Oxygen in cell media
klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Sat Nov 25 09:44:35 EST 2000
In article <8vlre7$2a6$1 at wsc10.lrz-muenchen.de>, "Markus Guetlich" <markus.guetlich at ch.tum.de> wrote:
>We want to increase the protein yield of our insect cell culture.
>Therefore we want to oxygenate the culture.
> Who knows what kind of air-bubbles are the best ?
I imagine a simple aquarium type air pump with adjustable
pumping rate fitted with 0.2 micron air filter (I'd also include
a prefilter) will work fine. Make sure the bubbling is not
too agressive and perhaps add pluronic.
On the other hand, I personally find that careful optimization
of infection moi and times is the key in boosting expression
levels. Also, cloning the virus. On more than one occasion,
I've had viruses that showed steady decrease of expression
with every amplification round (that is despite the fact that
titer was high). In all cases, cloning the virus and selecting
a clone that gives higher expression helped tremendously.
The problem is particularly severe with Gibco's Bac-to-Bac
system. I believe in this case transfection yeilds plenty of
non- or poorly expression viruses that preferentially propagate
during amplification cycles.
I know it's not exactly your question but consider plastic
reusable 1 l spinners from Nalgene. They are 3X cheaper than
glass and _much_ better. Unbreakable, better surface to volume
ratio, better designed ports and geometry. Four "normal" spinners
sounds to me preferable to 1 spinner with bubbler. I am very
happy with them and only wish Nalgene had other sizes of them.
Cloning of Bac to Bac-produced virus + Nalgene spinners =
20 mg/l of 150 kDa protein in my case (used to be 1-2 mg/l).
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