HEK 293 Problem Adherence - (Jan/03/2007 )

I am trying to grow HEK293 cells and they are not adhering to the flask. I am using tissue culture treated flasks, I also tried gelatin and still no adherence. Does anyone have any suggestions? They cells are just growing in clumps but not forming a monolayer.

Try the following: — First, pool the floating and adherent cells in a tube and centrifuge at 1000 rpm for 5 min. — Then, replate the cells on collagen coated plates (Becton Dickinson Cat. No. 354401: 60 mm; Cat. No. 354450: 100 mm). — Use these plates for the first few passages. When the cell line's adherence improves, begin passaging cells on regular plates.

But you do know that 293 cells are only semiadherent, right??? It happened to my colleague that she wasn't too gentle when she got the flask out of the incubator and was always wondering why she had big chunks that weren't adhering.

Just one stupid other thing: did you make sure that when you were thawing them (which i think you did) that you did that quickly enough so that they weren't harmed by the DMSO in the freezing medium???

When 293 cells are allowed to become overgrown, they loose some of their adherence. Then are also very sensitive to residual DMSO after thwoing, so make sure you do a wash after a thaw.