Thawing of hPSCs grown on MEFs v1

This protocol describes the standard procedure of thawing human pluripotent stem cells (hPSCs) on inactivated mouse embryonic fibroblasts (MEFs). General notes 1. 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. 2. Until otherwise indicated, 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. 3. While freezing hPSCs as single cell solution (using Rock Inhibitor) results in better cell recovery, some laboratories prefer freezing of hPSCs as cell clusters. We have used both approaches and do not observe obvious differences.