Loss of cell viability in primary culture - (Oct/02/2006 )

Hai,

I have been trying to establish primary culture in my lab .Now i am facing some problems in culturing the cells.I have tried with breast cancer tissue using dispase (2units/ml) for 14 hours in cold and also tried cold trypsnisation method( 0.3% under cold condition for 14 hours follwed by heating for another 20 minutes). I got good number of cells in both the ways.Cells are attaching after 24 hours but its not growing further.I am using 20% DMEM with additional growth factors. What may be the problem.Is anything further i have to do for he good nourishment of cells.

These procdure were less effective when I use cervical and oral cancer tissues.I mean the cell density were also very low with those tissues.Should I use any other alternate procedure for those tissues.

Can anyone help me resolving these problems as i have a very limited access to the tissues

by quuoating 20% DMEM what I mean is that DMEM containing 20% serum and I think that concentration works well while we thaw the cells from freezing medium and wherever cell needs more nourishment to grow.

when we do epithelial primary culture, we do it by a general protocol with collaganease + pronase; we found that pronase alone will do; I am not familiar with your type of cells but what I can tell is that concentration and incubation time are very critical; moreover, we were faced with problems of different charges of the the same protease of the same supplier; for us, it took a while to optimize incubation time and protease concentration; use the lowest possible amount of protease and the shortest possible incubation time; after isolation, offer an optimum ECM, f.i. a mixture such as Matrigel

Thank you very much. We are also thinking the same way to reduce the incubation time.

Thank you