Use healthy cells which have been fed within 24 hours of freezing; use flasks that are nearly confluent.
Trypsinize cells in usual manner; spin out cells in 15 mL tube for 5-10 minutes.
Remove supernantant and resuspend pellet in 1 mL cell freezing medium.
Quickly transfer cell mixture to sterile tube labeled with the cell name, passage number, date and initials of the person freezing the cells.
Wrap tube(s) in a few layers of paper towels and place in the -80°C freezer in ISB 262A.
Leave in the freezer for 24-48 hours, then remove cells and place in a cane in the liquid nitrogen tanks.
Fill out the freezing log
Be sure liquid nitrogen freezer does not run out of nitrogen!
Cells will remain “viable” for at least a year, usually several.
Pre-warm 10 mL of medium in a small flask in 37°C CO2 incubator.
Remove the tube of cells that you wish to thaw from the liquid nitrogen tank.
Rapidly bring to 37°C by holding in the water bath and swirling.
As soon as cells are thawed, bring tube to hood, wipe tube with 70% ethanol.
Using sterile technique, transfer cells to the flask of warm medium.
Incubate in CO2 incubator overnight; next day change the medium, removing dead cells and DMSO. (Viable cells will have stuck to the flask.)
Transfer thawed cells to tube containing 10 mL of warm medium
Spin in clinical centrifuge to pellet cells
Resuspend pellet into 5 mL of warm medium
place cells in a flask and grow in incubator as usual.
Make normal culture medium at 85% volume (ie medium components for 100 mL, but only bring the volume to 85 mL) AND with 20% serum.
Freezing Medium: for one flask of cells, take 850 µL of freezing medium and add 150 µL of DMSO (sterile).
Mix well and leave on ice. Make this fresh every time you freeze cells
Freezing S2 cells
S2 Cells Spin down a 3-4 day old flask.
Resuspend in 4 mL of S2 medium with serum and antibiotic.
Add 1mL DMSO (20% final).
Make 1mL aliquots.
Put in -80 in styrofoam for 1 day.
Transfer to liquid nitrogen.