Expansion of mouse embryonic fibroblasts (MEFs) for hPSC cultures
Hanqin Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner
Abstract
This protocol describes the expansion 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
Wash the plates twice with DPBS
Add Trypsin and incubate for 0h 5m 0s (37°C; 5% CO2)
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 at 250x g
Remove the supernatant. Re-suspend the cell pellet in fresh MEF medium to plate on gelatin-coated plates (Dilution ratio 1:4) and maintain in a humidified incubator (37°C; 5% CO2).
MEFs need to be passaged once confluent (every 3-4 days) and can be expanded up to passage 4 (P4) and frozen before or after inactivation using irradiation or Mitomycin C treatment.
For more information on freezing MEFs, as well as irradiation and Mitomycin C treatment of MEFs, refer to 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
