Controlled-rate freezing of human ES cells

Abstract

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° to -10°C), a freeze rate between 0.3° and 1.8°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.