Table 2.

Decellularization Methods for Central Nervous System (CNS) Applications.

Application	Study model	Source Tissue	Additional Components	Post Processing	Decellularization Method				Notable Results	References
					#	Step	Time	RPM
SCI	in vitro	Rat spinal cord	N/A	N/A		Mechanical tissue separation Tissue segmented			Good ECM structure preservation. Constructs contained laminin, collagen, and fibronectin and show increased neural cell adhesion and proliferation.	[130]
					2x	1% Triton X-100	3 h	yes*
						1% Deoxycholate Stored at 4 °C in PBS	3 h	yes*
	in vitro	Rat spinal cord	Genipin crosslinker and glutaraldehyde crosslinker	Lyophilized, immersed in crosslinker solutions, lyophilized again, and sliced into discs for culturing		Mechanical tissue separation Tissue segmented			Genipin was equivalent in structural properties and superior in biocompatibility to glutaraldehyde. Degradation of the genipin crosslinked scaffold was around 20% in trypsincontaining buffer over 14 days. Genipen increased proliferation and ECM secretion.	[15,130]
					2x	1% Triton X-100, 1 h media changes	3 h	100
						1% Deoxycholate, 1 h media changes Lyophilization	3 h	100
						5 mg/mL crosslinker immersion at 37 °C Lyophilization Cobalt-60 irradiation for sterilization Stored dry at −20 °C	24 h
	Rat spinal cord hemisection up to 8 weeks	Rat thoracic spinal cord	human umbilical cord MSCs	Freeze dried and gamma irradiated		Mechanical tissue separation			hUCB-MSC addition to ECM scaffolds showed increased motor function recovery. Increases in axon sprouting, myelination, and oligodendrocyte migration were observed.	[130,133]
					2x	Tissue segmented 1% Triton X-100	3 h	yes*
						1% Deoxycholate	3 h	yes*
						Freeze-dried Gamma irradiation	24 h
	Rat spinal cord hemisection	Rat paravertebral muscle	N/A	N/A	1x	3% Triton X-100	2d		Parallel and linear axonal spouting was observed and axon survival was increased.	[118,181]
						0.1% SDS	2d
						PBS	1d
	Subcutaneous rat implant model up to 1 week	Rat sciatic nerve	N/A	N/A	Method 1: Detergent				For methods 1 and 2, nuclear content was eliminated but some myelin was detected. Immunogenicity was lower than untreated sciatic nerve in methods in all 3 methods with method 3 showing the least MHC II presentation. Mechanical properties of normal sciatic nerve were preserved.	[138]
					2x	3% Triton X-100	12 h
						4% Deoxycholate Stored at 4 °C in PBS	24 h
					Method 2: Detergent/Enzyme
						0.5% Triton X-100	48 h	yes*
						DNase and RNase at 37 °C	12 h
					Method 3: Enzyme
						Hypotonic solution rinse at 4 °C	12 h
						Frozen at −80 °C	6 h
						Thawed at 37 °C	30 m
						0.05% pancreatin	6 h
						DNase and RNase	12 h
	in vitro	Rat spinal cord	N/A			Triton X-100 Deoxycholate		Varied	This study observed effects of agitation on decellularization and found 120 RPM to be optimal for spinal cord.	[141]
								Varied
	Rat spinal cord hemisection model up to 14 days	Rat spinal cord	bFGF and HP (heparin modified poloxamer)	lyophilized and mixed with bFGF and HP	1x	1% triton X-100 at 4 °C	12 h	120	Inclusion of HP-bFGF increases PC12 cell survival by 50%. bFGF reduced glial scarring and increased recovery.	[140]
							8 h	120
						4.0% sodium deoxycholate at 4 °C
					10x	0.01% PBS elution rinse at 4 °C	10m	60
	in vitro, PC12 cell line up to 7 days in culture	Porcine brain	N/A	Comminuted into < 1 mm particles, pepsin/HCl solubilized, gelled via neutralization and dilution	1x	Freeze/Thaw cycle at −80 °C Longitudinal quartering of tissue	16 h		This study observed the effects of various nerve tissue sources. Brain derived ECM scaffolds showed significantly greater neurite extension length. Otherwise spinal cord, brain, and UBM showed equivalent benefits.	[128,134,139]
					1x	Type 1 water at 4 °C	16 h	60 120 for brain/200 for spinal cord/nerve
		Porcine spinal cord				0.02% Trypsin/0.05% EDTA at 37 °C	1 h
						3% Triton X-100	1 h
						1.0 M Sucrose	15 m
		Porcine optic nerve				4% Deoxycholate	1 h
						1% Peracetic acid in 4% ethanol Lyophilized and stored dry	2 h
	Rat acute SCI up to 8 weeks post-injury	Porcine spinal cord UBM	hMSCs	Same gelation process as Crapo et al. 2012 above	Same method as Crapo et al. 2012 above with additional steps.				This study compared SC ECM with UBM hydrogels. Equivalence was found in most categories. hMSCs added to SC ECM found to have little effect. SC ECM and UBM both found to significantly modulate the immune response.	[128,137]
						Digested 1.0 mg/mL pepsin, 0.01 M HCl Neutralized 0.1 M NaOH, 10x PBS
	in vitro with rat SCs, in vivo rat SCI for 14 days	Rat sciatic nerve	SCs	Lyophilized, digested, applied as hydrogel		SB-10 in 50 mM sodium/10 mM phosphate buffer 0.14% Triton X-200/0.06 mM SB-16 in 50 mM sodium/10 mM phosphate 200 uL chondroitinase ABC at 37 °C	16 h		The ECM hydrogel supported axon growth, SC survival, and axon myelination. Locomotive recovery was increased, and no chronic immune response was observed.	[150,151,163]
