Freezing and Thawing Cells

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Freezing Mammalian Cells

  1. In a T75 flask, grow cells to 80-90% confluency, or 75-80% density for suspended cells.
  2. Collect cells (trypsinize/scrape adherent cells, etc). Pellet using a 500g (1500RPM) spin, 5min at 4oC.
  3. Suspend cells in 4ml of culture media containing at least 10% FCS.
  4. Add 1ml of DMSO, mix well.
  5. Aliquot into labelled cryotubes, 0.5-1ml per tube.
  6. Place in a cell freezer that has equilibrated to room temperature
    • Mark the container (on tape, on the lid) to indicate a use
    • After the 5th use, the isopropyl alcohol must be replaced with fresh alcohol
  7. Put cell freezer in a -80oC freezer. This will cool the cells at ~1oC/min. Let sit in -80oC overnight.
  8. Transfer tubes to liquid nitrogen store
  9. Make sure to enter the cells into the cell storage database.

Thawing Mammalian Cells

  1. Warm 10ml of the appropriate tissue culture media to 37oC
  2. Remove a frozen tube from the liquid nitrogen store and transfer immediately to a 37oC waterbath
  3. Place 10ml of media into a T25 or T75 flask (T25 will achieve confluency faster). Pipette thawed cells into media
  4. Place in 37oC incubator. 12-18 hours later exchange the media in order to remove the DMSO