DEVELOPMENT OF IMMORTALIZED HUMAN CEREBROMICROVASCULAR ENDOTHELIAL-CELL LINE AS AN IN-VITRO MODEL OF THE HUMAN BLOOD-BRAIN-BARRIER

The objective of this study was to generate an immortal cell line repr esentative of specialized human brain microvascular endothelia forming the blood-brain barrier (BBB) in vivo, Human capillary and microvascu lar endothelial cells (HCEC) were transfected with the plasmid pSV3-ne o coding for the SV40 large T antigen and the neomycin gene, The neomy cin-resistant transfected cells overcame proliferative senescence, and after a 6-8 wk period of crisis produced immortalization-competent ce ll colonies, Single-cell clones of near-diploid genotype were isolated from these colonies, propagated, and characterized, Immortalized HCEC (SV-HCEC) exhibited accelerated proliferation rates, but remained ser um and anchorage dependent and retained the characteristic cobblestone morphology at confluence, SV-HCEC displayed a stable nuclear expressi on of SV40 large T antigen, lacked the invasiveness of transformed cel ls, and maintained major phenotypic properties of early passage contro l cells including expression of factor VIII-related antigen, uptake of acetylated low-density lipoprotein, binding of fluorescently labeled lectins, expression of transferrin receptor and transferrin receptor-m ediated endocytosis, and high activities of the BBB-specific enzymes a lkaline phosphatase and gamma-glutamyl transpeptidase, The diffusion o f radiolabeled sucrose across SV-HCEC monolayers was fivefold lower th an that observed with human lung microvascular endothelial cells, Furt hermore, media conditioned by fetal human astrocytes increased the tra nsendothelial electrical resistance of SV-HCEC monolayers by 2.5-fold, Therefore, this newly established human cell line expressing the spec ialized phenotype of BBB endothelium may serve as a readily available in vitro model for studying the properties of the human BBB.