Comparative effects of trypsin, collagenase and mechanical harvesting on cell membrane lipids studied in monolayer-cultured endothelial cells and a green monkey kidney cell line.

Experiments are presented in which membrane lipids of endothelial cells in monolayer culture were labelled with [14C]linoleic acid. Approx. 90% of the radioactive label were incorporated into phospholipids. A comparison of various harvesting methods showed that during the disruption of the labelled endothelial cell monolayer, 0.25% trypsin and 0.125% trypsin (+0.01% EDTA) released 650 and 470% more radioactivity, respectively, than did 0.01% collagenase (+0.01% EDTA). Parallel studies were performed on a green monkey kidney cell line. In this case, 0.25% trypsin released 520% more radioactivity than did 0.1% collagenase (+0.01% EDTA), although 0.125% trypsin in the presence of EDTA (0.01%) was much less traumatic than trypsin alone, the released radioactivity being of the same order of magnitude as that for collagenase. Morphological studies on endothelial cell cultures failed to reveal any distinctive differences in surface morphology following the various enzyme treatments. The results suggest that collagenase treatment of endothelial cell monolayers is the least traumatic harvesting or subculturing method as far as the integrity of the lipids in the cell membrane is concerned.