Cell Freezing

Reagents / Solutions

• Cells! (at least 5 x 10⁶).
• Foetal calf serum (FCS), heat inactivated at 65 癈 for 1 hr.
• Dimethylsulfoxide (DMSO).
• Freezing vials.
• Hi - Tech Cell freezer (polystyrene box wadded with cotton wool [!]).
• Liquid nitrogen bank.

Protocol

1. Spin down cells, remove and discard the supernatant.
2. Resuspend the cells at 5 x 10⁶ per ml in FCS.
3. Add DMSO to 10% (by volume) dropwise, agitating gently after each drop. Do this slowly.
4. Place 1ml aliquots into the vials. Remember to label the vials precisely.
5. Put vials into central positions of a polystyrene rack (from e.g. Boehringer Enzyme Storage box), and place that rack into an expanded polystyrene box (e.g. 'Eprack' as supplied by Scotlab) which is wadded with cotton wool.
6. Place lid on box and seal in place with tape.
7. Place box in the vapour phase of the liquid nitrogen bank. It must not be placed in the liquid phase. Leave for more than 4 hrs to freeze.
8. Remove vials from box and place in liquid phase in the cell bank.
9. Test the efficiency of the freezing procedure by thawing (method) and growing on one vial.

If the cells were healthy (> 95% viable) at the start of this procedure, then we obtain a culture 80-90% viable 24 hours after thawing and growing on (method) the test vial from point 9.