Freezing

Approximately 1 freezing vial per 25 cm2 flask.

3 vials from large size (75 cm2) 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 (~100-500xg) 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 in "Mr. Frosty" (actually a VWR 414004-284 CryoCooler Freeze Controller)
• Put into liquid nitrogen.

Fill out the freezing log

Frozen cell lines