Copyright ©The Author(s) 2020.
World J Stem Cells. Jul 26, 2020; 12(7): 585-603
Published online Jul 26, 2020. doi: 10.4252/wjsc.v12.i7.585
Table 1 Different studies of decellularization and sterilization methods for preparation of DAM
Decellularization methods
Sterilization methodsRefs.
Physical treatmentsChemical treatmentsBiological treatments
Freeze–thaw, 3 cycles (-80 °C to 37 °C)99.9% isopropanol0.25% trypsin/0.1% EDTA 15000 U DNase, 12.5 mg RNase, 2000 U lipase70% 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 benzonase70% ethanol/1% penicillin and streptomycin[63,66]
0.05% trypsin 500 U/mL benzonase0.1% peracetic acid in 4% ethanol[61]
99.9% isopropanol 1 mol/L NaCl1 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% isopropanol0.25% trypsin-EDTA; 1 mL DNase + 1 mL RNase + 2 mL lipase70% ethanol/1% penicillin and streptomycin[135]
0.5 mol/L NaCl/1 mol/L NaCl/isopropanol/Triton X-1000.25% trypsinEDTA1% penicillin and streptomycin[69,71,93]
Freeze-thaw, 3 cycles (-80 °C to 37 °C) + ultrasonic0.5% SDS + 100% ethanol100% 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 NaCl100 U/mL DNase 100 μg/mL RNase[68]
Freezethaw, 4 cycles (-80 °C to 37 °C) + ultrasonic96% ethanol 0.5% SDS0.05% trypsin/0.05 mmol/L EDTA + DNase[87]
Freezethaw, 5 cycles (-80 °C to 37 °C)Isopropanol0.25% trypsin/0.1% EDTA DNase I + RNase AEthylene oxide[136]
Freeze-thaw, 5 cycles (liquid nitrogen to 37 °C)99.9% isopropanol0.05% trypsin-EDTA 20 ng/mL DNase I + 20 ng/mL RNase1% 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% SDS0.05% trypsin + 0.05% EDTA + 20 ng/mL DNase I + 20 ng/mL RNase1% penicillin and streptomycin[89]
Freezethaw, 618 cycles (liquid nitrogen to room temperature)0.1% sodium azide + 1 mol/L NaCl + 4% sodium deoxycholate2000 K units DNase[78]
1% Triton X-1002000 K units DNase
Homogenization, 5 min (12000 rpm)1 mol/L NaCl/0.5% SDS0.2% DNase + 200 μg/mL RNase[27,28,81]
0.5% SDS + 100% isopropanol[138]
Homogenization, 5 min + ultrasonic0.25% PancreatinEthylene oxide[79]
Homogenization, 3 min (12000 r/min)SDS[90]
4 mol/L urea4 mol/L GuEthylene oxide[100,139]
Homogenization (twice)2 mol/L urea+70% ethanol2 U/mL dispase II + 4 mol/L GuHClDialysis against chloroform[23,24]
Homogenization2 mol/L urea buffer70% ethanol/1% antibiotic/antimycotic solution[65]
Constant stirring1% SDS or 2.5 mmol/L sodium deoxycholate2.5 mmol/L sodium deoxycholate + 500 U porcine lipase + 500 U porcine colipase365 nm UV light[62,85]
Constant stirring1% SDS2.5 mmol/L sodium deoxycholate + 100 μg/mL lipase + 50 ng/mL colipase; 50 μg/mL DNase + 50 μg/mL RNaseEthylene oxide[86]
Mechanical processing0.1%, 1%, 3%, or 5% Peracetic acid + 1% Triton X-100600 U DNase[31,94]
3% Triton X100 + 4% sodium deoxycholate + 4% ethanol/0.1% peracetic acid + 100% n-propanol0.02% trypsin + 0.05% EDTA4% ethanol + 0.1% peracetic acid[22]
SCCO2 (180 bar)EthanolSC-CO2[91]
1% sodium dodecylsulfate + 100% isopropanol2.5 mmol/L sodium deoxycholate + 500 U lipase + 500 U colipase5000 IU penicillin and 5 mg/mL streptomycin[32]
0.5% SDS + isopropanol + 0.1% peracetic acid + 4% ethanol0.1% peracetic acid+4% ethanol[92,103]
1% Triton X-10010, 20 and 100 IU/mL DNase I[82]
1-propanolSodium deoxycholatePeracetic acid[33]
Organic solvent + surfactant/ethanol-based solutionPeracetic acid[34,64]
Table 2 Comparison of each physical, chemical, and biological treatments in the adipose tissue decellularization protocols
Agent/methodFunction or advantagesImpact or disadvantages
Freezing thawingIce crystals destroy cell membranesIce crystals also destroy the continuity of
Preserve component integrityDAM composition and microstructure
Reduce immune response
HomogenizationFully destroy the cell membrane structure and promote dissociation from basement membraneMechanical shear forces break the microstructure and component continuity
Constant stirringCleave the cell membraneStirring forces destroy microstructure
Full exposure accelerates the effect of chemical agentsMechanical properties are affected
Mechanical processingPromote cell membrane rupture and release from the basement membranePressure directly destroys microstructure; ultrastructure and basement membrane integrity are destroyed
SC-CO2 treatmentSupercritical inert gas penetrates tissues to remove cell residues/sterilizationEntrainer may reduce structural composition; supercritical pressure may destroy the structure
UltrasonicUltrasonically break cell membrane-
Hypotonic/hypertonic solutionsDissociate DNA from proteins; Osmotic pressure ruptures cell membranesLittle influence on the structure and composition of DAM
IsopropanolCell dehydration, cell membrane lysisDAM protein components are precipitated; destruction of ultrastructure; degreasing alone has poor effect
EthanolEffectively remove lipid residue
Acids and bases
Acetic acidHydrolyze biomolecules to remove residual nucleic acids; little effect on the structure; better retention of GAGs componentsSome collagen components are destroyed and removed; reduced strength of DAM; collagen, growth factors, and GAGs are damaged
Peracetic acidLittle effect on the structure and composition of DAM
Nonionic detergents
Triton X-100Disturbing DNA–protein, lipid–lipid, and lipid–protein associations; moderate effect/stable in solutionDestruction of ultrastructure; remove GAGs
Agent/ MethodsFunction or advantageImpact or disadvantage
Ionic detergents
SDSEffectively remove cellular nucleic acid components/destruction of cell membrane phospholipids and lipoproteins/dissolving antigen and eliminating immune complexesDisturbing protein–protein association; growth factor removal; destroy ultrastructure, GAGs ingredients; residue of the reagent causes cytotoxicity
Sodium deoxycholate
Triton X-200
TrypsinCleavage of the C-side peptide bond of Arg and LysRemove fibronectin, elastin, and GAGs components; damage degree of DAM composition and microstructure is highly time-dependent
Nucleases (DNase, RNase)Cleavage nucleotides sequenceDifficult to remove residue from DAM; residual effects on host recellarization; causes host immune response
Lipase and colipaseRemove residual lipidsDestruction of ultrastructure; removes GAGs; efficiency of lipid removal is low
EDTADissociation of metal ions plays a supporting role in tissue decellularizationDestruction of protein–protein linkages; poor application alone