Isolation and culture of human neuromicrovascular endothelial cells for the study of angiogenesis in vitro

Neovascularization in the adult central nervous system occurs as a response to several pathophysiological conditions such as ischemia, wound repair, o r neoplasia, Endothelial cells from different blood vessel types, different organs, and different species are heterogeneous; therefore, the appropriat e cell type should be used to study specific aspects of vascular pathology. We have developed a method to isolate human cerebral microvascular endothe lial cells (CMECs) from small, freshly obtained specimens of normal brain a dherent to human arteriovenous malformations (AVMs). The isolation procedur e involves enzymatic digestions tend gradient centrifugations, yielding ove r 95% pure primary cultures. Alternative isolation methods using magnetic b eads, panning, or cloning were not superior with regard to cell purity or y ield. CMECs were identified by their immunoreactivity for vWF, CD34, EN4, b inding of Ulex europeus lectin, and uptake of DiI-Ac-LDL. They displayed ul trastructural features characteristic of blood-brain barrier endothelial ce lls and expressed GLUT-1. CMECs were subcultured; however, prolonged cultur e led to reduced culture purity, Vascular endothelial growth factor, basic fibroblast growth factor and hepatocyte growth factor/scatter factor stimul ated the directional motility of CMECs, with dose-response profiles similar to human umbilical vein endothelial cells (HUVECs). In contrast, to stimul ate proliferation, lower concentrations of growth factors tended to be nece ssary for CMECs than for the large vessel endothelial cells, CMECs formed c apillary tube-like structures in an in vitro angiogenesis assay using matri gel, This study expands the spectrum of available tissue sources for the is olation of human neuromicrovascular endothelial cells, which are essential for the in vitro study of blood-brain barrier function and cerebral angioge nesis. J. Neurosci. Res. 55:370-381, 1999. (C) 1999 Wiley-Liss, Inc.