TY - JOUR
T1 - A novel technique for culture of human dermal microvascular endothelial cells under either serum-free or serum-supplemented conditions
T2 - Isolation by panning and stimulation with vascular endothelial growth factor
AU - Gupta, Kalpna
AU - Ramakrishnan, Sundaram
AU - Browne, Paul V.
AU - Solovey, Anna
AU - Hebbel, Robert P
N1 - Funding Information:
We thank the nursery room staff of the Methodist Hospital (St. Louis Park, MN) for supplying newborn human foreskin after circumcision. This work was supported by grants from the National Institutes of Health (HL 30160 and HL 55174) and by funds from the Minnesota Medical Foundation.
PY - 1997/2/1
Y1 - 1997/2/1
N2 - Several physiological and pathophysiological events involving vascular endothelium occur at the microvascular level. Studies on human microvasculature require homogenous primary cultures of microvascular endothelial cells. However, procedures available for isolating and culturing human dermal microvascular cells (HDMEC) result in significant contamination with fibroblasts. To eliminate contamination with fibroblasts or other cells, we developed a procedure to isolate HDMEC from neonatal human foreskin by panning the cells using EN4, an anti-endothelial cell monoclonal antibody. Panned cells uniformly expressed von Willebrand factor and CD36, confirming their microvascular endothelial characteristics, 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-dependent manner in serum-free medium or in media supplemented with either human serum or newborn calf serum. Since differences exist between large vessel endothelial cells and microvascular endothelial cells, we compared the response to VEGF stimulation of HDMEC with human umbilical vein 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 using RT- PCR in both HDMEC and HUVEC. The procedure described in this study will make possible the culture of highly enriched HDMEC without contamination with fibroblasts and facilitate studies with these cells under defined assay conditions in a serum-free environment.
AB - Several physiological and pathophysiological events involving vascular endothelium occur at the microvascular level. Studies on human microvasculature require homogenous primary cultures of microvascular endothelial cells. However, procedures available for isolating and culturing human dermal microvascular cells (HDMEC) result in significant contamination with fibroblasts. To eliminate contamination with fibroblasts or other cells, we developed a procedure to isolate HDMEC from neonatal human foreskin by panning the cells using EN4, an anti-endothelial cell monoclonal antibody. Panned cells uniformly expressed von Willebrand factor and CD36, confirming their microvascular endothelial characteristics, 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-dependent manner in serum-free medium or in media supplemented with either human serum or newborn calf serum. Since differences exist between large vessel endothelial cells and microvascular endothelial cells, we compared the response to VEGF stimulation of HDMEC with human umbilical vein 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 using RT- PCR in both HDMEC and HUVEC. The procedure described in this study will make possible the culture of highly enriched HDMEC without contamination with fibroblasts and facilitate studies with these cells under defined assay conditions in a serum-free environment.
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U2 - 10.1006/excr.1996.3421
DO - 10.1006/excr.1996.3421
M3 - Article
C2 - 9024783
AN - SCOPUS:18844470284
SN - 0014-4827
VL - 230
SP - 244
EP - 251
JO - Experimental Cell Research
JF - Experimental Cell Research
IS - 2
ER -