TBI	Mouse CCI model up to 35 days	Porcine brain	N/A	Lyophilized and reconstituted with PBS	Same method as Crapo et al. 2012 above.				ECM showed reduced glial scarring post-injury as well as reduced neurodegeneration and reduced lesion volume.	[128,139]
	Controlled cortical impact rat model up to 26–28 days.	Porcine UBM	NSCs cultered in UBM hydrogel prior to injection	Same gelation process as Crapo et al. 2012 above		Mechanical separation of the luminal surface 1 M saline rinse			UBM hydrogels reduced CNS tissue loss and mitigated losses in memory, cognition, and motor faculties. UBM and UBM + NSCs showed significant benefits, with reduction of loss in memory and cognition being significant with the inclusion of NSCs.	[166,167]
					1x	0.1% Peracetic acid/4% ethanol Lyophilization	2 h
						Solubiliztion in 1 mg/mL pepsin/0.01 M HCl Frozen at −20 °C for storage	2d
	Chick embryo chorioallantoic membrane	Whole rat brain	N/A		2x	Antibiotic/Antimycotic rinse			The observed angiogenic response was comparable to FGF-2 application. Little to no inflammatory response was seen.	[135,182]
					4x	Freeze/Thaw cycle 2x	4% Sodium deoxycholate	1 h
						2000 kU DNase I in 1 M Na CL	15 m
		Storage at 4 °C in PBS
	in vitro	Whole Rat Brain	N/A	Lyophilized, mixed with gelatin, electrospun, and then genipin crosslinked		Antibiotic/Antimycotic rinse			A cytocompatible scaffold was produced that, when cultured with allogeneic rat mSCs, showed enhanced differentiation potential.	[127,135]
					4x	Freeze/thaw cycle
					1x	1% Triton X-100	1 h	60
						4.0% deoxycholate	1 h	60
						2000 KU DNase in 1 M NaCl Stored in PBS 1% antibiotic/antimycotic	1 h	60
	In vitro characterization with NSCs and subcutaneous and intracranial implantation in a rat model up to 4 weeks	Mouse Cerebellum	N/A	N/A		Transcardial 1% SDS infusion (pre-harvest)	5 m		Scaffolds retained neurosupportive proteins and increased migration and proliferation of NSCs in coculture. When compared to UBM intracranial implants, advantages were seen in cerebellum-derived ECM scaffolds.	[120]
					1x	1% SDS	1 h	100
						0.02% Trypsin/0.05% EDTA	30 m	60
						1% Triton X-100	1 h	100
						1 M Sucrose	15 m	60
						Antibiotic Rinse Stored in PBS	3d
	In vitro with iPS cells	Halved rat whole brain	N/A		3x	0.1% SDS in PBS with 1% antibiotic/antimycotic	24 h		Decellularized brain scaffolds induced neural differentiation pathways when cultured with iPS cells.	[119]
					1x	Centrifugation in 50 mL conical tubes	5 m	10,000
					10x	PBS solution centrifugation (SDS removal)	5 m	10,000
	3D in vitro culturing	Fetal and young adult porcine brain	N/A	Lyophilized and gelled following various manufacturer instructions for Hydromatrix, Puramatrix, and HyStem C		Mechanical separation of the dura mater			Neural network formation was enhanced in the presence of fetal and young adult ECM compared to collagen alone in 3D culture.	[128,136]
					1x	0.05% Trypsin/EDTA with 0.2% DNase at 37 °C	1 h
						3% Triton X-100 with 0.2% DNase	1 h/30 m
						1 M Sucrose	15 m
						4% sodium deoxycholate	1 h/15m
						0.1% peracetic acid/4% ethanol Lyophilization and storage at −20 °C	2 h
	in vitro 3D culturing with NSCs up to 7 weeks	Mouse brain 1.5 mm sections	N/A	N/A	1x	4% sodium deoxycholate	14 h	150	NSCs labeled with GFP showed continued growth over 7 weeks cultured on brain ECM sections, and, when exposed to mitogenic stimuli, retained their NSC phenotype.	[129]
						40 kU/mL DNase	1 h	150
							deionized water 150	4h
							3% Triton X-100	2 h
					150 40 kU/mL DNase 2x		1 h	150
							deionized water 150	4h
							4% sodium deoxycholate 150	12 h
	40 kU/mL DNase	1 h	150
	deionized water	2 h	150
	3% Triton X-100	2 h	150
	40 kU/mL DNase	1 h	150
	in vitro	Rat cerebral cortex	Genipin crosslinker	—		Freeze/thaw Triton X-100 Deoxycholate DNase and RNase Genipin crosslinker			Genipen increased pore sizes of scaffolds. Fibronectin and basement membrane were weakly retained but laminin was strongly retained.	[131]
Stroke	Rat stroke model up to 14 days and 12 weeks	Porcine UBM	N/A	Same gelation process as Crapo et al. 2012 above	1x	Mechanical delamination 0.1% Peracetic acid/4% ethanol Debris removal via PBS rinses Lyophilization Solubilization in 1 mg/mL pepsin/0.01 M HCl Frozen at −20 °C for storage	2 h	300	Injection of the UBM hydrogel increased cell proliferation and density at the injury site interface with some penetration. However, at 12 weeks there were no significant differences in scar formation.	[164,165]
Parkinson's	Parkinson's Disease model rats up to 20 days	Rat brain	bFGF	N/A		Transcardial PBS perfusion during harvest			Sustained release of bFGF from ECM scaffolds increased cell survival and behavioral recovery.	[127,132]
					1x	3% Triton X-100	12 h	120
						4% deoxycholic acid sodium salt	24 h	120
						0.1% peracetic acid/4% ethanol Stored in PBS 1% antibiotic/antimycotic	3 h	120