A NOVEL TECHNIQUE FOR CULTURE OF HUMAN DERMAL MICROVASCULAR ENDOTHELIAL-CELLS UNDER EITHER SERUM-FREE OR SERUM-SUPPLEMENTED CONDITIONS - ISOLATION BY PANNING AND STIMULATION WITH VASCULAR ENDOTHELIAL GROWTH-FACTOR

Several physiological and pathophysiological events involving vascular endothelium occur at the microvascular level. Studies on human microv asculature require homogenous primary cultures of microvascular endoth elial cells. However, procedures available for isolating and culturing human dermal microvascular cells (HDMEC) result in significant contam ination with fibroblasts. To eliminate contamination with fibroblasts or other cells, we developed a procedure to isolate HDMEC from neonata l human foreskin by panning the cells using EN4, an anti-endothelial c ell monoclonal antibody. Fanned cells uniformly expressed von Willebra nd factor and CD36, confirming their microvascular endothelial charact eristics, whereas cells cultured without panning showed a significant degree of contamination with fibroblasts. In the presence of vascular endothelial growth factor (VEGF), HDMEC could be cultured under serum- free conditions. VEGF stimulated the growth of HDMEC in a dose-depende nt manner in serum-free medium or in media supplemented with either hu man serum or newborn calf serum. Since differences exist between large vessel endothelial cells and microvascular endothelial cells, we comp ared the response to VEGF stimulation of HDMEC with human umbilical ve in endothelial cells (HUVEC). The dose response of the two cell types to VEGF was different. This effect of VEGF on endothelial cells may be mediated by the VEGF receptor kdr, since mRNA for kdr was detected us ing RT-PCR in both HDMEC and HUVEC. The procedure described in this st udy will make possible the culture of highly enriched HDMEC without co ntamination with fibroblasts and facilitate studies with these cells u nder defined assay conditions in a serum-free environment. (C) 1997 Ac ademic Press.