Protocols
Medium:
Standard human ES medium (500ml):
- 400 ml DMEM/F12
- 100 ml KoSR
- 5 ml GlutaMAX
- 5 ml MEM-NEAA
- 500 ul 2-Mercaptoethanol (1000x)
- 10 ug bFGF (final 20ng/ml)
Other commerically available hESC/iPSC medium:
- mTeSR1 & mTeSR2: Stemcell Technologies
- Essential 8 medium: Life Technologies/Invitrogen
- StemPro hESC SFM: Life Technologies/Invitrogen
- HEScGRO medium: Millipore
- NutriStem medium: Stemgent
- PSGro medium: StemRD
MEF medium:
- 450 ml DMEM
- 50 ml FBS
- 5 ml L-Glutamine
- 5 ml Pen/Strep
Human ESC/iPSC culture:
Thawing hiPSC derived from the core:
Generally, the human iPSC lines derived in the core are maintained in feeder-free culture medium and frozen 1 vial per well of a 6-well plate.
Quickly transfer the frozen vial into 37C water bath, swirl the vial gently
When only a small ice remains, remove the vial from water bath and sterilize the outside of the tube with ethanol
Transfer the cells gently into 15ml conical tube, and slowly add 4ml of hPSC medium and mix
Centrifuge the cells at 150xg for 4 mins
Aspirate the supernatant and re-suspend the cells with 2.5ml hPSC medium containing 10uM Y-27632 (rock inhibitor, increase the iPSC survival)
Plate the cells onto 1 matrigel-coated well of 6-well plate, incubate overnight
Feed the cells everyday with freshly warmed hPSC medium (without Y-27632)
More detailed protocols about human PSC maintenance can be found in WiCell website:
http://www.wicell.org/home/support/stem-cell-protocols/stem-cell-protocols.cmsx
Reprogramming protocols:
Lentivirus: Sigma-Aldrich
Sendai Virus: Life Technologies/Invitrogen
Episomal: Life Technologies/Invitrogen
mRNA transfection: Stemgent