Table 1.

Results of experimental studies on obtaining MSCs from cryopreserved UC tissues

Cryopreservation protocol (cryoprotectant; freezing; storage; thawing)	Method used for obtaining primary cultures	First migratory cells from tissue fragments or first cell colonies	MSC phenotype	Differentiating potential of MSCs (in vitro)	Other findings	Reference
10% DMSO; controlled freezing to − 180 °С; storage in liquid nitrogen for 5 years; thawing at room temperature for 30 s, followed by a complete thaw in a 37 °C water bath	Explants	After 10–14 days of culture	CD44+, CD90+, CD105+, CD34−, CD45−	Adipogenic, osteogenic	MSCs isolated from thawed tissue displayed lower plating efficiency, along with a prolonged cell doubling time and fewer total cell doublings, compared with MSCs from fresh tissue	[47]
10% DMSO + 0.2/0.5 М sucrose; freezing to − 80 °С (1 °C/min); storage in liquid nitrogen for 5–29 days; thawing in a 37 °C water bath	Explants	–	CD73+, CD90+, CD34−	Adipogenic, chondrogenic	It took longer to obtain MSCs from cryopreserved UC explants compared with the corresponding fresh explants from the same donor	[43]
					Cryostorage time influenced neither the terms of MSC outgrowth nor their mean population doubling time
10% DMSO; programmed freezing to − 90 °С; storage in liquid nitrogen for 2–3 months; thawing in a 37 °C water bath	Explants pre-incubated in collagenase IV solution for 30–45 min on ice and covered with a stainless steel mesh to protect the tissue from floating	After 7–9 days of culture	CD90+, CD105+, CD73+, CD13+, CD29+, CD44+, CD54+, CD117+, CD71+, CD146+, HLA−ABC+, CD34−, CD45−, HLA-DR−, CD309/VEGF−R2/KDR−	Adipogenic, osteogenic	MSC cultures were obtained from all examined tissue samples irrespective of the time of incubation with the cryoprotectant (5–60 min)	[48]
					Proliferative activity of MSCs isolated from fresh and frozen biological material did not differ
10% DMSO (Cryo Sure-DEX40, 55% w/v DMSO + 5% w/v Dextran); slow freezing to − 90 °С; storage in liquid nitrogen for 1 month; thawing in a 37 °C water bath	Explants	After 10–14 days of culture	CD90+, CD105+, CD34−, CD45−	Adipogenic, chondrogenic, osteogenic	MSCs from cryopreserved UC explants and corresponding fresh explants from the same donor showed similar adipogenic, chondrogenic, and osteogenic in vitro differentiation capacities regardless of growth media used for their isolation and expansion	[49]
					The time required to reach 60% confluence for the post-thaw cultures was longer
					No difference in doubling population time was observed for the cells derived from pre-freeze tissues vs post-thaw tissues
STEM-CELLBANKER (contains DMSO and anhydrous dextrose); freezing to − 80 °С (2 °C/min); storage in liquid nitrogen for 2 weeks; thawing in a 37 °C water bath	Explants covered with a stainless steel mesh to protect the tissue from floating	–	CD73+, CD105+, CD90+, CD44+, HLA-ABC+, CD45−, CD34−, CD14−, CD19−, HLA-DR−	Adipogenic, chondrogenic	Cells derived from UCs after cryostorage retained their immunosuppressive properties, as assessed by allogeneic mixed lymphocyte reactions, and their potential to differentiate into adipocytes and chondrocytes was comparable with cells derived from fresh UC tissue	[44]
Recovery™ Cell Culture Freezing Medium (contains 10% DMSO); programmed freezing to − 150 °С; storage in liquid nitrogen for 1 month; thawing in a 37 °C water bath	Post-thaw WJ was syringed through an 18 G needle a few times to loosen the gelatinous material and dislodge the stem cells; explants for entire cord segments	After 6 days of culture for cell suspensions, after 12 days of culture for explants	CD29+, CD44+, CD73+, CD90+, CD105+, HLA-ABC+, CD14−, CD34−, CD45−, HLA-DR−	Chondrogenic, osteogenic	Freezing of freshly dissected WJ worked better than freezing of entire UC segments in terms of higher MSC viability and proliferation rates, lower numbers of annexin-V-positive and sub-G1 cells, and enhanced osteogenic and chondrogenic in vitro differentiation	[50]
10% DMSO or 0.05 M glucose + 0.05 M sucrose + 1.5 M ethylene glycol cocktail; conventional freezing to − 80 °С (1 °C/min) or programmed freezing to − 140 °С; storage in liquid nitrogen for 3 months; thawing in a 37 °C water bath	Enzymatic digestion with DPBS containing 1 mg/ml collagenase type I at 37 °C for 15 min	After 5 days of culture	CD73+, CD90+, CD105+, CD34−, CD45−	Adipogenic, chondrogenic, osteogenic, hepatogenic	A new cocktail cryoprotectant ensured better preservation of WJ tissue than DMSO	[42]
					Proliferation capacities of the cells isolated from fresh and programmed freezing WJ samples were comparable, proliferation capacity of the cells from conventional freezing WJ samples was significantly lower
					MSCs from the conventional freezing samples showed upregulated expression of pro-apoptotic factors (BAX, p53, and p21) and downregulated expression of anti-apoptotic factor (BCL2), compared with MSCs from the fresh and programmed freezing samples
10% DMS; freezing to − 80 °С (1 °C/min); storage in liquid nitrogen for 3 days	Explants or “conditioned explants” (cultured for 2 weeks allowing adaptation to the culture conditions prior to freezing)	After 14 days of culture for explants, after 2–8 days of culture for “conditioned explants”	CD73+, CD90+, CD105+, CD14−, CD31−, CD34−, CD45−	Adipogenic, chondrogenic, osteogenic	Cryopreserved “conditioned explants” could be repeatedly cryopreserved to show repeated outgrowth MSCs with the same properties for at least four freeze/thaw/explant culture cycles	[51]
					Increasing the number of freeze/thaw/explant culture cycles to 7 and 10 associated with significant decrease in proliferation capacity
CryoStor CS10 (contains 10% DMSO); freezing to − 80 °С; storage in liquid nitrogen for 1 month; rapid thawing at 37 °C	Explants (tissue pieces were allowed to rest without medium for 10 min to ensure adherence to the culture plate)	After 14 days of culture	CD73+, CD90+, CD105+, CD34−, CD45−, CD14−, CD19−, HLA-DR−	–	Two newly developed approaches for quantitative evaluation of UC tissue as a cell source were suggested (a standardized explant approach and a metabolic activity assay)	[52]