Controlled-rate freezing of human ES cells.
A significant obstacle to using human embryonic stem cells (hESCs) arises from extremely poor survival associated with freezing, typically in the range of 1%. This report describes a slow controlled-rate freezing technique commonly used for mammalian embryo cryopreservation. Using a combination of surviving colony number and colony diameter; survival was determined relative to untreated hESCs. Using a dimethyl sulfoxide (DMSO) cryoprotectant and either a homemade controlled-rate freezing device or a commercial freezing device, survival rates of 20%-80% were obtained. To achieve the highest levels of survival, the critical factors were an ice crystal seed (at -7 degrees to -10 degrees C), a freeze rate between 0.3 degrees and 1.8 degrees C/min, and a rapid thaw rate using room temperature water. Slow controlled-rate cooling allows a rapid, simple, and reproducible means of cryopreserving hESCs.
Ware
Carol B
CB
Department of Comparative Medicine, University of Washington, Seattle, WA 98195-7190, USA. cware@u.washington.edu
Nelson
Angelique M
AM
Blau
C Anthony
CA
D002470:Cell Survival; D015925:Cryopreservation; D019610:Cytoprotection; D004121:Dimethyl Sulfoxide; D004622:Embryo, Mammalian / Q000166:cytology*; D005615:Freezing; D006801:Humans; D007621:Karyotyping; D013234:Stem Cells / Q000166:cytology*
D004121:Dimethyl Sulfoxide