Freezing and Thawing Cells in a 96-well Plate
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
Accutase Innovative Cell Technologies AT104
Cryostor CS10 Stemcell Technologies 07930
STEM-CELLBANKER® Thermo Fisher / Life Technologies 11897
Required Equipment / Consumables:
  • Biosafety Cabinet
  • CO2, O2, humidified, water jacketed incubator
  • Sterile, glass, borosilicate pipets (Fisherbrand)
    • Freezing
    1. Add 50μl of Cryostor CS10 directly to the wells of a 96-well plate.
    2. Using a P200 pipet tip, gently scratch the bottom of the well to suspend the cells in the freezing media.
    3. AWrap the plate with parafilm, and store directly in -80°C in a freezing container.
    Thawing Directly to 24-well Plate:
    1. Remove plate from -80°C and thaw for about 5 minutes in a 37°C incubator.
    2. Add 200 μl of basal media directly to the 96-well plate and pipet up and down gently.
    3. Add 250 μl of basal media to 1 well of a Matrigel-coated 24-well plate.
    4. Add the resuspended cells into the well of a Matrigel-coated 24-well plate (total volume: 500 μl). Incubate at 37°C.
    5. Feed cells every day until confluent.
    Thawing and Centrifuging:
    1. Remove plate from -80°C and thaw for about 5 minutes in a 37°C incubator.
    2. Add 200 μl of basal media directly to the 96-well plate and pipet up and down gently.
    3. Transfer resuspended cells to a 1.5mL eppendorf tube.
    4. Add 250 μl of basal media to resuspended cells.
    5. Spin at 300g for 3 minutes.
    6. Aspirate the supernatant, leaving a trace amount to prevent the pellet from drying.
    7. Resuspend cell pellet in 250 μl of basal media.
    8. Add the resuspended cells into a well of a Matrigel-coated 96-well plate. Incubate at 37°C.
    9. Feed cells every day until confluent.
    Which way is the best way?
    Thawing directly to a 24-well plate is a much faster way, but thawing and centrifuging the cells yields a higher cell survival rate.