Freezing cultured cells

Dr Engelbert Buxbaum engelbert_buxbaum at
Fri Dec 6 05:33:34 EST 2002

Sean Shaw wrote:

> Using an adherent cell-line at about 90% confluence:
> Spin down the harvested cells, remove the old medium and resuspend in 
> the DMSO/FBS at 1ml per 75cm2 flask. Place 1ml aliquots in freezing 
> vials and place in ice for 1 hr. Transfer to pre-cooled polystyrene box 
> at -20C and leave overnight. Next day move box to -80C freezer or place 
> vials in liquid nitrogen storage tank.

I found it better to count cells first in a haemocytometer, with the
usual trypan blue exclusion test for viability. Cell density should be
about 3-5 million cells per ml, with better than 98% viability.

Some cell lines respond better to 10% glycerol in FBS rather than DMSO,
others don't like that.

> Defrosting: prepare a 75cm2 flask with 15ml warmed medium. Warm a vial 
> in the waterbath until the ice block inside can move freely then tip the 
> contents into the flask (don't forget to wipe vial with ethanol first). 
> Gently rock flask until ice is melted and then place in incubator 
> overnight.

The remaining DMSO can cause problems. Better to transfer the
half-molten contents of the vial into a tube with 10 ml of cold medium,
spin down the cells and discard supernatant. Resuspend cells into fresh
medium and transfer to flask.

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