Method for culture of a bovine pulmonary endothelial cell strain (CPAE) in a serum-free medium and on microporous membranes coated with matrigel ™.

In order to optimize culture conditions of bovine pulmonary endothelial cells (CPAE), we have compared different culture media, supports and extracellular matrices. Cell biomass was estimated by protein assay using Lowry's method. Different constituents including albumin, hydrocortisone, insulin, transferrin, triiodotyronine, Epidermal Growth Factor (EGF) and Fibroblast Growth Factor were separately added to Basal Defined Medium (BDM). Among them, BDM supplemented with EGF (1 ng/ml), hydrocortisone (100 nM), insulin (1 mu g/ml), triiodotyronine (2 nM) and linoleic acid complexed to albumin (10 mu g/ml) also named `synthetic BDM' appeared to be the best serum-free nutritive medium and showed similar results as compared to DMEM supplemented with 10\% Fetal Bovine Serum (FBS). Several supports have then been tested including tissue culture polystyrene (as a reference), teflon, polycarbonate or poly(ethylene terephthalate) track-etched membranes. Among them, cells cultivated on surface treated membranes in poly(ethylene terephthalate) exhibited the highest protein content with a significant increase in comparison to tissue culture polystyrene, probably because cells are fed on the two faces instead of one. On treated poly(ethylene terephthalate) membranes, cells kept their endothelial morphology and ultrastructure. Finally, cell biomass on several exogenous extracellular matrices was studied. Cells were cultivated in `synthetic BDM' or DMEM supplemented with 10\% FBS and on poly(ethylene terephthalate) membranes. Among fibronectin, matrigel(TM) (solubilized tissue basement membrane), laminin, collagen (type I) and polylysine; matrigel(TM) appeared to be the optimal extracellular matrix.