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
