ECACC recommends that a stock of the cell line(s) should be frozen as soon as possible after receipt at between 2 - 4 x 10⁶ cells/ml at a minimum of 80% viability.
The following guide is offered for the preparation and freezing of cell lines.

1. Harvest cells in the same manner used for routine subculture.
   
2. Prepare freeze medium by allowing 1ml for each ampoule to be frozen. ECACC uses 90% serum + 10% cryoprotectant for all of its cells unless otherwise specified on the data sheet. Most cell lines can be frozen in the appropriate culture medium supplemented with 20% serum and 10% cryoprotectant. This is usually DMSO but in certain instances (see datasheet) glycerol is recommended. This should be tested for each cell line before use. Do not include antibiotics in the freeze medium.
   
3. Resuspend the cell pellets in the freeze medium to give a final cell concentration of between 2 - 4 x 10⁶ cells/ml and aliquot 1ml into each ampoule. The ampoules should be clearly marked. If there is any doubt about rapid exposure of the cryoprotectant causing toxic shock to the cells, resuspend the pellets in half the required volume of freeze medium without cryoprotectant, mix and then add equal volume of medium to which double the volume of cryoprotectant has been added.
   Freeze the cells at a cooling rate between 1-3^oC/min using a programmable freezer. When the temperature reaches -150^oC, transfer ampoule to a gas phase liquid Nitrogen storage vessel. As an alternative to this a polystyrene box or a 'Mr Frosty' (BDH cat no. 215/0884/00) or similar in a -80^oC freezer for 24 hours prior to transfer to gaseous phase liquid nitrogen.
   
4. It is advisable to test the cell viability by thawing one ampoule after freezing. Viability below 70% may cause problems in starting new cultures, due to low cell numbers and the presence of debris.

Ref. Cryopreservation of Animal Cells in Advances in Biotechnology Processes (1988), 7, A.R. Liss