Effects of storage media, supplements and cryopreservation methods on quality of stem cells

Table 1 Comparison of different protocols used during cryopreservation of hematopoietic stem cells

HSC source	Storage period and temperature	Cryopreservation	Viability post freezing	Engraftment in days	Results	Ref.
< 600 x 106 cells/mL autologous PBSC	5-15 yr, -150 °C	10% DMSO and 23.3% Plasma Lyte A	66.4%	12	Viable CD34+ cells or CFU-GM is a reliable predictor of rapid engraftment	[13]
< 300 x 106 cells/mL autologous PBSC	< 6 mo, -80 °C	3.5% DMSO, 1% HSA and 2.5% HES	72%	14	Low DMSO conc allows successful engraftment and reduces toxicity (8%); Similar engraftment after combination of DMSO with or without HES and HSA	[115]
< 100 x 106 cells/ mL autologous PBSC	< 6 mo, -80 °C	5% or 10% DMSO, autologous plasma, 5% ACD	85%	14	19.1% infusion-related toxicity in the 10% DMSO group vs 6.8% in the 5% DMSO group, lowering DMSO results in reduction in infusion toxicity and lower costs with a similar hematopoietic reconstitution	[116]
Autologous PBSC	< 11 yr, -80 °C	3.5% DMSO + 1% HSA and 2.5% HES vs 6% DMSO + 6% HES	no significant change	11-12	Uncontrolled-rate freezing and cryopreservation with 5% DMSO/HES at −80 °C supports hematopoietic reconstitution comparable to that of controlled-rate freezing and liquid nitrogen storage	[117]
< 4000 x 106 cells/mL autologous PBSC	1-98 wk, -80 °C	3.5% DMSO, 2.5% HES and 1% HSA	60.8%	11-20	Reduction in DMSO concentration decreases transfusion-related adverse events. PBPCs cryopreserved in low DMSO/HES/HSA at -80°C allow successful engraftment	[24]
50 x 106 cells/mL autologous PBSC and BM	PB: 35 mo (26-78); BM 16 mo (27-71), -90 C	5% DMSO, 6% HES and 4% HSA in RPMI1640	93%		DMSO-associated toxicity during infusion, storage of HSCs at -90°C in DMSO/HES/HSA did not cause loss of cell numbers, viability, and clonogenic activity	[118]
Autologous PBSC	Controlled rate freezing at -186 °C	5% or 10% DMSO and 6% HES		10-20	Two patients who received components cryopreserved with DMSO alone experienced serious neurological toxicity, none of the recipients who received components frozen in DMSO/HES experienced serious infusion-related toxicity, better hematopoietic recovery in presence of HES independent of DMSO concentration	[14]
100 x 106 cells/mL – 200 x 106 cells/mL autologous PBSC	5-6 yr, controlled rate freezing at -160 °C	2%-10% DMSO, 10% ACD	73% with 5% DMSO	10-14	Cryopreservation using 5% instead of 10% DMSO improves CD34 + cell and leukocyte viability, but has only minor effects on supernatant levels of leukocyte- and platelet-derived soluble mediators	[61]
75 x 106 cells/mL - 250 x 106 cells/mLautologous PBSC	32-180 d, controlled rate freezing, -196 °C	5% or 10% DMSO	84%-95%	10-14	The use of 5% instead of 10% DMSO was associated with a decrease in side effects, cryopreservation with 5% DMSO followed by storage in nitrogen is a simple, highly standardized, and safe procedure for cryopreservation of autologous stem cell graft	[119]
UCB	1-2 mo, uncontrolled vs controlled rate freezing at -90 °C	5% or 10% DMSO	Uncontrolled 84.2%; controlled 92.5%		Best recovery of UCB cells when controlled-rate freezing and 5% DMSO were combined	[120]
15 x 106 cells/mL UCB	> 2 wk, controlled rate freezing at -170 C	5%, 10% or 20% DMSO and 2% HSA or autologous plasma	89%		Optimal conditions for cryopreservation were 10% DMSO and 2% HSA with fast addition and removal of DMSO	[121]
800 x 106 cells/mL UCB	10 yr, controlled rate freezing at -196 °C	10% DMSO and 5% Dextran	83.7%		Long term storage of UCB units does not affect the quality of the HSCs	[122]
Autologous BM	4 mo, -80 °C	5% DMSO and 6% HES	82.2%	21	BM cells can be rapidly and inexpensively cryopreserved in DMSO/ HES, without need for rate-controlled freezing or storage in liquid nitrogen	[123]
20 x 106 cells/mL BM or 17 x 106 cells/mL PBSC	Controlled rate freezing at -196 °C	10% DMSO or 0.25-1 mol/L TH with or without 0.25 IU/mL insulin (I)	DMSO: 33%TH: 32%; TH/I: 30%		DMSO-cryopreserved cells exhibited the best median viability-rate after thawing. Comparable results could be achieved with trehalose 0.5 mol/L with/without insulin	[45]
200 x 106 cells/mL autologous BM or PBSC	BM: 11.8 yr vs PB: 33 d controlled rate freezing at -196 °C	10% Medium 199 , 80% autologous plasma and 10% DMSO	BM: 81.5%; PBSC: 68.0%		BM can be cryopreserved for more than a decade without apparent loss of progenitor activity in comparison to short-term cryopreserved PBSC	[124]

