The ROCK inhibitor Y-27632 enhances the survival rate of human embryonic stem cells following cryopreservation.

After slow freezing, the survival rate of human embryonic stem (hES) cells is poor and inconsistent. The aim of this study was to increase the freeze-thaw survival rate of hES cells by utilizing the ROCK inhibitor Y-27632. hES cell colonies were first treated with Y-27632, followed by collagenase IV and TrypLE Select dissociation whereupon small clumps were slow frozen using 90% Knockout serum replacement and 10% dimethyl sulfoxide. After thawing at 37 C, the clumps were cultured in medium supplemented with 10 microM Y-27632 for 1 day. Our results show that the use of Y-27632 significantly increases the survival of hES cells after thawing compared with that of the control group. Y-27632-treated freeze-thawed hES cells retain morphology, stable karyotype, expression of cell surface markers, and the potential to differentiate into derivatives of all three germ layers after long-term culture. We have concluded that conventional slow freezing with Y-27632 treatment is efficient and convenient for the cryopreservation of hES cells.