Problems with Neuronal Culture

Matt Jones jonesmat at ohsu.edu
Sat Aug 30 20:23:37 EST 1997


In article <3401CC87.3FD1 at nwu.edu> Bill Marszalec, billmars at nwu.edu
writes:
>My question relates to a 
>seemingly cyclic fluctuation of the quality of these cells.

Oh no. It's the "cyclical cultured cell health problem" again.

We've had similar problems in the recent past, although we use slightly
different culture methods. The problem is characterised by declining
recording quality (sometimes the cells look fine but are really leaky,
sometimes they're just dead). This occurs even though there's been no
identifiable change in methods, and it eventually goes away, once again
with no identifiable change in methods. 

The important elements of your problem and ours, as I see it, are the
following:

1) You said: "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". This means that, for the most part, ANY
aspect of your method which you actually have some control over (and know
that you have control) is probably not the culprit (provided that you've
been careful, etc...). So enzyme concentrations, serum lots, media, etc
are unlikely to be the cause, as they usually work properly for you and
only sometimes fail.

2) The problem is intermittent. In our case, I can't say that it is
cyclical exactly, although I have an inkling that it might be. Last year
it lasted from about early autumn through mid-winter. This intermittency
makes it very difficult to systematically isolate the critical variables.
Basically, very few things that we've tried in the past proved to be the
right answer. More often than not, the problem just seemed to go away.
Obviously, this is not a satisfying situation since you want to know
_why_.

Unfortunately, I have no clear solution to this dilemma, other than
providing a sympathetic ear. I can, however, tell you what we believe was
the cause of this problem on at least one occassion: parvo virus. Last
year we pestered our animal supplier (B &K) to test the animals for
viruses, bacteria, diseases of all sorts (this was only after exhausting
all other possibilities, and banging our heads against a wall for
months). They swore up and down that they had done this, and found
nothing. We sent some animals to an independent lab for testing (because
we didn't know what else to do, and could imagine no further options),
and they found a pretty widespread parvo infection. When confronted with
this finding, B&K owned up and admitted that they could (now) also detect
parvo. We changed suppliers and within a few weeks things were back to
normal. Needless to say, we don't purchase from them anymore. 

I _am not_ saying that this is the source of your problem, or that this
is a common problem with this particular supplier. But in our case,
sending the animals to an independent lab for testing revealed a
significant variable which had changed without our knowing it. 

Other things that I will test independently next time (and I'm sure that
there will be a next time) are a) water and media purity, b) CO2 purity
in the incubators, c) air, food and water quality in the animal house,
and basically anything else that we receive from someone else and whose
quality we take for granted.

I sincerely hope you resolve this problem soon.

Cheers,

Matt Jones
The Vollum Institute
Portland, OR



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