Cryopreservation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells

Until now, it has been problematic to obtain high survival rates after thawing of human embryonic stem cells (hESCs) and later also induced pluripotent stem cells (iPSCs). Already in 1994, Freshner and �coworkers established the conventional slow freezing/rapid thawing methods by using a culture medium supplemented with 10% dimethylsulfoxide (DMSO). The cell survival using this method was low. It has been improved since then in various ways, for example, using a ROCK inhibitor to freeze dissociated hESCs. Later, Reubinoff and coworkers published a completely new method called vitrification, wherein an open pulled straw was used. It showed good results by using vitrification in some cell types including hESCs; yet it has been problematic and difficult to handle. Since it is mostly suitable for small amount of cells, this is inconvenient for researchers because they often need large amount of cells. A novel, chemically defined effective xeno-free cryopreservation system has recently given excellent results for the cryostorage of both hESCs and iPSCs, frozen in cell aggregates or as single cells. It is based on an optimal constitution of cryoprotectants, 10% DMSO, glucose, and a high polymer described in the Japanese Pharmacopoeia (JP), plus NaCl, KCl, Na₂ HPO₄ , and NaHCO₃ as pH adjustors for maintenance of the cell function, all dissolved in phosphate-buffered saline (PBS). The procedure is slow freezing without any extra equipment. Over 90% survival has been achieved.