293T adherence problems - (May/27/2008 )

Hi guys,

I recently received a batch of 293T cells stabily expressing a vector containing an MLH1 minigene under the control of a TetOff promoter system. However, i'm having huge problems with the adherence of these cells. I know this is a common problem, but the person who provided these cells has them growing fine and I cannot work out what I am doing wrong. Even after allowing the cells 2-3 days of uninterupted growth in a flask, the next time I look at them probably 90% are still in suspension. The cells are also growing extremely badly (Slow), although I attribute this to the fact they are not attaching?

Does anyone here have any advice? I've listed the things i've tried below... any ideas as to why my 293T cells aren't adhering whereas ones from the same batch in another lab are would be very appreciated!

1) Never pipette liquids directly onto cells (carefully turn flask upside down to remove and replace media)
2) General careful handling (making sure liquid does swish back and forth during transport to and from incubator)
3) Using pimeria-coated flasks/plates (there is lots of literature where these coats were used successfully with these cells)
4) Being careful to minimise their time outside incubator so as to not let the temperature drop (apparently their attachment is temperature sensitive)
5) Using PBS containing calcium and magnesium for PBS washes

The only thing I can think of as maybe affecting adherence specifically in my lab would be possible vibrations. The incubators my cells are stored in are quite heavily used and so the door is opening and shuting throughout the day. Coupled to this, the incubators are next to the main door into the tissue culture suite, and so there are some possible vibrations going on there as well.

Thanks guys!

Vibrations may be a possibility, but I would suggest that you get a vial of normal 293t cells and see if they attach ok. You must have a vector control cells anyways.

your stable cell line, although inducible, may be leaky and that may be reducing the well-being of the cells.

293 typically don't adhere very well. It may well be that the selected cells were not tested for similarity to the parent line so they have lost some attachment ability.

I would also have a look for contamination - especially mycoplasma.

I know it sounds patronising: but go back to basics and check the conditions that the cells were growing under back in the lab you got them from, someone may have been using a different medium or a different antibiotic (assuming you are keeping them under maintenance pressure so that the keep the transfected plasmid)

Also, many FCS sources have tetracycline in them (it's fed to cows to keep them healthy), which may be switching the target gene off.