293t Cell Attachment Issues - (Oct/16/2008 )

I'm really new to cell culture and I got thrown into mammalian culture recently for one of our contracts. I'm culturing the cells in order to transfect plasmid DNA to express a protein of interest. I'm having issues with cell attachment. The first time I washed one of my flasks with DPBS (W/O calcium and magnesium) every cell detached from the flask. I flipped out and called invitrogen and they said, that's just what happens with 293t cells, they don't adhere well, and that PBS without Mg and Ca begins facilitating cell detachment.

Also, when I put ANYTHING in the flasks, no matter how slow or careful I'm trying to be, some cells get thrown off the bottom of the wells. I'm doing it as slowly as I can, but it doesn't seem to be working well.

So my question is:

How do you keep 293t cells attached while still being able to wash them and pipette new media and so forth?

Please feel free to respond to anything I brought up, such as if PBS with Mg and Ca would work better (which is a whole 'nother issue, I keep getting precipitation even though I have two separate homogeneous 20x solutions, and when I mix them together, instant precip.)

Any help or advice would be appreciated.

may wash the cell with medium instead of PBS.

could you use say a 15cm plate instead of a flask, seems to work a lot better at least for me, the cells stick a lot better.

What brand of flask do you use?

I think Corning is better than Starstedt.

As invitrogen said, 293 do not attach well, but they are one of the easiest cells to culture. The best thing to do is to treat the cells really gently, dribble the media/PBS slowly down the side of the flask/well so that it is not being directly pipetted onto the cells will help some. If you are passaging your cells, you don't really need to use PBS, the trypsin will work fine on the cells so long as you have removed almost all of the medium, and the FCS in the medium you replace/dilute your cells in will neutralise the trypsin at the end.

If you are having really bad attachment problems (i.e whole flasks lifting off) then try coating the growth surface with collagen/gelatine or poly-L-lysine before adding the cells.

There are instances in which I have to wash with PBS or some kind of wash buffer, so I think that is unavoidable.

I am using Nunc flasks with some proprietary coating on them. I am currently doing a test transfection in 6 well plates (corning) and the major issue is that every time I aspirate the solutions off of the cells and then add something new a portion of the cells come off of the plate. PBS seems to be the worst culprit because I need the cells attached after I wash them. I am effectively neutralising the trypsin, I have great attachment when they're growing in the incubator, it's just when I use them for anything I lose a ton of cells.

Is there a different kind of wash buffer I can use that won't promote cell detachment, or is it worth it to try to coat the flasks with something?

I use PBS to wash and later Trypsin to detach cells. DO not add anything on cells directly. You can completely tilt the flask 90 degrees and add PBS on sides, gently rock twice and discard. 2 minutes of incubation of trypsin at 37 will detach all cells. 293Ts are very finicky so treat them very tenderly.

I'm solving my problem as I'm working through the project. The cells are very easily detached, so I'm being as ginger as I can. I'm going at meditation speed when I tilt or add media. It seems necessary and it's not all that bad, I get paid by the hour