Thawing Cells (General)

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Thawing cells properly is crucial to cell culture health. Usually, specific cell lines will have specific thawing protocols provided by the manufacturer. This is a general protocol for thawing cells that do not accompany a manufacturer’s thawing protocol. Please note that not following manufacturer instructions may result in cell death or improper function.



  1. Warm cell medium to 37˚C in the water/bead bath;
  2. Remember to spray everything down with 70% ethanol and wipe it with Kimwipes before moving into the BSC;
  3. Check the number of cells that are in the cryopreserved vial and prepare the appropriate number of flasks with the corresponding amount of warm cell medium. Cell seeding density will be specific to each cell type, but it is usually 2,000-5,000 cells/cm2. Amount of media will also vary by flask size, but it is recommended to use 0.2 mL of media per cm2. Find useful formulae below;
    • Number of flasks = number of cells/cell seeding density/flask area in cm2
    • Amount of media = (flask area in cm2)*0.2 mL
  4. Remove cell vial from liquid nitrogen and submerge it in the water/bead bath up to the cap for no longer than 2 minutes;
  5. Remove vial when its sides are thawed but its center remains frozen;
  6. Spray down all media and pipets and move them into the BSC;
  7. Pipette 1 mL of medium into the cell vial;
  8. Pipette solution up and down to ensure homogeneous cell distribution;
  9. Pipette corresponding amounts of cell solution into each cell medium-containing flask. Use the formula below to calculate how much cell solution to add to each flask;
    • Amount of cell solution to be seeded = volume of cell solution/number of flasks
  10.  Gently rock the flasks without allowing the solution to touch the filter cap. If the filter comes in contact with the liquid, the sterile barrier might be broken, and you may have exposed your cell culture to external pathogens;
  11.  Label your flasks. It is good practice to include the following information;
    •  Your initials and date of passage (e.g.: if your initials are YN, and the date is MM/DD/YYYY, label it as YNYYYMMDD)
    • Cell line and passage number (if your vials were Pn, they will now be Pn+1).
  12.  Place flasks in the incubator;
  13.  Feed cells every two to three days, following this protocol;
  14.  Passage cells following this protocol.

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