PC12 cell variation and differentiation

[Michael Holloway]

Are there any folks out there in Netland working with PC12 cells?  I'm 
interested in hearing about your experiences.  Specifically, have you had 
trouble with variability of the cells with continuous passage?

Apparently there are major differences in media used and a suspicion that a 
large amount of differentiation has occurred between lab groups as the cells 
are being passaged.  The directions I've received from Simon Halegoua here at 
Stony Brook call for splitting 1:5 when the cells reach 6 to 7 X 10^6 in a 10cm 
dish.  When this is followed religously, the cells are supposed to be able to 
be passaged nearly continuously.  The practical problem I'm having as I start 
to culture them though is that getting a good cell count is impossible.  They 
do not come apart in trypsin and no amout of pipetting will separate them.  
Apparently, the way cell density is followed on a practical basis is to split 
the cells when a certain degree of confluence is reached.  The problem with 
this though is that the cells NEVER actually become confluent, they clump.  
I've learned then that the upshot is that successfully keeping PC12 cells going 
relies entirely on a qualitative "feel" for the look of the plate and the 
cells.  When they get "too clumpy" its time to go back to the frozens.

This is of particular importance to me now because of the results I've gotten 
in preliminary observations of PC12 cells differentiated -- not with NGF -- but 
with Na butyrate.  This is supposed to send the cells off toward a mature 
chromaffin cell morphology.  As I've tried to repeat observations I'm getting 
unexpected variability in the results which I attribute to the number of 
passages through which these cells have gone.

If anyone has any suggestions or similar experiences I'd be glad to hear from 
you. 

Mike