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Much is determined by the cell density at which the cells were cryopreserved, the cryopreservation solution used, and whether recovering feeder-dependent or feeder-free iPSCs. If recovering feeder-dependent iPSCs, then it can take 3-4 days to recover and show nice emergence of colonies. If recovering feeder-free iPSCs, then I would discontinue the culture and start again. I would recommend using Thermo Fisher Scientific’s new RevitaCell™ Supplement (Cat #A26445-01) to assist in recovery of your cryopreserved PSCs. It is compatible with both feeder-free and feeder-dependent medium systems and should only be added for the 1st 24 hours post-thaw, with subsequent feedings with growth medium in the absence of RevitaCell™ Supplement. This supplement contains a ROCK inhibitor coupled with compounds having antioxidant and free radical scavenger properties, providing for maximum post-thaw recovery.
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