Freezing

Approximately 1 freezing vial per 25 cm2 flask.

3 vials from large size culture flask.

Feeding Cells before freezing

  • When cells are 90% confluent (~4 days), replace medium
  • Draw out old medium, replace with 5 mL new (warm) medium.

Freezing cells

  • Make freezing medium (15% DMSO, 20% serum) in advance
  • Remove medium from flask
  • Rinse once with ~3 mL sterile PBS. (Squirt in, then suck out.)
  • Add 10 – 15 drops trypsin (0.5 – 0.75 mL)
  • Incubate (37C) 5 – 30 min
  • Add 3 mL cold medium (regular)
  • Pour into 15 mL centrifuge tube
  • Optional – rinse with another 2 mL, to capture as many cells as possible
  • Spin 5 – 10 min (until supernate is clear and pellet is hard)
  • Pour off supernate (if pellet is solid – otherwise pipette it off)
  • Resuspend in 1 mL freezing medium (pipette up and down)
  • Transfer to freezing vial (should be slightly more than 1 mL)
  • Put on ice
  • Store overnight in -80C
  • Put into liquid nitrogen.