VERO cells die after a while. There isn't evidence of contamination (bacteria and fungi). In addition, other cell lines aren't killed and with the same culture medium VERO. Which can be?
What's the base media you are using and what are you supplementing the media with? Can you give me the exact catalog number?
It sounds like this 'dies after a while' has happened several times. I assume you've thawed out new vials of frozen cells after each death? How many passages do they last?
It's possible you have mycoplasma contamination (although usually that doesn't get so severe that it kills the host cells) and you can find out with an easy screen of the culture media that has been growing atop the cells. I use Lonza's MycoAlert kit routinely; it's very sensitive and takes about 30 minutes to do.
Supplement DMEM medium (Embriolife) with 10% fetal bovine serum (Cultilab, Lt 014/13), L-glutamine, Amphotericin B and penicillin / streptomycin. Already thawed out new vials of frozen cells. Or the cells do not adhere to the plate or die after 10 days. Already switched to the culture medium Gibco, but the problem persists. The cells continue to die after a while. Interestingly, using the same culture medium in another cell line (Huh7.5-SGR-Feo) and they don't die.
Hey,
do I get you right, the cells do not even attach to the plate after seeding? Then it appears there is a problem with your cryostocks. How are they stored? Liquid nitrogen or 'only' -80°C freezer? At -80°C, viability can suffer in the course of months or years, as chemical processes are only slowed but not stopped at this temperature. If stored in nitrogen, can you be 100 % sure the container did not run dry at any time? In the freezing medium, cells will die in a matter of hours if the medium ever thaws.
Anyways, you might try to assess membrane intactness by trypan blue staining right after thawing and washing the cells. If most / all cells are stained, you know your stocks are done...
Hello, cells adhered to the plate, but loosen after some time. I staining by trypan blue and some cells stained.
Given the symptoms, the problem does seem to be restricted to this cell bank. I recommend getting a fresh source of Veros from ATCC or Sigma or a colleague. Commercial sources test their lines to confirm the absence of contaminants, so while they're more expensive in the short term, they might be worth it.
As I said, while mycoplasma contamination is a possibility (it's not detectable by eye) it's usually not culture-lethal and is probably not what's killing your cells.
I'd be most likely to suspect starvation (i.e. your l-glut is bad/expired/missing) except that this control cell line doesn't seem to be affected.
When the Veros die, do they do so in patches? Or all at once? Can you attach photos of what the cells look like before each passage?
Viral infection is a possibility-- Veros can support a variety of viruses (indeed that's what I use them for) and a small quantity of virus could start out undetectable and take a few passages of the host cells before there was enough to wipe them out.
I performed a staining DAPI and could be detected mycoplsm. How eliminate this problem?
Though there are methods for mycoplasma elimination, from my experience it is much more safe and less cumbersome to just get some fresh and clean stocks, like Katherine recommended. The cultures may appear cured for a few weeks after treatment and can be reinfected if only a few bacteria survive the treatment (mycoplasma has a very long doubling time). Don't fall for the '100 % success' promises many mycoplasma removal kit suppliers make...
Also be sure to perform a thorough cleaning of your laminar flow hood and incubators, decontaminate the pipets used for cell culture work and discard ALL already opened media, supplements and other solutions, otherwise you will be risking reinfection if any of your reagent stocks was contaminated, which takes you back to the start.
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