A SIMPLIFIED METHOD FOR GROWTH OF HUMAN MICROVASCULAR ENDOTHELIAL-CELLS RESULTS IN DECREASED SENESCENCE AND CONTINUED RESPONSIVENESS TO CYTOKINES AND GROWTH-FACTORS

Human dermal microvascular endothelial cells are used to analyze the f unctions of microvascular endothelium in vitro. However the low yield and short lifespan of these cells in culture has limited the types of analysis that could be performed. Human microvascular endothelial cell s are typically grown in media containing supplements such as dibutyry l cyclic AMP, hydrocortisone, bovine brain extract, and antifungal age nts, each of which increase the complexity of experimental design and interpretation of results. Tn the present study, endothelial cells wer e transferred after Ulex europeus-I selection into a simplified medium consisting of Endothelial Basal Medium 131, 10% fetal bovine serum, a nd 2 ng/ml basic fibroblast growth factor and analyzed over 3 mo. The human microvascular endothelial cells expressed the endothelial marker s von Willebrand factor, CD31, P-selectin, and E-selectin. In addition , the cells showed increased proliferation in the presence of basic fi broblast growth factor (0.5 ng/ml) or vascular endothelial cell growth factor (10 ng/ml). Tumor necrosis factor-alpha-induced expression of E-selectin was similar in cells at Passages 3, 6, and 12, indicating t hat the cells maintained responsiveness to cytokines over several week s. Furthermore, the endothelial cells attained a typical cobblestone m orphology with increased cellular density and also formed capillarylik e tubes on Matrigel. In summary, the human dermal microvas cular endot helial cells display the expected endothelial characteristics when gro wn for several passages and, therefore, provide a valuable in vitro mo del for human microvascular endothelium.