Harvesting and irradiation 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 harvesting and irradiating mouse embryonic fibroblasts (MEFs) to use 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
Wash the plates once with 10 ml DPBS for each 15-cm plate.
Add 3 ml Trypsin and incubate for in 37°C; 5% CO2 for 0h 10m 0s
Add 9 ml MEF medium to neutralize the Trypsin. Collect cell suspension in a 50 ml 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
Rinse the plate with 9 ml of new MEF medium to collect the remaining cells.
If there are cell clumps that not fully dissociated, let the clumps settle by gravity for 3 min then transfer the single cell suspension to a new conical tube. Cells in clumps are usually not uniformly irradiated and that may lead to proliferating feeders.
Parafilm the conical tubes and bring cells to irradiation.
Irradiate the cells in conical tubes at 3500 cGy on Precision X-RAD 320 irradiator.
