Problems with Neuronal Culture

Bill Marszalec billmars at nwu.edu
Mon Aug 25 13:18:47 EST 1997


Please Help:

   I would like to pose a problem to those members of this group who 
have worked with cultured neurons or cultured cells in general.  For the 
past three years, we have been performing whole cell voltage clamp 
experiments using a primary culture of isolated frontal cortical neurons 
prepared from 17 day embryonic rat pups.  My question relates to a 
seemingly cyclic fluctuation of the quality of these cells.  I don't 
mean to bore you, but what follows is an account of our usual 
procedures.  I hope that one of you may perceive problem in the 
preparation that we have overlooked.

The Procedure:   We prepare these cells once a week and usually use them 
for our electrophysiology experiments two to three weeks later.  Small 
wedges of frontal cortex are rapidly excised from the isolated brains in 
magnesium- and calcium-free physiological balanced salt solution (PBS) 
and incubated at 37o C in 2.5 mg/ml of trypsin (Sigma Type XI) for 25 
min.  Following this incubation the cells are washed twice and 
resuspended in PBS containing DNase.  The cells are then gently 
triturated with a  glass pipette. 
   The dissociated cells are then resuspended in a culture media 
containing (vol/vol) 80% Dulbecco's Modified Eagle's Medium (DMEM), 5% 
Fetal Bovine Serum (heat inactivated, and 5% HAM's F- 12 Supplement. The 
amino acid glutamine is also added to the media just before use to a 
final concentration of 2 mM. No antibiotics are used in this 
preparation.  The cells are diluted to 100,000 cells per ml media. Then 
3 ml containing 300,000 cells, are plated into 35 mm culture wells, each 
containing five 12 mm L-polylysine-coated glass coverslips. 
    Several weeks previous, a glial culture derived from post- natal rat 
cortex was established on these same coverslips so that the embryonic 
neurons are plated on a preexisting confluent glia layer. The cells are 
incubated at 37 degrees C in a moist atmosphere containing 10% CO2. 
After three days, cytosine arabinoside (8 uM) is added to inhibit 
further glial growth.  For every subsequent week, 0.5 ml of media is 
added to each 35 mm well as a nutritive supplement, no media is ever 
removed.

The Problem:  Over the last three years I have noticed that the quality 
of this culture goes from good to excruciatingly bad, and then seem to 
pick up again to repeat the cycle about every six months.  Recently, 
during June and early July 1997, these cells were looking very good and 
many electrophysiological experiments were performed. Then in mid July 
through August, the quality of the culture began to gradually 
deteriorate, with an increasing percentage of dead cells and cells 
having rough-looking granular membranes and swollen nuclei. 
   Over the last two weeks the culture has reached rock bottom!  The 
neurons did not survive even one day in culture!  As in the past, we are 
replacing all our media, serum and trypsin with new material and are 
hoping for the best.  In the past, the cultures usually have returned to 
a better quality with time, although we have never actually discovered 
the true nature of the initial problem. I will explicitly state that 
were did not knowingly change any of the materials or methods of our 
procedure including; the media, the trypsin, the animal supplier, the 
technician, the coverslips etc.  All media is less than 6-8 months old 
and is kept refrigerated.  The serum (purchased Jan 1997) is kept in 
frozen aliquots at -20 C.



The Question: Does anybody have a ready answer to the source of our 
problem?  Have we overlooked something?  Have any of you encountered a 
similar problem with any of your preparations. What about this 
mycoplasma contamination I keep hearing about? Could it be the problem? 
(our media is supposedly mycoplasma tested).


						Thanks in Advance,
						Bill Marszalec 

Bill Marszalec
Northwestern University, Evanston, IL.  USA
billmars at nwu.edu



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