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Freezing and Thawing Feeder-free Cells
ReagentVendorCatalog Number
Corning™ Matrigel™ hESC-Qualified Matrix Fisher Scientific 354277
Falcon™ Polystyrene Microplates Fisher Scientific Various
Corning™ Cell Culture PBS (1X) Thermo Fisher / Life Technologies MT21040CV
DMEM-F12 Thermo Fisher / Life Technologies MT10092CV
Essential 8 Medium Thermo Fisher / Life Technologies A1517001
Stemflex Medium Thermo Fisher / Life Technologies A3349401
mTesR Medium Stemcell Technologies 85850
Rock Inhibitor Stemcell Technologies 72302
Accutase Innovative Cell Technologies AT104
Cryostor CS10 Stemcell Technologies 07930
STEM-CELLBANKER® Thermo Fisher / Life Technologies 11897
Nunc™ Biobanking and Cell Culture Cryogenic Tubes Thermo Scientific 12-565-164N
Required Equipment / Consumables:
  • Biosafety Cabinet
  • CO2, O2, humidified, water jacketed incubator
  • Sterile, glass, borosilicate pipets (Fisherbrand)
    • Freezing:
    1. Remove plate to be passaged from incubator and place it in the biosafety cabinet.
    2. Remove the spent medium from the wells to be passaged by aspirating with borosilicate pipet.
    3. Rinse each well to be passaged with 1ml room temperature PBS.
    4. Add Accutase to each well to be passaged.
      1. For a 6-well plate, add 1mL Accutase to each well.
      2. For a 24-well plate, add 500μl Accutase to each well.
    5. Incubate for 6-10 minutes at 37°C until cells detach.
    6. Once incubation is complete, add the desired medium to neutralize the Accutase, and collect cells to be frozen using fresh media using a 5mL pipet.
      1. For a 6-well plate, add 3mL media to each well.
      2. For a 24-well plate, add 1mL media to each well.
    7. Spin the cells at 120g for 3 minutes.
    8. Aspirate the supernatant, leaving a trace amount to prevent the pellet from drying.
    9. Resuspend the cell pellet with an appropriate amount of freezing media for freezing.
      1. For a 6-well plate, add 2mL freezing media.
      2. For a 24-well plate, add 500μL freezing media.
    10. Aliquot 500μL to each cryogenic tube.
    11. Place cryogenic tube in freezing container and store in -80°C.
    Thawing:
    1. Retrieve cryogenic tube from either -80°C or LN2.
    2. Thaw the cryogenic tube quickly in a 37°C water bath.
    3. Transfer thawed cells to a 15mL tube.
    4. Add ~500μL of media of choice (E8 or mTESR1 or Stemflex) drop by drop, swirling the tube while doing so.
    5. Add an additional ~2mL of media of choice to the tube.
    6. Spin the cells at 120g for 3 minutes.
    7. Aspirate the supernatant, leaving a trace amount to prevent the pellet from drying.
    8. Resuspend the cell pellet with an appropriate amount of media for passaging (E8+ Rock inhibitor/ or mTESR1 + Rock inhibitor/ or Stemflex).
      1. For a 6-well plate, add 2mL media.
      2. For a 24-well plate, add 500μl media.
    9. Transfer cells to a matrigel-coated plate.
    10. Incubate at 37°C.