Table 1.
Cryopreservation protocols for human embryonic stem cells
| Cell lines | Culture condition | Cryo-method | Cryo-medium | Cryo-container | Results after thawing | Reference |
|---|---|---|---|---|---|---|
|
| ||||||
| hES-1,2 | MEF, Dispase DMEM+20%FBS | Vitrification Slow freezing | 20% DMSO+20% EG+0.5M S 10% DMSO+90% FBS | OPS CV+Container | 100%a 16% | Reubinoff et al. 2001 (60) |
| hES-2,3,4 | Fetal skin fibroblasts, M DMEM+20%FCS+ITS | Vitrification | 20%DMSO+20%EG+0.3M S+12% HSA | CS | ~80%b | Richards et al. 2004 (61) |
| Vitrification Slow freezing | 20% DMSO+20% EG+0.5M S 10%DMSO+90% FCS | OPS CV+Freezer | ~80% ~10% | |||
| hES-1 | MEF, M KO-DMEM+20% SR or FBS | Vitrification | 10% DMSO+90% SR+0.2M trehalose | CV+Container | ~48%c | Wu et al. 2005 (69) |
| SNUhES-3 | STO, M DMEM/F12+20% SR+bFGF | Slow freezing | 5% DMSO+10% EG+50% FBS | CV+Container | ~30%d | Ha et al. 2005 (74) |
| H1 | MEF, Dispase KO-DMEM+15% SR | Slow freezing | 10% DMSO+25% FBS | CS+Freezer+ seeding | ~70%e | Ware et al. 2005 (65) |
| HS-207,401 | Foreskin fibroblast, M KO-DMEM+20% SR+bFGF | Slow freezing (single cell) | 10% DMSO+culture media (ROCK inhibitor pre-incubation) | CV+Container | 6~7%f | Martin-Ibanez et al. 2008 (70) |
| CHA-hES 3,4 | STO, M DMEM/F12+20% SR+bFGF | Vitrification | 5.5M EG+1M S+10% FBS (pre-incubation on feeder/CM) | EM grid | ~80%b | Cha et al. 2008 (62) |
| VAL-3 VAL-5 | Foreskin fibroblast, M /Coll IV KO-DMEM+20% SR+bFGF | Slow freezing | Culture media+addition of increased concentration of DMSO | CV+Container/ Feezer | 41~68%d 8~15% | Valbuena et al. 2008 (75) |
| α-ES-C | MEF, Coll IV KO-DMEM+20% SR+bFGF | Bulk vitrification | 20%DMSO+20%EG+0.5M S | Cell strainer (nylon) | ~94%g | Li et al. 2008 (76) |
| Royan H5,6 hiPSC1,4 | Feeder-free, Coll IV/Dispase DMEM/F12+20% SR+ITS+bFGF | Slow freezing (single cell) | 10% DMSO+90% FCS+ROCK inhibitor (ROCK inhibitor pre-incubation) | CV+Container | ~90%h | Mollamohammadi et al. 2009 (77) |
| H1, H9 | MEF/Matrigel coated Microcarrier Coll IV/Dispase, CM+bFGF | Slow freezing | 10% DMSO+30% FCS+60% CM/bFGF (adherent on microcarriers) | CV+Container | 1.5~1.9 timesi | Nie et al. 2009 (73) |
| CHA-hES 3 H9 | MEF, Dispase DMEM/F12+20% SR+bFGF | Slow freezing | 10% DMSO+ 90% SR (stepwise addition) | CV+Container/ Feezer | 42~51%j 20~30% | Lee et al. 2010 (67) |
| Shef-4,5,6,7 | MEF, Coll IV/Accutase KO-DMEM+20% SR+bFGF | Slow freezing | 10% DMSO+ 90% FCS (attached on culture cassette) | Culture cassette /Container | 8~200 foldsk | Amps et al. 2010 (72) |
MEF: mouse embryonic fibroblasts; DMEM: Dulbeco's modified Eagle medium with high glucose; FBS: fetal bovine serum; FCS: fetal calf serum; SR: Knockout serum replacement; ITS: insulin transferrin selenite; M: mechanical passaging; Coll IV: collagenase type IV; CM: feeder conditioned media; DMSO: dimethylsulfoxide; EG: ethylene glycol; S: sucrose; HSA: human serum albumin; OPS: open pulled straw; CS: closed straw; CV: cryovial; Freezer: programmable cell freezer.
a count colonies including completely differentiated colonies, b count colonies with >50% undifferentiated, c count AP positive colonies after alkaline phosphatase (AP) staining at day 7 after thawing, d count developing colonies at 10 days after thawing, e calculated relative to the control using the formula (see the reference in detail), f colony formation efficiency of dissociated hES cells, g number of adhered clumps/number of thawed clumps after 48 hour of plating, h cell viability by the Trypan Blue exclusion, i recovery of hESCs cryopreserved on microcarriers/recovery of hESCs cryopreserved as suspended clumps (recovery by the Trypan Blue exclusion), j count AP positive colonies after alkaline phosphatase (AP) staining, k relative proliferation efficiency compared to cryovial-culture flask control (see the reference in detail).