Freezing of mouse embryonic fibroblasts (MEFs) for hPSC cultures
Hanqin Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner
Abstract
This protocol describes the freezing of mouse embryonic fibroblasts (MEFs), which can later be used as feeder cells for human pluripotent stem cell (hPSC) culture.
General notes
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This protocol can be used to freeze MEFs before or after inactivation.
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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)
Steps
Wash the plates twice with DPBS
Add Trypsin (5 ml for 15-cm plate) and incubate for 0h 5m 0s (37°C; 5% CO2)
Add 10 ml 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 MEF Freezing Medium I at a concentration 2X the desired final freezing concentration (Freezing concentration will vary with MEF usage.)
MEF Freezing Medium I
| A | B |
|---|---|
| FB Essence/FBS* | 5 ml |
| MEF medium | 5 ml |
*We have successfully used either FB Essence or FBS and have not observed an obvious difference. Final volume: 10ml
While swirling, slowly and drop-wise, add an equal volume of MEF Freezing Medium II to the cell suspension.
MEF Freezing Medium II
| A | B |
|---|---|
| FB Essence/FBS* | 8 ml |
| DMSO | 2 ml |
*We have successfully used either FB Essence or FBS and have not observed an obvious difference. Final volume: 10ml
Dispense cell mixture into pre-labeled cryovials (10x106) including type of cells, passage number, date frozen, and who handled cells.
Place cryovials into Styrofoam microtube freezer box or NALGENE™ Cryo 1°C Freezing Container pre-filled with 250 ml room temperature isopropanol.(This ensures that a -1°C/min rate of cooling is achieved, which is critical to cell viability.)
Freeze at -80°C
For long term storage, store cryovials in liquid nitrogen (-196ºC).
