Abstract
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 μM ROCK inhibitor (Y-27632) and 1 μM pifithrin-μ 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. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010
Highlights
Due to their potentially unlimited capacity for self-renewal and unique developmental potential to differentiate into all somatic cell types of the human body, human embryonic stem (hES) cells have opened a new door for drug discovery, regenerative medicine, and tissue replacement after injury or disease.[1,2] an essential prerequisite for successful applications of hES cells is to develop efficient cryopreservation methodsVC 2009 American Institute of Chemical Engineers to overcome the current low cell recovery rate after cryopreservation.Two freezing protocols are currently applied to cryopreserve hES cells, including slow-freezing and vitrification
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
The cells after cryopreservation were cultured in the presence of ROCK inhibitor, or in the presence of ROCK inhibitor and pifithrin-l
Summary
Two freezing protocols are currently applied to cryopreserve hES cells, including slow-freezing and vitrification. Slow-freezing using 10% DMSO as a cryoprotectant is commonly used successfully to cryopreserve primary cells,[3] human mesenchymal stem cells,[4] and mouse embryonic stem cells.[5] this protocol has not been successfully transferred to hES cells. It leads to poor cell survival rate after freezing.[6,7] On the other hand, vitrification of hES cells by the open pulled-straw method is much more effective than the slow freezing. A higher cell survival rate, 70–90%, is reported after vitrification.[8,9,10] the fast-freezing
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