Isolation and culture of microvascular endothelial cells.

The cultivation of endothelial cells from large vessels, predominantly from human umbilical veins (1,2), has become a routine procedure in many laboratories and has contributed to the development of modern vascular biology. However, there is convincing evidence that microvascular endothelial cells display a number of important functional differences, compared to large vessel-derived endothelial cells (3), in particular, with regard to their growth factor response (4,5) and their regulation of adhesion molecule expression (6-8). Since endothelial cells involved in the pathogenesis of tumor angiogenesis, wound healing, and acute and chronic inflammation are predominantly of microvascular origin, techniques have been developed to isolate endothelial cells from small vessels, most frequently from the skin (5,9-13). The culture of human dermal microvascular endothelial cells (HDMEC) has remained problematic because of difficulties in cell isolation, low cell yields, and short lifespans of the isolated cells. In particular, potential contamination of HDMEC cultures with fibroblasts required time-consuming density-gradient centrifugations (5,12) or mechanical removal of fibroblasts (10), and remained problematic after several cell passages. We established a simplified protocol that allows the rapid and reliable immunomagnetic isolation of a well characterized, 100% pure population of HDMEC from neonatal human foreskins. This technique is based on the endothelial cell-specific induction of E-selectin by tumor necrosis factor-alpha (TNF-α) (14), predominantly in postcapillary venule endothelial cells (15), and selection of E-selectin-expressing cells by Dynabeads coupled with an anti-E-selectin monoclonal antibody.