Isolation and growth of endothelial cells from the microvessels of the newborn human foreskin in cell culture

Abstract

A procedure for the isolation and in vitro cultivation of endothelial cells from the microvessels of the newborn human foreskin dermis is described. The epidermis was removed from foreskin tissue using a Castroviejo keratotome (0.1 mm shim). Endothelial cells were released from the dermal vessels by trypsinization of 5 mm2 sections of dermis at 37°C for 40 min. Cells were expressed into Minimal Essential Medium (MEM) containing 10% pooled human serum, collected by centrifugation and plated onto either a plain plastic or a fibronectin treated culture surface. In primary culture the rate of endothelial cell proliferation was dependent upon serum type and concentration being optimal in 50% pooled human serum. High serum concentration in combination with pretreatment of the culture surface with fibronectin was required for maximal proliferation rate, for the cells to achieve confluence and for subcultivation. Primary and subcultured cells were characterized as endothelial by light microscopic, immunofluorescent (Factor VIII associated protein) and ultrastructural (Weibel-Palade body) criteria.