thawing & plating cell lines

Curt Ashendel ashendel at
Mon Jun 24 14:19:33 EST 1996

On 21 Jun 1996 05:01:17 -0700, cram at PIPELINE.COM  wrote:

>    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? 

It depends on the cell line. Cells that are fairly density dependent 
for growth (NIH 3T3 cells, for ex.) do not thaw well if plated at 
lower densities that you would usually plate them after splitting 
them. Also, if your freeze killed a lot of the cells, the viable cell 
plating density after thawing is even lower. I always plate freshly 
thawed cells at very high densities (unless there is a good reason not 
to) to get the fastest recovery from the freeze.

Curt Ashendel
Purdue University, West Lafayette, IN
ashendel at

More information about the Methods mailing list