage	Impact or disadvantage
Ionic detergents
SDS	Effectively remove cellular nucleic acid components/destruction of cell membrane phospholipids and lipoproteins/dissolving antigen and eliminating immune complexes	Disturbing protein–protein association; growth factor removal; destroy ultrastructure, GAGs ingredients; residue of the reagent causes cytotoxicity
Sodium deoxycholate
Triton X-200
Biologics
Trypsin	Cleavage of the C-side peptide bond of Arg and Lys	Remove fibronectin, elastin, and GAGs components; damage degree of DAM composition and microstructure is highly time-dependent
Nucleases (DNase, RNase)	Cleavage nucleotides sequence	Difficult to remove residue from DAM; residual effects on host recellarization; causes host immune response
Lipase and colipase	Remove residual lipids	Destruction of ultrastructure; removes GAGs; efficiency of lipid removal is low
EDTA	Dissociation of metal ions plays a supporting role in tissue decellularization	Destruction of protein–protein linkages; poor application alone

SDS: Sodium dodecyl sulfate; EDTA: Ethylenediaminetetraacetic acid; SC-CO₂: Supercritical carbon dioxide.