passage of HUVEC - passage of huma  umbilical vessel endothelium (Apr/03/2002 )

I failed in the passage of HUVEC. I have used 1% gelatin to coat the culture plate; the mixture of 0.25% trypsin and 0.02% EDTA to disgest the cell ; but the cell cann't spread and grow. Who can tell me some clue? Thanks very much.

More details would help clarify the possible causes. Can you answer the following:

1) what are you culturing the cells in (serum concentration, presence of ascorbic acid etc.)? I have found with primary ligament cells that the presence of ascorbic acid in the medium immediately after isolation OR after trypsinization tends to kill them !

2) how long did you trypsinise the cells ? 0.25% trypsin is very strong. Many cell types will come off in much lower concentrations (0.05%) after 5-10minutes.
Trypsin works by chewing up cellular and ECM proteins eliminating cell-matrix adhesion. In the process it also chews up cell membrane proteins such as receptors, and I believe opens holes in the membrane itself. Consequently the longer the digestion the less happy your cells will be and the longer it take for them to recover.

3) Did you recover the cells and check whether they were alive by trypan blue exclusion. How long after seeding did you examine them ?

4) Whay are you seeding them on gelatin ?
Is the cell culture surface tissue culture polystrene, bacteriological grade polystyrene, a well insert membrane ? If it was bacteriological grade polystyrene or some of the well inserts and your gelatin coating was at fault then cells wouldn't attach.

Please note I have no experience with endothelial cells so some of these questions may be irrelevant.