This collection describes the maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell (hPSC) culture.

Collection overview

Thawing of mouse embryonic fibroblasts (MEFs) for hPSC cultures

Expansion of mouse embryonic fibroblasts (MEFs) for hPSC cultures

Freezing of mouse embryonic fibroblasts (MEFs) for hPSC cultures

Harvesting and irradiation of mouse embryonic fibroblasts (MEFs) for hPSC cultures

Mitomycin C inactivation of mouse embryonic fibroblasts (MEFs) for hPSC cultures

Preparation of mouse embryonic fibroblast (MEF) feeder plates for hPSC cultures

A. Starting with frozen irradiated or Mitomycin C inactivated MEFs (optional)

B. Starting with fresh irradiated or Mitomycin C inactivated MEFs

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. Either fresh or frozen stocks of irradiated or Mitomycin C inactivated MEFs can be used to prepare hPSC feeder cells.

3. The indicated MEF densities are recommended starting densities and might have to be adjusted for each hPSC line and hPSC media formulation (KSR, serum-free versus serum-containing media).

4. MEFs were obtained as described in Manipulating the Mouse Embryo: A Laboratory Manual, Third Edition (ISBN: 0879695919)