This collection contains protocols which describe the standard procedure of culturing human pluripotent stem cells (hPSCs) on inactivated mouse embryonic fibroblasts (MEFs).

Collection overview

Thawing of hPSCs grown on MEFs

Passaging of hPSCs grown on MEFs

Freezing of hPSCs grown on MEFs

A. Freezing of hPSCs as single cell suspension using trypsin

B. Freezing of hPSCs as cell aggregates using collagenase

General notes

1. Throughout these protocols, 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.

4. While bulk/collagenase passaging is used for routine maintenance of hPSC cultures,

manual/microdissection passaging is used to enrich for undifferentiated hPSC colonies (“clean-up” of culture based on undifferentiated hPSC colony morphology) or to expand (“pick”) individual colonies (e.g.,for clonal expansion of individual targeted cells in the process of establishing genome edited cell lines). Manual passaging/ microdissection requires (i) the identification and discrimination of undifferentiated and differentiated hPSC colonies and (ii) the capacity to excise the undifferentiated cells and transfer them to a new plate and can be performed using various approaches as established in many hPSC laboratories.