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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

-Anand Krishnan-

QUOTE (Anand Krishnan @ Oct 2 2006, 03:26 PM)
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


only 20% DMEM should kill your cells ┬┤cause of hypo osmolarity

-The Bearer-

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.

-Anand Krishnan-

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

-The Bearer-

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

Thank you

-Anand Krishnan-