HSC: Hematopoietic stem cell; DMSO: Dimethyl sulfoxide; CFU-GM: Colony Forming Unit-Granulocyte/Macrophage; ACD: Acid citrate dextrose; RPMI: Roswell Park Memorial Institute Medium; HES: Hydroxyethyl starch; HSA: Human serum albumin; BM: Bone marrow; UCB: Umbilical cord blood; TH: Trehalose.

Table 2 Comparison of different protocols used during cryopreservation of mesenchymal stem/stromal cells

MSC source, passage	Culture medium	Storage period and temperature	Cryopreservation	Viability	Phenotype	Results	Ref.
BM-MSC/P3	MEM, 15% FBS, 1% P/S, 1% L-glutamin	7 wk at -196 °C	90% FBS and 10% DMSO		Osteogenic and adipogenic differentiation, high expression of CD44, CD73, CD90 and CD105	No effects of freezing on function, differentiation and phenotype of the cells	[125]
1 x 106BM-MSC/P3, P4, P8, P13, P18	MEM, 10% FBS, 1% P/S, 1% L-glutamin	12 mo, controlled rate freezing at -80 °C	30% FBS, 60% MEM and 10% DMSO	85%-100%	Chondrogenic, adipogenic, neurogenic differentiation, no difference in expression of cell surface markers between passages	No differences in phenotype or differentiation between different cryopreserved MSCs from different passages	[82]
0.5 x 106/mL; BM-MSC	MSC growth medium, 10% FBS	1-5 mo, controlled rate freezing at -196 °C vs 4 d at 4 °C	Freezing medium (FM): 10% DMSO, 10% FBS, MSC growth medium, 30% BSA vs CryoStor (CS) animal component free freezing medium with 2%, 5% or 10% DMSO vs storage in HypoThermosol-FRS medium (HTS-FRS) at 4°C	FM 10% DMSO: 102.8%; CS 2% DMSO: 91.7%; CS 5% DMSO: 95.6%; CS 10% DMSO: 95.4%; HTS-FRS: 85.0% (rapid loss of viability after > 6 d)	Osteogenic differentiation, high expression of CD44, CD90, CD105, CD166, loss of expression of CD9 after hypothermic storage	No difference in differentiation or phenotype before and after freezing; HTS-FRS preserved MSC marker expression, proliferation and osteogenic differentiation after storage for at least 4 d	[81]
1 x 106/mL; BM-MSC	MEM, 10% FBS, 1% P/S	7 wk at -196 °C	10% DMSO ± 10% or 90% FBS, 7.5% DMSO, 2.5% PEG ± 2% BSA, 5% DMSO, 5% PEG, 5% DMSO, 2% PEG, 3% Trehalose ± 2% BSA, 2.5% DMSO, 7.5% PEG ± 2% BSA, 10% Propanediol, 2%BSA, 7.5% Propanediol 2%BSA, 2.5% PEG	Highest viability with 7.5% DMSO, 2.5% PEG and 2% BSA: 82.9% ± 4.3% vs 10% DMSO: 82.7% ± 3.7%	Adipogenic, osteogenic and chondrogenic differentiation	In comparison to 10% DMSO, best results with 7.5% DMSO, 2.5% PEG and 2% BSA. In presence of and 2% BSA also good results with 5% DMSO, 5% PEG or 7.5% propanediol with 2.5% PEG	[84]
BM-MSC/P1-6	MEM, 10% Human Serum, 1% L-glutamine, 1% P/S	1 yr at -196 °C	MEM, 40% Human Serum, 5% DMSO		Osteogenic, adipogenic and myogenic differentiation, before and after thawing high expression of CD73, CD90 and CD105, no expression of CD16, CD34, CD45 and HLA-DR	Cryopreserved MSCs show slightly lower proliferation rate, no differences in differentiation, senescence markers, CFU-F or karyotype between frozen and fresh cells	[89]
5 x 105/mL; BM-MSC/P1	MEM, 15% FBS, 1% P/S	< 6 mo vs 33-37 mo	CELLBANKER cryopreservation medium (contains serum and DMSO)	90%	Osteogenic differentiation, both fresh and cryopreserved MSCs were negative for CD14, CD34, CD45 and HLA-DR and positive for CD29 and CD105	No difference in osteogenic potential between fresh and cryopreserved cells. Long-term cryopreserved MSCs retained high osteogenic potential, no difference in phenotype	[86]
