METHOD FOR CULTURE OF A BOVINE PULMONARY ENDOTHELIAL-CELL STRAIN (CPAE) IN A SERUM-FREE MEDIUM AND ON MICROPOROUS MEMBRANES COATED WITH MATRIGEL(TM)

In order to optimize culture conditions of bovine pulmonary endothelia l cells (CPAE), we have compared different culture media, supports and extracellular matrices. Cell biomass was estimated by protein assay u sing Lowry's method. Different constituents including albumin, hydroco rtisone, insulin, transferrin, triiodotyronine, Epidermal Growth Facto r (EGF) and Fibroblast Growth Factor were separately added to Basal De fined Medium (BDM). Among them, BDM supplemented with EGF (1 ng/ml), h ydrocortisone (100 nM), insulin (1 mu g/ml), triiodotyronine (2 nM) an d linoleic acid complexed to albumin (10 mu g/ml) also named 'syntheti c 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(et hylene terephthalate) track-etched membranes. Among them, cells cultiv ated on surface treated membranes in poly(ethylene terephthalate) exhi bited the highest protein content with a significant increase in compa rison 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 wa s studied. Cells were cultivated in 'synthetic BDM' or DMEM supplement ed with 10% FBS and on poly(ethylene terephthalate) membranes. Among f ibronectin, matrigel(TM) (solubilized tissue basement membrane), lamin in, collagen (type I) and polylysine; matrigel(TM) appeared to be the optimal extracellular matrix.