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Freezing cells

Cells should be confluent (85-95%). For a T75 culture flask. Prepare from 2-3 criotubes for a confluent T75 culture flask.

1. Aspire the culture media.
2. Rinse cells with 3 ml of 1X PBS.
3. Aspire 1X PBS.
4. Add 2.5 ml of warm TRIPSINA-EDTA (1X), 0.05% and incubate at 37 ºC during 3-5 minutes (time depends of the cell confluency, it is ready when cells are detached). It is also possible to use TrypLE™ Express Enzyme (1X).
5. Neutralize with 2.5 ml of culture media and resuspend the cells.
6. Spin 1000 rpm during 5 minutes.
7. Aspire culture media and add from 1 to 3 ml of COLD descomplemented FBS with 10% DMSO (it must be as cold as possible as DMSO is toxic for the cells).
8. Carefully resuspend and transfer 0.5-1 ml of cells to each criotube (as fast as possible).
9. Freeze the cells at -80 ºC. Cells can be viable at this temperature for around 4-6 months if the process has been followed as indicated. For a long time storage, move the cells to LN2 after 1 or 2 days.