ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells

Efficient slow freezing protocols within serum-free and feeder-free culture systems are crucial for the clinical application of human embryonic stem (hES) cells. Frequently, however, hES cells must be cryopreserved as clumps when using conventional slow freezing protocols, leading to lower survival rates during freeze-thaw and limiting their recovery and growth efficiency after thawing, as well as limiting downstream applications that require single cell suspensions. We describe a novel method to increase freeze-thaw survival and proliferation rate of single hES cells in serum-free and feeder-free culture conditions. hES cells maintained on Matrigel-coated dishes were dissociated into single cells with Accutase and slow freezing. After thawing at 37 degrees C, cells were cultured in mTeSR medium supplemented with 10 mu M of Rho-associated kinase inhibitor Y-27632 for 1 day. The use of Y-27632 and Accutase significantly increases the survival of single hES cells after thawing compared with a control group (P < 0.01). Furthermore, by treatment of hES cell aggregates with EGTA to disrupt cell-cell interaction, we show that Y-27632 treatment does not directly affect hES cell apoptosis. Even in the presence of Y-27632, hES cells deficient in cell-cell interaction undergo apoptosis. Y-27632-treated freeze-thawed hES cells retain typical morphology, stable karyotype, expression of pluripotency markers and the potential to differentiate into derivatives of all three germ layers after long-term culture. The method described here allows for cryopreservation of single hES cells in serum-free and feeder-free conditions and therefore we believe this method will be ideal for current and future hES cell applications that are targeted towards a therapeutic end-point.