Mitomycin C inactivation of mouse embryonic fibroblasts (MEFs) for hPSC cultures
Hanqin Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner
Abstract
This protocol describes the process of using Mitomycin C to inactivate mouse embryonic fibroblasts (MEFs), which can then be used 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
Steps
Grow MEFs to 90-95% confluency in 15-cm cell culture dish
Aspirate the medium and add 15 ml of Mitomycin C solution to cover the surface
Incubate in Mitomycin C for 2h 30m 0s 37°C
Aspirate Mitomycin C solution from the plates and wash 4 times with DPBS (with Ca/Mg) and final wash with DPBS (w/o Ca/Mg).
Add Trypsin and incubate in 37°C; 5% CO2 for 0h 5m 0s
Add MEF medium to neutralize the Trypsin and collect the solution into a conical tube.
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
Centrifuge the cell suspension at 250x g
Discard supernatant and re-suspend the cells in fresh MEF medium
Count cells using trypan blue solution
Mitomycin C treated cells can be either freshly plated as feeder cells for hPCS cultures or frozen as Mitomycin C inactivated stocks at 10x106 cells/vial.
A protocol on freezing MEFs can be found in the collection "Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell culture." A link to this collection can be found in the title section of this protocol, located above
