I have a IgG secreting hybridoma. Unexpectly, when the cell number increase, the culture turn red and the cells are not happy. Any suggestions?
I have seen this behaviour in the past with some hybridoma cells. Obviously they are not producing an acidic product of metabolism but instead are producing a product which is more neutral to basic. I think this can be the case when instead of relying on glucose metabolism as an energy source that the cells are overly dependent on other media components such as L-glutamine see the link below for a discussion of this type of metabolism seen with some tumour cells.
http://en.wikipedia.org/wiki/Glutaminolysis
Mike, thank you so much. Do you think I should put more glutamine to the medium to make cells happy? We have been using Ad-RPMI-1640 plus 2% FBS to culture the hybridoma.
You certainly good try supplementing the media to see if this improves growth or yield of antibody. However in the longer term, particularly if this antibody is considered useful and unique, I would suggest you consider whether it might be more worthwhile to sequence the antibody and then to produce a recombinant version, perhaps expressing it in another cell line which would then hopefully grow under more optimal metabolic conditions.
If the medium is red with a hint of purple, the cells may not be getting enough CO2.
Hi Linda, yes of course if all the cultures in an incubator are turning purple it can be a sign of low CO2 in the incubator. However I think in this case it sounds like a more specific phenomena associated with only some hybridoma clones and not others, even when they're being cultured in the same incubator and in the same media and conditions. That was certainly my experience in the past. If the cells are breaking down glutamine or other amino acids as a principle source of energy, then they may be producing some ammonia or other basic metabolites, which means the media doesn't acidify and turn yellow as expected.
Hi Mark, you are right. Only one of my hybridomas behaves like that. Other cells are fine in the same incubator.
By my experience such clones have never been good antibody producers. I think it will be better to leave an attempt to do it useful)))
Good point, Mark. I also would try boosting the glutamine. L-glutamine is unstable and decomposes even in the fridge. Freezing it in "just enough" aliquots is a good practice that has always worked for me. You can buy L-g supplemented media, but I'd rather just add it as I go.
I fully endorse testing adding additional glutamine to glutamine supplemented medium. Freeze down small aliquots (5 ml or less) glutamine and store at -20o until required. Throw out unused after 2 weeks. Another one to try is UltroSer G . This was always added to the culturing medium of hybridomas and worked well even in those growing suboptimally.
L-Glutamine is very unstable at 2-8C temperature, it should be better stored at -20 C by small aliquotes. Pl try Glutamax from Life Technlogies for better performance.
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