Defrosting Eukaryotic Cells Frozen In Liquid N2

Reagents / Solutions

• Frozen cell line.
• Growth media (e.g. RPMI1640)
• Foetal calf serum (FCS).

1. Make 20ml growth media/10% FCS for each vial of cells to be thawed.
2. Place a small sandwich box containing warm (~ 37癈) water in the CO₂ incubator.
3. Remove vial(s) of cells and thaw rapidly by placing in an expanded polystyrene 'floater' in the water bath. Transfer cells to a universal.
4. Add 10ml media/10% FCS very slowly. First ml should take ~ 1 min, second ml should take ~ 45 seconds etc.
5. When cells are resuspended in 10ml spin down in bench-top centrifuge and resuspend in 10 ml media/10% FCS (this does not need to be added slowly).
6. Place cells in large flask and allow to settle for >= 2 hours.
7. Take a sample, assess % viability and cell number.
8. Allow to grow overnight.
9. Assess cell number and viability, if the cell density is too high for the normal grwoth of the cell line add further normal growth media (e.g. RPMI1640/10% FCS.