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:

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:

Reprogramming protocols: