Protocols available for the cryopreservation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells are very inefficient and laborious compared to those for the cryopreservation of murine ES/iPS cells or other general cell lines. While the vitrification method may be adequate when working with small numbers of human ES/iPS cells, it requires special skills and is unsuitable when working with large cell numbers. Here, we describe a simple and efficient method for the cryopreservation of hES/hiPS cells that is based on a conventional slow freezing method that uses a combination of Pronase/EDTA for Stem™ and CP-5E™ [final concentrations: 6 % hydroxyethyl starch, 5 % DMSO, and 5 % ethylene glycol in saline]. CP-5E™ is highly effective for the cryopreservation of small cell clumps produced by hES/hiPS colony detachment in the presence of Pronase and EDTA (Pronase/EDTA for Stem™, a formulation containing multiple digestive enzymes from Streptomyces griseus). This novel method would be quite useful for large-scale hES/iPS cell banking for use in clinical applications.
Keywords: Cryopreservation; Dimethyl sulfoxide; Ethylene glycol; Human embryonic stem cells; Human induced pluripotent stem cells; Hydroxyethyl starch; Pronase; Slow freezing.