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Cryopreservation of Mammalian Cells

Karmella Haynes & Ben Nyer, 2014


  • 70% ethanol spray bottle
  • Ice
  • 1x Phosphate-buffered saline (PBS)
  • Trypsin-EDTA buffer/ media
  • Complete cell culture medium (e.g., 10% FBS, 1% pen-strep)
  • Fetal Bovine Serum (FBS) with 10% DMSO (in freezer)
    • For MCF 10A cell lines: prepare complete cell medium w/ 7.5% DMSO added
  • 15mL Falcon tubes
  • 2mL cryo vials
  • 100% confluent (dense) cell culture in T-75 flask(s) (check confluency under the light microscope)


  1. Warm up the PBS, Trypsin-EDTA and complete cell culture medium to 37°C
  2. Thaw the FBS/DMSO solution and then place on ice.
  3. Label one 15mL conical per T-75 flask you are turning into frozen stock.
  4. Turn on the vacuum pump.
  5. Label five 2mL cryo vials per T-75 flask you are turning into frozen stock. Label them with the cell line, date, and your initials.
  6. 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.
  7. Harvest cells from the flask in the same manner you would for passaging cells. You will end up with cells suspended in 10ml growth medium (includes the trypsin-EDTA).
  8. Transfer the 10 mL of cells into the 15 mL Falcon tube. Pellet the cells at room temp for 5 min at ~200 g (typical spin). Use a balance if you have an odd number of tubes.
  9. Aspirate off the growth medium from the pellet, but leave a bit of liquid covering the pellet. Flick the tube with your finger to break up the pellet.
  10. Sterilize the chilled FBS/DMSO bottle with the ethanol spray bottle and place it in the biological safety cabinet.
  11. Resuspend the cells in 5 mL chilled FBS/DMSO. Pipet up and down a couple of times to make sure there are no cells left in a pellet at the bottom.
  12. Aliquot 1 mL of cells into each cryo vial. Place closed vials on ice. Chill for 5 min.
  13. Get a styrofoam box with a lid and place a multi-tube insert from a cardboard freezer box into it. This will serve as a slow-cooling unit for the cells.
  14. Place cell vials into the slow cooling unit. Place this in the -80 C freezer for two days (48 hrs).
  15. Transfer tubes to -150 C for long term storage.