Contamination of unique cell line (eeek!) - (Oct/30/2006 )

I've recently started a new job and was left in charge of a colleague's fibroblast cell line derived from a patient sample. There are no frozen stocks of either of the cell lines I am looking after , unfortunately. He split them last Monday and they were v v v v v v v thin when I looked at them on Thursday, and not really growing. My experience is mainly with epithelial cells, but I anticipate subculturing about once a week? I was also advised that fibroblasts like to have neighbours (but not too many neighbours!) to grow. So I split some of the flasks (175cm) into smaller flasks on Thursday and on Friday I fed the remaining flasks new medium. There was also another fibroblast line that had been in the same flask since March (!) and was just being fed now and then, so I fed that too (on Thursday). I had also gotten a seperate non-fibroblast cell line out of the freezer and gave it a change of media on Friday for the weekend. This non fibroblast line is in DMEM:F12 and the others are in BEM plus Earle's salts. So different pots of media.

This morning I came in to find the cell line which had been growing in the same flask since March was totally overwhelmed by some sort of infection. There were slimy lumps and bumps all over the flask - maybe fungal? I have never seen anything like it! Obviously threw that away. The other fibroblast lines were OK-ish. Some growing but not lots even in the more concentrated flasks. Some dodgy bits floating around in the media - but nothing too terrible yet. Non-fibroblast cell line nothing wrong at all. I really don't trust them but since I have no frozen stock I don't want to throw them away. I have put the non affected cells in another part of the incubator (I only have one to use).

I am totally stumped. I routinely cultured epithelial and insect cells for 4+ years and never had anything like this happen to me. We did have mycoplasma but never a gross fungal or bacterial infection. I have cleaned everything zillions of times. paid special attention to my technique, the hood has been serviced recently etc, etc. There a few things I am not sure about though.

1) Would you expect your fibroblasts to be needing subculturing every week. I've done a bit of reading and this seems reasonable. Here they just seem to put some media on once a month or something if the cells are not growing and leave it. Seems odd to me? Especially if you are trying to grow up enough cells to extract DNA/RNA which they are.

2) Is it necessary to put water in a tray at the bottom of the incubator. I was considering this as a source of contamination, but it has sigmaclean in it now and nothing obvious growing. Wondered if I should remove it.

3) Has anyone tried to remove/contain an infection from cells? I have put a bit of fungizone on to see what happens but I understand this just contains rather than eliminates fungus. I would normally chuck them away immediately, just I have no more.

Thoughts greatly appreciated, sorry its so long!

I have just asked our technician, who is quite experienced with priamary human fibroblasts. She says, that she changes the medium of the cells once every week. As they grow very slowly, she only moves them to a bigger flask, when the small one is quite full and cells have plenty of cell-cell contacts. As to the contamination she says, that she was never able to get rid of contamination. *sorry* but advises to try remove the contamnation by multiple washing steps with PBS. And not use antibiotics, as fibroblasts don't really like that.
As to the water in the incubator: if you use sigmaclean and the water is clear it is most probably not the source of contamination. However, fibroblast cultures often get contaminated, because they are left for long periods in the same flask, due to the slow growth. So don't blame yourself. It's something that happens quite often (not only in our lab, as we have checked with other labs!).

I'm sorry, I can't answer all of your questions but I'll try to give some considerations.

They are primary cells, right??? Derived from a patient sample??? Are they on feeder cells or growing on their own???

1)if they were "normal" fibroblasts that are immortalized and so on, yes, I would think they need a change of media at least once a week. However if the cells you were about to take care of weren't confluent or not even close to being confluent, I would not have split them, cause as you said-they like neighbors. And if they are primary cells, I would maybe only move them to a bigger vessel cause they grow very slow.

2)Yes it is necessary to have water in the bottom of the incubator, cause otherwise-where is the humidity to come from. If you are very unsure about the water, change it and add some inhibitors of fungi etc. But I wouldn't think it's the source of contamination. Especially not if the cells are in a flask and not in a dish.

3) other than mycoplasma, I have not tried to get rid off any contaminations, sorry.

Hope it helps at least a little bit.

Dear Rachelle,

In 30 years of growing both primary and cell lines, I have never successfully cleaned up a bacterial or fungal contamination.
Saying that there are products as you have mentioned that are aggressive agents which may. However you have to consider that these agents are NOT ONLY ANTIBACTERIAL/ANTIFUNGAL, but do themselves have other PROFOUND actions. For example Fungizone, active ingredient Amphotericin B, which in my hands helped reduced fungal contamination, BUT COMPLETELY INHIBITED THE MOLECULE I WAS INTERESTED IN.

You do need water to humidify the CO2 Incubator. By changing the water every week, completely negates the need to use products such as sigmaclean.

DO NOT USE THE SAME FLASK FOR LONG PERIODS OF TIME.... what is the point of using a flask for such a long period of time. These days flasks are very cheap.

I am sorry but the only thing to do is chuck the cells.

Thanks all for your advice! I was sure I was correct about the water - we always used it in my old lab - but they had none when I started here so I wanted to check and make sure. I'm really beginning to doubt my own abilities. Maybe I was confusing, but I didn't split the fibroblasts as such, just took them all off the bigger flask into the smaller flask to attempt to give them neighbours. The ones still in the big flasks have no close enough cells to form cell-to cell contacts with. They are growing on their own, not on feeder cells. I don't know a lot about them - apparently they were sent to us from another group and can probably be replaced that way. I know in one case they are the only source of DNA for a particular patient and they hope to extract DNA and RNA from the cells. It is not my project so I am not 100% sure what the aim is.

I am going to carry on with the fungizone this week, until the preson who is using these cells gets back and can decide what to do about it. But if they were 'my' cells I would definately throw them away now.

coming back to my fibroblast like synoviocytes.
I used penicillin streptamycin, because I was not really sure that the explants were perfectly sterile. Of course you have to check it doesn't interfer with your experiments.
Twice a week I either changed the medium (only the 2/3, I always let 1/3 of medium) or split the cells if they are confluent (they were confluent once a week).