Isolation, characterization, and long-term cultivation of porcine and murine cerebral capillary endothelial cells

We present a simple method for isolation and long-term cultivation of porcine and murine cerebral capillary endothelial cells (cEC). Two major points are made. First, that the "characteristic" morphology of the endothelial cells depends mainly on the presence of endothelial cell growth factors in the culture medium and second, that the identification of the cells as endothelial cells requires a special lectin instead of criteria used for large vessel endothelial cells, such as factor VIII staining or LDL uptake. Pure cerebral capillaries were isolated by means of a series of centrifugation steps; endothelial cells were released by collagenase treatment and cultivated on plastic petri dishes, which proved to be better for cell attachment than collagen or gelatin coating. The microvascular cells were cultivated in either the presence or absence of growth factors. Medium 199 + 10% FCS produced mainly spindle-shaped cells, growing in the "hills and valleys" pattern, which, if not passaged for weeks, showed three dimensional tubular structures. Cells of the "cobblestone" phenotype were promoted in medium 199 + 10% FCS, enriched with endothelial cell growth supplement (ECGS) and heparin (referred to as complete medium). These cells retained their phenotype for months and could be passaged up to 35 times till now. If ECGS and heparin were omitted from these cultures, the cells became elongated and resembled smooth muscle cells. This effect was reversible when the cells were transferred to complete medium. With cEC, cloned by limiting dilution, we noticed this reversal phenomenon as well. We used several markers to characterize the microvascular cells and could show that the lectin of Bandeiraea simplicifolia is a highly reliable marker for endothelial cells and that the monoclonal antibody alpha-sm-1 (anti-smooth muscle cell actin) is excellent for determining smooth muscle cells.