thawing & plating cell lines

Andreas Jordan jordan at ukrv.de
Fri Jun 21 13:20:48 EST 1996


In most cases we observed those effects on any mammalian cells, there 
were some uncertainities during freezing procedure. For example often 
a slight increase of the temperature during freezing procedure above 4 
degr.C cause damage to cells by DMSO. Furthermore, the freezing speed 
from 4 degr. C to -80 degr. C to liquid nitrogen is critical. We 
always envelop the cryo vial boxes with thick cotton or paper towels 
at the time interval between 4 and -80 degr. C to achieve at least a 
temperature decrease of 1 degr C per minute. Faster freezing will also 
damage the cells physiologically so that they come out dividing later 
as usual. My last concern is speed of centrifugation after thawing 
procedure. You should try to use the lowest speed possible for 
sedimentation and avoid pipetting the cell suspension more than 
absolutely necessary in order to minimze membrane damage of the 
freshly thawed (mostly sensible) cells. Use 25 or better 12.5qcm 
flasks (Falcon) for first passage to obtain best cell density, i.e. 
cell growth.

cram at PIPELINE.COM wrote:
> 
> 
> Hello,
>     my team and I are having trouble producing healthy-looking cell
> cultures at plating time.  We thaw quickly, spin to remove DMSO and then
> plate.  After one night, the cells are dividing slowly, and have a strange,
> small, sort of compact morphology.  Overall, they are half the size as they
> appear when healthy.  Only after another day or so do they have the nice
> flattened look to them, where you can see the cytoplasm and their nuclei
> are large, etc.  Then they grow to confluency, no problem.
>     Could this be due to a low density at plating, perhaps too low?  Or is
> it more likely caused by our cryopreservation?
> 
>     Any input would be appreciated.
>     Thank you in advance.
> 
> Marc Rothman
> <cram at pipeline.com>
> Technician
> Neurosurgery Research Labs
> Dept. of Neurosurgery
> NYU Medical Center
> 550 First Ave.
> NY, NY  10016
>



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