1 x 106/mL; WJ-MSC	ADMEM, 10% FBS, 1% P/S, 1% L-glutamine	3 mo, controlled rate freezing at -196 °C	A: ADMEM, 10% PVP ± 10% FBS, B: ADMEM, 10% FBS, 0.05 mol/L glucose, 0.05 mol/L sucrose, 1.5 mol/L ethylene glycol ± 10% FBS, C: ADMEM, 10% DMSO ± 10% FBS	A: 62.9% ± 0.4%; A without FBS: 6.8% ± 0.2%; B: 72.2% ± 0.23%; C: 81.2% ± 0.6%	Adipogenic and osteogenic differentiation, both fresh and cryopreserved MSCs were negative for CD34 and CD45 and positive for CD73, CD90 and CD105	Complete elimination of FBS in cryoprotectants resulted in drastic reduction in cell viability. Cryopreservation did not alter basic stem cell characteristics, plasticity and multipotency, except for proliferation rate	[83]
1 x 106/mL; tgMSC	DMEM, 10% FBS, 1% P/S/A	1 d or 6 mo, freezing at -196 °C	20 μg/mL NaB, 20% FBS, 1% P/S/A , 10%, 7%, 5%, 3% or 0% DMSO	First cycle: > 90%; Second cycle: > 70%; Third cycle: > 80%; Fourth cycle: > 80%	Osteogenic, chondrogenic, and adipogenic differentiation, high expression of CD29 and CD73, medium expression of CD90, CD105 and CD166, no expression of CD14, CD45, CD34	< 5% DMSO in freezing medium resulted in increased cell death, NaB improved cellular viability after freeze-thaw cycles, addition of NaB to the freezing medium did not affect differentiation capacity of MSCs	[85]
5 x 105/mL ADSC/P2	DMEM-LG, 10% FBS	2 wk, freezing at -196 °C	0.9% NaCl containing 10% DMSO HSA, HS, KSR or 90% FBS	DMSO + 9%; HSA: 78.0%; DMSO + 90%; HS: 72.4%; DMSO + 90%; KSR: 77.0%; DMSO + 90%; FBS: 78.5%; DMSO alone: 19.6%	No differences in adipogenic, osteogenic, and chondrogenic differentiation, gene expression of CD73, CD90, CD105, CD106, CD166, SCF, REX1 and NANOG. All ADSCs were positive for surface expression of CD44, CD73, CD90, CD105, CD166 and HLA-ABC and negative for CD31, CD34 and HLA-DR	ADSCs frozen with HSA, HS, or KSR showed similar growth kinetics as cells frozen with FBS. Multilineage differentiation of ADSCs did not differ between groups	[88]
1 x 106/mL DPSC/P5-7	MEM, 15% FBS, 1% P/S/A, 100 uM L- ascorbic acid 2-phosphate	1 wk, freezing with Mr. Frosty (NMF) vs magnetic freezing (MF)	Serum-free cryopreservation medium (SFM) containing 3% DMSO, SFM + 10% DMSO, FBS + 3% DMSO, FBS + 10% DMSO	SFM + 3%; DMSO: 75%; SFM + 10%; DMSO: 78%; FBS + 3%; DMSO: 70%; FBS + 10%; DMSO: 73%	CD29, CD44 and STRO-1 expression did not differ between the NMF and the MF groups, whereas levels of CD73, CD90, CD146 and CD166 in the MF group increased compared to the NMF group.	DPSC viability using MF was significantly superior to that of the NMF using 2%–10% DMSO; Post-thaw MF-DPSCs expressed MSC markers and showed osteogenic and adipogenic differentiation similar to fresh DPSCs	[90]
ESC-derived MSC	MEM, 10% FBS, 1% NEAA	Controlled rate freezing at 196 C	Sucrose, glycerol, creatine (SGC) and sucrose/ glycerol/isoleucine (SGI) solutions were incubated for 1h before freezing, Sucrose, mannitol, creatine (SMC) solutions were incubated for 2 h before freezing	SGI>SGC>SMC	Osteogenic and chondrogenic differentiation, all groups were positive for CD73, CD90 and CD105, and negative for CD45	Osmolyte-based cryopreservation formulations retain MSC post-thaw viability, cell surface markers expression, proliferation, and osteochondral differentiation potential	[31]

MSC: Mesenchymal stem/stromal cell; FBS: Fetal bovine serum; DMSO: Dimethyl sulfoxide; ESC: Embryonic stem cell; NEAA: Non essential aminoacids; MEM: minimal essential medium; KSR: Knockout serum replacement; BSA: Bovine serum albumin; P/S: Penicillin/Streptomycin; DPSC: Dental pulpa stem cells; ADSCs: Adipose derived stem cells.

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.