ECACC recommends freezing a stock of your cell line(s) soon after receipt (between 2 - 4 x 106 cells/ml) as a precaution.
The following guide is offered for the preparation and cryopreservation of cell lines.
1. Harvest the cells in the log phase of growth in the same manner used for routine subculture. For adherent cell lines harvest as close to 80-90% confluency as possible.
2. The standard procedure is to use 90% serum + 10% cryoprotectant for all cell lines unless otherwise specified on the data sheet. Allow 1ml for each ampoule. 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 glycerol is recommended. If DMSO is not suitable an alternative will be specified on the cell line data sheet.
3. Pellet cells by centrifugation e.g. 150 x g for 5 minutes. Resuspend the cell pellets in the appropriate freeze medium to give a final cell concentration between 2 - 4 x 106 cells/ml and pipette 1ml into each ampoule.
4. Freeze the cells at a cooling rate between 1-3oC/min using a programmable rate controlled freezer or suitable alternative1. When the temperature reaches at least -130oC, transfer the ampoule to a gas phase liquid nitrogen storage vessel.
It is advisable to test cell viability by thawing one ampoule after short term storage in gas phase nitrogen.
Ref. Cryopreservation of Animal Cells in Advances in Biotechnology Processes (1988), 7, A.R. Liss
1 A polystyrene box, or alcohol bath (e.g. a Nalgene 5100 'Mr Frosty' box Sigma cat no. C1562) can be used in a -80oC freezer for up to 24 hours prior to transfer to gaseous phase nitrogen.
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