A Controlled-Cooling Protocol for Cryopreservation of Human and Non-Human Primate Embryonic Stem Cells

Freeze storage of human embryonic stem (hES) cells has not proven effective using the methods employed for mouse ES (mES) cells, while rhesus ES (rhES) cells are only modestly effectively frozen using common mES cell methods. Because human and rhES cells are passaged and frozen in clusters that approximate the size of embryos, we employed a mammalian embryo freezing method to cryopreserve primate ES cells. This protocol involves freezing in a dimethylsulfoxide cryoprotectant using straws. An ice crystal seed is induced at −10^� C followed by controlled cooling at −1^� C per minute down to −33^� C with a plunge from there directly into liquid nitrogen (LN₂ ) at −196^� C. Thaw is effected rapidly by moving the frozen cells directly from LN₂ into a water bath and placing directly into culture medium without step-wise cryoprotectant removal. Using this protocol, we have increased the survival of human ES cells from ≤ 1 to ~ 80% and rhES cells from ~ 30 to ≥ 90%. Thus, this protocol describes a technically simple but effective means of long-term storage of primate ES cells.