Due to widespread applications of human embryonic stem (hES) cells, it is essential to establish effective protocols for cryopreservation and subsequent culture of hES cells to improve cell recovery. We have developed a new protocol for cryopreservation of dissociated hES cells and subsequent culture. We examined the effects of new formula of freezing solution containing 7.5% dimethylsulfoxide (DMSO) (v/v %) and 2.5% polyethylene glycol (PEG) (w/v %) on cell survival and recovery of hES cells after cryopreservation, and further investigated the role of the combination of Rho-associated kinase (ROCK) inhibitor and p53 inhibitor on cell recovery during the subsequent culture. Compared with the conventional slow-freezing method which uses 10% DMSO as a freezing solution and then cultured in the presence of ROCK inhibitor at the first day of culture, we found out that hES cell recovery was significantly enhanced by around 30 % (P < 0.05) by the new freezing solution. Moreover, at the first day of post-thaw culture, the presence of 10 mu M ROCK inhibitor (Y-27632) and 1 mu M pifithrin-mu together further significantly improved cell recovery by around 20% (P < 0.05) either for feeder-dependent or feeder-independent culture. hES cells remained their undifferentiated status after using this novel protocol for cryopreservation and subsequent culture. Furthermore, this protocol is a scalable cryopreservation method for handling large quantities of hES cells. (C) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 26: 781-788, 2010