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Thawing Cells

Karmella Haynes, 2013


  • 70% ethanol spray bottle
  • 100% FBS (50 mL aliquot), thawed at 37°C
  • Complete cell culture medium with 20% FBS, if available (e.g., 20% FBS, 1% pen-strep)
  • Sterile tissue culture-treated vent-capped T-25 flask, one per new culture
  • One frozen vial of cells (1 mL) from the -150°C freezer
Three flask sizes: T-25 (small, for starting cultures from frozen stocks), T-75 (medium, for routine passaging and expanding cell cultures), and T-150 (large, for growing & harvesting large amounts of cells). A flask must be properly laid flat (where the opening tilts upward) so that the cells can adhere to & grow on the bottom inside surface.


  1. Pre-warm the growth medium and 100% FBS (NOT the frozen vial of cells) to 37°C in the bead bath.
  2. After reagents are warmed, spray bottles down with 70% ethanol and prepare the biosafety hood as for routine work. Do all work in the biosafety hood.
  3. Transfer 5 mL of 100% FBS into a 15 mL conical tube (one per frozen cell vial).
  4. Retrieve the frozen cell vial from the -150°C freezer.
  5. Quick-thaw the frozen vial in the 37°C bead bath just until the last of the ice in the tube is thawed. Do this for no more than 2 minutes.
  6. Use a 5 mL pipette to transfer the 1 mL of thawed cells into the 5 mL of FBS. Do not mix by pipetting.
  7. Spin the cells + FBS at room temperature at 200 g for 3-5 minutes. If you have just one tube, use a counter balance of equal volume (6 mL water).
  8. Get a new T-25 flask and label it with the label it with the cell line name, your initials, the date, "thawed" to indicate that this is a new culture.
  9. After the cells have pelleted, bring them back to the biosafety cabinet and aspirate off the FBS, but leave about 100 uL of FBS covering the pellet.
  10. Flick the tube to gently resupend the pellet in the ~100 uL FBS.
  11. Stand the t-25 flask up and remove the cap.
  12. Add 4 mL of growth medium to the cells. Mix by gently pipetting up and down (about 3 times).
  13. Transfer the cells + medium into the T-25 flask.
  14. Lay the flask flat and push it back-and-forth and side-to-side to spread the cells evenly. Do not swirl it in a circular motion.
  15. Place the flask into the 37°C incubator. The cells should adhere to the growth surface overnight.
  16. Discard all plastics, etc. that have come into contact with growth medium and cells as biohazard waste.

Follow-up expansion: After the cells have become 100% confluent in the T-25 flask, aspirate off the growth medium, wash the cells with 2 mL 1xPBS, trypsinize them with 0.5 mL of Trypsin buffer and transfer the entire culture into a T-75 flask with 10 mL total growth medium (10% FBS, 1% pen-strep). After the t-75 is 100% confluent, you can make new frozen stocks or passage the cells at 1:10 (or 1:5 if they are slow-growing).