Human macrovascular endothelial cells: Optimization of culture conditions

The purpose of this study is to identify optimal culture conditions to supp ort the proliferation of human macrovascular endothelial cells, live cell l ines were employed: human saphenous vein endothelial cells (HSVEC) and huma n umbilical vein endothelial cells (HUVEC). The influence of basal nutrient media (14 types), fetal bovine serum (FBS), and mitogens (three types) wer e investigated in relation to cell proliferation. Additionally, a variety o f extracellular matrix (ECM) substrate-coated culture dishes were also test ed. The most effective nutrient medium in augmenting cell proliferation was MCDB 131. Compared to the more commonly used M199 medium, MCDB 131 resulte d in a 2.3-fold increase in cell proliferation. Media containing 20% FBS in creased cell proliferation 7.5-fold compared to serum-free media. Among the mitogens tested, heparin (50 mu g/ml) and endothelial cell growth suppleme nt (ECGS) (50 mu g/ml) significantly improved cell proliferation. Epithelia l growth factor (EGF) provided no improvement in cell proliferation. There were no statistical differences in cell proliferation or morphology when en dothelial cells were grown on uncoated culture plates compared to plates co ated with ECM proteins fibronectin, laminin, gelatin, or collagen types I a nd IV. The culture environment yielding maximal HSVEC and HUVEC proliferati on is MCDB 131 nutrient medium supplemented with 2 mM glutamine, 20% FBS, 5 0 mu g/ml heparin, and 50 mu g/ml EGGS. The ECM substrate-coated culture di shes offer no advantage.