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

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.