Cells dying in serum-free medium

nishir at ohsu.edu nishir at ohsu.edu
Mon Sep 27 15:27:41 EST 1993


In article <CDz03F.BFs at dartvax.dartmouth.edu> knight at grafton.dartmouth.edu
(John  Boswell) writes:
>
>Hi,
>	Our lab is new to cell culture, and we are having a bit of a problem.
>We have a cell line (HEK) containing a plasmid encoding a protein we wish
>to overexpress.  The cells *do* express the protein, as we detect it via
>Western blot and activity assays.  The problem is that though the cells
>do quite nicely in DMEM with fetal calf serum (also with pen/strep), when
>we switch to serum-free medium the cells last a day or two at most, then
>detach and die. :(  The serum free medium has no fcs (obviously), and we
>also add trypsin inhibitor, insulin, transferrin and bacitracin.  Otherwise
>it is the same as with serum.  We've increased the insulin and transferrin
levelto see if that was the problem, but they had no effect.  We can't leave
the
>trypsin inhibitor out, as our protein would be chewed up.  So, that leaves
>bacitracin.  We aren't sure *why* bacitracin is used (as the medium also
>has pen/strep).  However, we are reticent to leave it out or increase
>it, as it may be used to somehow induce the plasmid to express our protein
>(or is that nonsense?)
>	Anyway, does someone know if we are doing something stupid, based
>on the above quick description?  I know debugging (so to speak) such things
>is difficult with all the things that can go wrong, but maybe we are doing
>(or not doing) something that is obvious to someone with more experience.
>	Well, thanks a lot in advance,
>-John Boswell
>
>--
>Dr. John Boswell	 			knight at grafton.dartmouth.edu 	
>Oregon Graduate Institute, Portland, OR		503-690-1086
>
Hi.  Here's my two cents worth from down hwy 26...Bacitracin is a nasty
antibiotic because it blocks glycosylation of protein and therefore could have
some bad side effects on eucaryotic cell lines if the concentration is too
high.  I would suggest leaving it out and having just pen/strep in the medium. 
If your cells are contaminated by a bug that is resistant to pen/strep, then
try gentamycin as a different antibiotic.  I don't know of a plasmid that needs
bacitracin for expression, but I'm ignorant on that front.  Perhaps someone
else would like to comment.

Other things to check are the components of your "serum free" medium and the pH
of your medium.  Many cell lines can grow quite well in serum-free medium, but
they differ in what they need.  The original Sato lab formulations always
required a polyamine, a steroid hormone, and trace amounts of selenium dioxide
in addition to insulin and transferrin.  Perhaps you need to add these.  Also,
insulin in particular is very labile.  It dies at neutral pH pretty rapidly so
you cannot keep stocks of medium in the fridge for long periods of time with
the insulin in it.  Better to keep it at acid pH and add it fresh to your
medium every time you feed.  Your cells might also need a growth factor to
stimulate cell division. Different cells require different growth factors, but
some that are commonly added to serum free medium are EGF (epidermal growth
factor) and FGF (fibroblast growth factor).  Finally, check the pH of your
DMEM.  The original formulation of DMEM was for extra bicarbonate so that the
medium was pH 7.2-7.3 in 10% CO2.  Most of us are used to buffering culture
medium with 5% CO2.  If the DMEM you are using has the extra bicarbonate in it,
then the pH might be too alkaline, something that might not bother the cells in
FCS, but could when you switch to the more minimal serum-free conditions.

Rae Nishi
OHSU
Portland OR



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