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HomeArrowResearch TopicsArrowStem Cell Research at NIHArrowNIH Stem Cell UnitArrowThawing Pluripotent Stem Cells

Thawing Pluripotent Stem Cells

  • Prepare 2 wells of irradiated MEFs or Matrigel the day before thawing the cells (Note: cells cultured on MEFs prior to freezing may NOT thaw well on Matrigel).
  • Thaw the cells quickly by swirling the vial in a 37oC waterbath until only a small chip of frozen material remains.
  • Swab with alcohol and place in the tissue culture hood.
  • Transfer the contents to a 15ml tube and slowly add 5ml of pre-warmed culture medium. Mix gently.
  • If there are many visible colonies, allow them to sediment at room temperature for 5 min. If colonies are not visible, spin at 200g for 5 min.
  • Aspirate the supernatant and resuspend in 2ml of culture medium.
  • If the vial is precious or you have had issues thawing in the past, 10µM of Y27632 (ROCK inhibitor) may be added to aid survival.
  • Wash 1 well of MEFs with D-PBS or aspirate 1 well of Matrigel and add thawed cells
  • Distribute the colonies evenly by gentle agitation and incubate overnight in a tissue culture incubator (37oC/5% CO2).
  • The next day wash the second well of MEFs with D-PBS or aspirate the second well of Matrigel and transfer the supernatant from the first well onto it. Add 2ml culture medium to each well.
  • Distribute the colonies evenly by gentle agitation and incubate overnight in tissue culture incubator (37oC/5% CO2).
  • Feed both wells as usual. (Note: Sometimes colonies attach to the second well and sometimes not but it’s worth trying—sometimes it may even be better than the first well).