hPSCs culture (MEF Feeder Layer)
|Falcon™ Polystyrene Microplates||Fisher Scientific||Various|
|Cytotune-iPS 2.0 Sendai Reprogramming Kit||Thermo Fisher / Life Technologies||A16517|
|CF-1MEF Feeder Cells||Applied Stemcell Inc.||ASF-1223|
|Corning™ Cell Culture PBS (1X)||Thermo Fisher / Life Technologies||MT21040CV|
|DMEM-F12||Thermo Fisher / Life Technologies||MT10092CV|
|Knockout Serum Replacement||Thermo Fisher / Life Technologies||10828028|
|MEM-NEAA||Thermo Fisher / Life Technologies||11140050|
|L-glutamine||Thermo Fisher / Life Technologies||25030081|
|2-mercaptoethanol||Thermo Fisher / Life Technologies||21985023|
|DMEM||Thermo Fisher / Life Technologies||11330057|
|5 U/mL Dispase||Stemcell Technologies||7913|
|0.05% Trypsin-EDTA||Thermo Fisher / Life Technologies||25300054|
Required Equipment / Consumables:
DMEM/10% FBS (500 mL)
- DMEM - 450 mL
- FBS - 50 mL
hESC (1000 mL)
- DMEM/F12 – Fill up to 1000 mL
- KoSR – 200 mL
- MEM-NEAA – 10 mL
- L-glutamine – 5mL
- 2-mercaptoethanol – 1 mL
- 6 ng/mL bFGF
- 60% FBS
- 20% hESC media
- 20% DMSO
- The day before passage, coat tissue-culture treated dishes with 0.1% gelatin/PBS for at least 15 minutes.
- Plate primary mouse embryonic ﬁbroblasts in DMEM+10% FBS at a density of ~12,000 cells/cm2.
- Incubate at 37°C for at least 24 hours prior to hESC plating
- Aspirate DMEM+10%FBS media used to plate MEFs, and wash with PBS to eliminate traces of serum. Aspirate PBS and add fresh hESC media. Place dish in the incubator to pre-warm and correctly pH the media.
- Remove hPSCs from liquid nitrogen tank and move it rapidly to a 37°C water bath.
- Transfer cells into 9 mL of hESC media in a 15 ml conical tube.
- Spin at 200xg for 5 minutes.
- Resuspend pellet with media pre-warmed from the MEF plate and add cells directly back to the same MEF plate/wells. Incubate 37°C
- Feed daily with hESC
- Aspirate human ES media and add 2 ml dispase (per 6 cm dish). Place in incubator (37°C) for ~7 minutes. Check colonies on the microscope after 7 minutes. If the edges have started to detach, you are ready to proceed. If not, you can place them back in the incubator for another minute before checking again.
- Aspirate the dispase. Add 3 ml (per 6 cm dish) of hESC media for wash.
- Aspirate hESC media and add fresh hESC media. Gently dislodge the colonies from the surface of the dish by pipetting with a 5 ml pipette. Transfer the colonies/media to a 15 ml conical containing 10 ml hESC media.
- Spin at 200xg for 5 minutes. Aspirate media and resuspend in 10 ml hESC media as above. Split accordingly. Depending on the growth characteristics of the cell line and plate density, cells can be split between ~1:3-1:12.
- Incubate 37°C
- Feed cells daily
- Cells are ready to be split in 6-9 days.
- Pre-label cryotubes and place in the freezer prior to freezing cells
- Aspirate human ES media and add 2 ml dispase (per 6 cm dish). Place in incubator (37°C) for ~7 minutes.
- Aspirate dispase and add 3 ml hESC media for wash
- Aspirate and add fresh hESC media to gently dislodge colonies while pipetting media over the surface of the dish. Add to 15 mL conical tube with 10 mL of media
- Centrifuge at 200xg for 5 minutes
- Aspirate, and gently resuspend colonies in hESC media, 500 uL per cryotube intended on freezing
- Add of 2x freezing media dropwise to colonies in hESC media slowly, 500 uL per cryotube intended on freezing. Gently invert to mix, then dispense into the appropriate number of tubes
- Place in freezing container and store at -80°C overnight. The next day, transfer to liquid nitrogen. DO NOT LEAVE AT -80°C FOR EXTENDED PERIODS OF TIME
Our protocols were largely derived from the Thomson Lab’s protocol (http://ink.primate.wisc.edu/~thomson/ ). Another wonderful resource is the WiCell Research Institute protocols (www.wicell.org).