N2 storage of murine cell-lines

Klaus.Matthaei at anu.edu.au Klaus.Matthaei at anu.edu.au
Wed Aug 26 17:28:33 EST 1992


>I've had problems in successfully freezing macrophage cell cultures so they 
>can be successfully resuscitated. The problem does seem to lie in the actual freezing. The cells are harvested in the log phase, suspended in a glycerol freezing mixture at five-fold concentration then left overnight (well insulated) in a -70C freeze
>Can anybody suggest alternative procedures? The alternatives facing me at present are constructing a "freezing box" which will cool at the correct rate in a -70C freezer (feasible) or persuading the department to buy a commercial cell freezer (less f
>
>Sean Shaw
>Microbiology, Reading University
>skrshaw at sksscsc1.rdg.ac.uk


G'Day

Forget the need for commercial freezing machines.

For many years I have been able to successfully freeze a wide range of
mammalian cells including mouse and cattle cells, monoclonals etc. with
excellent retrieval rates (sometimes > than 90%). The cells are simply
suspended in sterile 90% Foetal Calf serum, 10% DMSO, frozen overnight at
-70*C in a styrofoam container and then stored in liquid nitrogen (some
have been down more than 10 years and still revive).  Retrieval is by quick
thawing in a 37*C water bath until JUST thawed (do not warm to 37) and
IMMEDIATE resuspension in fresh COLD medium (DO NOT pellet the cells before
resuspension in the fresh medium).  If the dilution is more than 10 fold
i.e. the DMSO is reduced to below 1% you do not even need to spin down and
wash the cells, just plate directly.  If not pellet gently, resuspend
gently and then plate as per normal.

I hope that this works for you as it does for me.

Cheers, Klaus.
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Klaus Matthaei
Gene Targeting
The John Curtin School of Medical Research
The Australian National University
E-mail: Klaus.Matthaei at anu.edu.au

"Don't try and fix it if it ain't broke".
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