HEK 293 cell culture cytotoxicity? - (Nov/19/2007 )

Hi,

I'm a newbie working with HEK cells. I've been able to get some good results for a while now, but I have had a recurrent problem for several weeks.

Basically, after a passage, I plate my cells as I would normally in flasks, 200-300k cells/mL. About 2 days after plating, the culture medium appears cloudy and turbid, without having changed in color. It looks like a milky cloud floating on top of the cells, getting denser where the cells are more confluent. At that point, if I look at the cells, they are normal appart from a little bit of cell death and some little black irregular debris floating around. As the days go by, the amount of debris increases and so does the turbidity, and my cells start to detatch and die about 4 days after passage. If I remove the medium and replace it with some fresh one, the turbidity comes back within hours. Oh, and one more detail: the turbid medium absolutely stinks...

So first I thought it was contamination. Looking and asking around, the closest hypothesis was yeast contamination. However, I don't see anything that looks like yeast in the culture, just those small black debris. I've tried to work with some other cells newly thawed, but the problem reappeared at the very first passage. I tried serum-depriving those cells, and it seems to potentiate the problem. Has anyone encountered such a problem before?

Help would be very much appreciated!

Thanks

It must be contamination, bacterial. probably the media or some other culture stuff is contaminated. Throw all old media and get fresh ones or makes new media.

Or maybe the frozen stocks are contaminated.

If you want to check the medium, put some ml in a flask and place it in a 37°C incubator. Check after one day if it looks cloudy or normal.