Unexplained cell death - (Dec/09/2007 )

I have this problem with unexplained cell culture death. All my cultures mysteriously expires within a day to a week. I am currently culturing various strains of VERO, and COS-7. They had been fine 2 months ago until they start dying without any reasons. I am currently sharing an incubator with 2 other lab members and they have very healthy cells. I received cells from them and placed them at my own shelf in the incubator and those cells died without me doing anything to them. I had even took cells from another lab member and cultured it for her (I placed it in her rack after that) and her cells was extremely heathly while mine just floated and the few surviving ones wont divide.

It looks like only my cells were affected so I changed to another incubator. Same problem. The cells float and the ones left attached to the flask look sick (blebbing, strangly cytoplasm, large vacuoles). The strange thing is that when I place newly received cells with the sickly ones in the same rack overnight in the incubator, the heathly ones start dying as well. The medium looks fine, no cloudiness and does not smell. No moving spots could be observed except for cell debris.

And as to help the dying cells. I changed the medium daily, some with replacement of 1:1 old medium:new medium, added high concentrations of FBS (20-30%), added antibiotics (for mycobacterium as well). Nothing seems to help.

Please help me on this. It has been driving me crazy for 2 months as I cannot do any other experiment but watch my cells die everyday. I had thawed dozens of stocks but none of them survived.

Any advice would be of a great help. Thanks in advance.

Fortunately I've never seen a mycoplasma infection, but it would be the first thing I'd try to test for (in media, shelf, cultures etc). Now I see you added antibiotics against it, but a check would not hurt.. I assume that you used the right type of media and so on. As a last resort, maybe the FBS is not compatible with your COS cells? I'd try heat-inactivation or using a different batch. How about heat-regulation in your incubator? Maybe your cells are more sensitive to changes in temperature, and the mixing is not homogeneous or something like that?

Dont know about vero, but Cos 7 are very hardy cells. You dont really need to use heat inactivated FBS.

*At any point during the culture, have you let them become too confluent without passing when they should be?

**If someone lese uses the same incubator and is fine with their cells, than that is not a problem.

*** !!!! Check if your DMEM contains L-Glu. if not, you need to add it. L-Glu helps cells to make GSH, a very important antioxidant. Cells grown in L-Glu-free medium can manage to survive for several generations, but not more than that, because, all the endogenous one are depleted. then funny thing like what you described can happen. good lcuk.

Thank you all for your quick replies.

To The Bearer:
Yes, I too think that something is toxic to the cells but I still do not know what is causing the cells to die so quickly. The FBS used was what everyone in my laboratory is using and they do not seem to have any problems with cell culturing. I received another batch of cells from another lab member and cultured them with my own media. The cells in her shelf grew right up to confluency while mine died.

To Kupac:
Perhaps the culprit is heat regulation or erratic CO2 levels. I placed another batch of cells in another shelf and they seem fine today. The cells in my shelf had died within 48 H while those which I cultured and placed in another shelf (not mine but same incubator) grew to confluency...well I need to wait for another week to see wheter they survive that long or not. I had read plenty on mycoplasma but no one in my lab does any testing for them. I really wonder.....but kanamycin, penicillin-streptomycin, ciprofloxacin and the myco-killa sold by Roche didnt work anyway. I think my cells has a streak of sadism cause they love to watch me suffer while they happily blast themselves to millions of pieces. Maybe someday I can write a paper on "Sadistic experiments: How to deal with it" and publish it in Science.

And GeneHunter-1:
I use this site alot and had seen plenty replies written by you. Glad that you had answered mine : )
COS and VERO are very hardy and they can grow for 10 days without feeding them with fresh medium(that was last autumn). What was really puzzling was why the culture died within 48 hours. Same incubator but different shelfs seem to effect cell growth(?) Yes, I have never heard of that but there are always things which puzzles you in science, and at the same time, drives you crazy too. And yes, the DMEM contains L-glutamine and the medium was new.

I've been having the EXACT same problem and cannot figure out what is happening! The only thing that has changed is the incubator, but it is new, and when I check temp and fyrite it is always 37/5% CO2. Did your incubator turn out to be the problem?

1) Can you tell by the sound if the fan is ON when you open the door? 2) Does the color of the medium looked alright to you- not too yellow, not too pink?