Thawing of feeder-free hPSCs v1

This protocol describes the procedure of thawing feeder-free human pluripotent stem cells (hPSCs) using mTeSR-plus or StemFlex General Notes Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs. Until otherwise indicated, feeder-free hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions. We have routinely maintained feeder-free cells in either mTeSR-plus or StemFlex. However, these two mediums are not interchangeable. Pick one and stick to it. We have routinely maintained feeder-free hPSC cultures on VTN, Matrigel and Geltrex--coated cell culture plates without observing obvious differences.