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 endothelial cells (CPAE), different culture media, support and extracellular matrices have been compared. Cell biomass was estimated by protein assay using Lowry's method. Different constituents including albumin, hydrocortisone, insulin, transferrin, triiodothyronine, Epidermal Growth Factor (EGF) and Fibroblast Growth Factor were separately added to Basal Defined Medium (BDM). Among them, BDM supplemented with EGF (1 ng per ml), hydrocortisone (100nM), insulin (1 microgramme per ml), triiodothyronine (2 nM) and linoleic acid complexed to albumin (10 microgrammes per 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 per cent Fetal Bovine Serum (FBS). Several supports have then been tested including tissue culture polystyrene (as a reference), teflon, polycarbonate or poly(ethylene terephtalate) track-etched membranes. Among them, cell cultivated on surface treated membranes in poly(ethylene terephtalate) 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 terephtalate) 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 per cent FBS and on poly(ethylene terephtalate) membranes. Among fibronectin, matrigel TM (solubilized tissue basement membrane), laminin, collagen (type I) and polylysine; matrigel TM appeared to be the optimal extracellular matrix. In conclusion, bovine pulmonary endothelial cell cultures can be optimized in a serum-free medium and on microporous membranes with matrigel TM as an extracellular matrix without alterations of cell morphology.