Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell culture
Hanqin Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner
Abstract
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
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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.
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Either fresh or frozen stocks of irradiated or Mitomycin C inactivated MEFs can be used to prepare hPSC feeder cells.
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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).
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MEFs were obtained as described in Manipulating the Mouse Embryo: A Laboratory Manual, Third Edition (ISBN: 0879695919)
