Thawing of mouse embryonic fibroblasts (MEFs) for hPSC cultures
Hanqin Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner
Abstract
This protocol describes the thawing of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell (hPSC) culture.
General notes
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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.
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MEFs were obtained as described in Manipulating the Mouse Embryo: A Laboratory Manual, Third Edition (ISBN: 0879695919)
Before start
All cell culture plates which are used as feeders to maintain hPSCs are coated for at least 1 hour with autoclaved 0.2% gelatin solution at room temperature. Remove gelatin solution immediately before plating MEF cells.
0.2% Gelatin Solution
| A | B |
|---|---|
| Sterile H2O | 1L |
| Gelatin powder | 2g |
After preparation, the gelatin solution should be autoclaved. Final volume: 1L
Steps
To recover frozen stocks for MEF expansion, P0 MEF tubes will be thawed in a water bath at 37°C by gently shaking
Thawed cells are transferred into a 15 ml conical tube containing 9 ml pre-warmed MEF medium. Centrifuge the tube at 250x g
MEF medium
| A | B |
|---|---|
| DMEM | 435 ml |
| FB Essence/FBS* | 75 ml |
| 200mM L-Glutamine | 5 ml |
| Penicillin & Streptomycin (100x) | 5 ml |
| MEM Non-Essential Amino Acids | 5 ml |
*We have successfully used either FB Essence or FBS and have not observed an obvious difference. Final volume: 500ml
Resuspended MEFs (10x106 cells/10 cm plate) are plated in fresh MEF medium and maintained in a humidified incubator (37°C; 5% CO2)
3-4 days after thawing, the cells should be ready for passaging (90-95% confluent)
