Human dermal microvascular endothelial cells: an improved method for tissue culture and a description of some...

Tissue culture of human large vessel endothelium is now routine in many laboratories but tissue culture of human microvascular endothelium remains a difficult procedure, preventing study of features of endothelial function that may be peculiar to the microvasculature. This report describes an improved method for tissue culture of human dermal microvascular endothelium derived from foreskin. The method is rapid, reproducible, avoids contamination with nonendothelial cells, and does not require the use of a tumor-conditioned medium. The major modifications over existing techniques are the use of a Percoll density gradient to remove the majority of nonendothelial cells followed by a simplified weeding procedure that removes residual nonendothelial cells and leaves large numbers of endothelial cells to grow rapidly to confluence. The cells are identified as endothelial by their morphology and by positive immunofluorescence for Factor VIII. Proliferation experiments demonstrate their requirement for an exogenous matrix and for a high concentration of human serum. Whole serum was required as platelet-poor plasma serum had poor growth stimulatory activity. Proliferation could be enhanced by dibutyryl cyclic AMP or endothelial cell growth substance and was maximal with the combination of endothelial cell growth substance and heparin. However, the use of these agents did not remove the requirement for an exogenous matrix. Fibroblast growth factor, platelet-derived growth factor, epidermal growth factor, nerve growth factor, and thrombin did not increase proliferation.