OVCAR Culture Problems

[cellbioS]

I need to pick some brains: We have been using OVCAR-3 cells in our lab for years. All of a sudden, they are dying after a few days in culture. This happens a few days after trypsininzation and reseeding. The culture conditions are the same, the media are all the same (fresh). The other cell lines are doing fine. We have received new aliquots from ATCC and it happens. This also happens to cells that we thaw from cryopreservation.

 

It's very frustrating because this has been going on for some time and has caused havoc.

 

Any ideas?!

[CharonY]

Do you use glasswares to cultivate them or disposable plastics? Also do you buy the medium or do you make it yourself (are the chemicals fresh?).

[cellbioS]

Do you use glasswares to cultivate them or disposable plastics? Also do you buy the medium or do you make it yourself (are the chemicals fresh?).

 

We use dipsosable plastic, cell culture treated. Dishes from the same bags have been used with other cell lines with no problems. I made the complete media from freshly purchased components. Complete media was sterile filtered. Again, components are used in media for other cell lines that have had not problems.

[CharonY]

Hmm that is a tricky one. The fact that it works for different cell line tends to be a a slightly weak indicator that everything is all right, as some are more susceptible to, e.g. certain contaminations.

However if the same protocol suddenly does not work with the same cell line that worked before, that is strange. Especially if you use the same equipment. Disposable plastic wares also eliminate a lot of contamination problems. One final thing that I can think of at the moment is whether you have changed anything on the water system? For instance whether pyrogen and TOC levels are still low.

I have not cultivated OVCAR cells myself (the closest I have cultivated are HeLa cells) so I am not 100% sure how sensitive they are.

[cellbioS]

One final thing that I can think of at the moment is whether you have changed anything on the water system? For instance whether pyrogen and TOC levels are still low.

I have not cultivated OVCAR cells myself (the closest I have cultivated are HeLa cells) so I am not 100% sure how sensitive they are.

 

Water source is still the same. That's why I'm so frustrated with this; nothing's changed in the procedure but the growth rate (or lack of it).

[Cap'n Refsmmat]

What sort of water source do you use? (I have no idea what a lab might use.) If tap water is used at any point, remember that some municipalities add extra chlorine and such to their water during the summer to keep it from stagnating in the heat.

[CharonY]

For cell cultures only ultra-purified water is used. What I am asking is whether the catridges and filters for the water system are still good, as they have to be cleaned/replaced in certain intervals. Another contamination source is the outlet, especially if some kind of tubing is used.

In other words, when were the filters and cartridges exchanged and/or decontaminated? Old filters can contain bacteria that might either directly contaminate the medium directly, or, what is more common, they may release pyrogens that may cause cell death.

 

Another thing might be contamination e.g. in the incubator. Most bacterial and fungal contaminations can be seen, but especially viral not. It is not very typical, but somewhat possible (and also somewhat dangerous.).

Edited July 14, 2008 by CharonY

[cellbioS]

What sort of water source do you use? (I have no idea what a lab might use.) If tap water is used at any point, remember that some municipalities add extra chlorine and such to their water during the summer to keep it from stagnating in the heat.

 

We only use di-water that is brought in from a supplier since we have no plumbing in the lab. Our buffers are already hydrated so it's not an issue anyway. The only thing I use the di-water for is to keep the incubator hydrated.

[CharonY]

Then the last idea I have is contamination in the incubator or maybe even in the biosafety bench. As the rest you have are disposables. (Unless the supplier changed something in their medium. But I assume that you buy a standard medium for the cells).