Table 3.

Comparison of different protocols used during cryopreservation of induced pluripotent stem cells

Source of cell	Storage periode and temperature	Cryopreservation	Viability	Parameters	Results	Ref.
1.5 x 106-2 x 106 hiPSC line UMN PCBC16iPS	Controlled rate; -196 °C	NEAA, sucrose, glycerol, isoleucine and albumin in a P188 in HBSS vs 7.5% DMSO; Aggregates vs single cells		Viability, adherence and intracellular ice formation	P188 was found here to not only inhibit ice formation significantly but also soften the solid-liquid interface of ice and increase the distance between adjacent ice crystals; The cryoprotective effects of the DMSO- free CPA cocktail could be capitalized only with the optimized composition. Deviation from the optimum may result in less desirable outcomes	[96,97]
H9 hESC and hiPSC	3-6 d,controlled rate; -80 °C	10% DMSO, 10% EG, 10% PG, 10% glycerol, clumps vs single cells; ROCK inhibitor after thawing	EG-DMSO> PG>>glycerol	Toxicity of CPAs, expression of NANOG by hiPSCs	Freezing single cell iPSCs in the presence of a ROCK inhibitor and EG and programmable freezing drastically improved the yield of iPSCs in comparison to standard freezing in clumps without ROCK inhibitor	[98]
1-2x106 hiPSC	-196 °C	A: 10% DMSO/90% FBS; B: 10% DMSO/90% KSR; C: 10% DMSO/ESC medium + 20%KSR + ROCK inhibitor; Single cells	A: 90%; B: 70%; C: 70%	Viability, karyotype, expression of pluripotency markers TRA-1-60, TRA-1-81, Oct4, SSEA-3, and SSEA-4, embryoid body formation, neuronal differentiation, colony formation	Addition of ROCK inhibitor to pre- and post-thaw culture media increased survival rate, hiPSCs retained typical morphology, stable karyotype, expression of pluripotency markers and the potential to differentiate into derivatives of all three germ layers after long-term culture	[103,105,108]
hiPSC	-196 °C	10% DMSO in KO DMEM, 20% KSR, 1% NEAA, 1% L-glutamine, 0.2% b-mercaptoethanol, 1% antibiotic/ antimycotic and 8 ng/mL bFGF; ROCK inhibitor after thawing; Single cells		Colony number and size	ROCK inhibitor Y-27632 significantly improves the recovery of cryopreserved human iPS cells and their growth upon subculture	[104]
hiPSC line 253G4 and 201B2	7 d, Vitrification in; -196 °C	VS2E vitrification solution (40% EG, 10% PEG in Euro-Collins medium), DAP213 vitrification solution (1.2% DMSO, 22% PG, 5.9% acetamide); Single cells	VS2E>DAP213	Proliferation, expression of pluripotency markers Oct3/4, SSEA4, ALP, pluripotency in teratoma assay	Higher recovery rate of hiPSCs with DMSO and serum-free VS2E vitrification medium, cells after vitrification expressed Oct-3/4 and SSEA-4 and alkaline phosphatase and retained their pluripotency	[114]

iPSC: Induced pluripotent stem cells; NEAA: Non-essential amino acids; DMSO: Dimethyl sulfoxide; CPA: Cryoprotective agents; ESC: Embryonic stem cell; bFGF: basic Fibroblast Growth Factor; ROCK: Rho Kinase; ALP: Alkaline phosphatase; KSR: Knockout Replacement; FBS: Fetal Bovine Serum; HBSS: Hank’s Balanced Salt Solution.