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cell culture help
Thanks in advance for any advice offered!
Our lab has just suffered the departure of our long time cell culture technician and now we are experiencing problems with our cell cultures. Most apparent is the pH change in the cultures that appear to be converting to acidity to quickly. We generally change media every three to four days depending on cell line. Have tested for bacterial contamination-negative. I am concerned the quality of the media has been compromised. The new technician began turning very bright flourescent lighting on in the storage area that was not lit previously. Does anyone think this could cause our dramatic lack of thriving cells? I have read that deterioration of components could create toxic by products. This has been going on over the course of a month. Some cell lines ok, some totally dead, generally not thriving.
Re: cell culture help
A fluorescent light in a storage area is not an effective way of controlling bacterial growth - the intensity of the light drops off with the square of the distance, meaning unless something is close to the bulb it'll not get sanitised.
The UV could be breaking down your media, although for the above reason's I suspect that's not the issue.
Did you do microplasma testing? Your problem sounds like micro, and they wouldn't appear on normal bacterial tests. If you haven't done that, I'd recommend MP testing.
Also, have you tried replacing any of the cell lines with frozen stocks? If so, are the frozen stocks also not growing well?
Finally, are you sure you're using the right media for the right cell lines? Some cells are quite sensitive to the type of media and serum concentration.
Oh, and one last thing, are you heat-inactivating your serum first?
Re: cell culture help
Thanks for the ideas!
The lighting was not intended for sanitizing purposes-the tech just liked the additional lighting and left them on nine hours a day-quite intense.
We have gone back to our frozen lines, with similar problems.
I suspect a problem in the production of our MEM. We add Sodium Bicarb and RO water to a powdered MEM and go through a filtering process. We store this and make up a 10% or 2% FBS media as needed.
I am also concerned about the mycoplasma contamination potential.
Re: cell culture help
Have you considered trying prepared MEM? You'd still have to add serum, but it would eliminate any preparation issues in terms of the pH, filtering, etc.
Personally, I make up my media with the serum added to the desired concentration, which eliminates the possible measuring error when adding serum to smaller amounts of media. But I doubt that kind of error would cause the problems you describe.
I'd check for MP before anything else. That, and maybe try prepared media.
Oh, and one last thought - are you certain your plastics are tissue-culture grade? Bacterial and chemical grade plastics often contain components toxic to mammalian cells, and may also be poor substrates for cell adhesion. You may want to double-check that they are tissue-culture quality.
Oh, and one other last thought - in the past we've had issues with some batches of serum not supporting good cell growth. If you haven't done it already, it may be a good idea to try serum from another company, or at least a different lot from the same company. I think the quantities of growth factors can vary largely batch-to-batch...
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