title: Development of immortalized human cerebromicrovascular endothelial cell line as an in vitro model of the human blood-brain barrier.
abstract: The objective of this study was to generate an immortal cell line representative of specialized human brain microvascular endothelia forming the blood-brain barrier (BBB) in vivo. Human capillary and microvascular endothelial cells (HCEC) were transfected with the plasmid pSV3-neo coding for the SV40 large T antigen and the neomycin gene. The neomycin-resistant transfected cells overcame proliferative senescence, and after a 6-8 wk period of crisis produced immortalization-competent cell 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 serum and anchorage dependent and retained the characteristic cobblestone morphology at confluence. SV-HCEC displayed a stable nuclear expression of SV40 large T antigen, lacked the invasiveness of transformed cells, and maintained major phenotypic properties of early passage control 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-mediated endocytosis, and high activities of the BBB-specific enzymes alkaline phosphatase and gamma-glutamyl transpeptidase. The diffusion of radiolabeled sucrose across SV-HCEC monolayers was fivefold lower than that observed with human lung microvascular endothelial cells. Furthermore, media conditioned by fetal human astrocytes increased the transendothelial electrical resistance of SV-HCEC monolayers by 2.5-fold. Therefore, this newly established human cell line expressing the specialized phenotype of BBB endothelium may serve as a readily available in vitro model for studying the properties of the human BBB.
authors.affiliation: Cellular Neurobiology Group, Institute of Biological Sciences, National Research Council of Canada, Ottawa, Ontario.
authors.forename: A
authors.initials: A
authors.lastname: Muruganandam
authors.forename: L M
authors.initials: LM
authors.lastname: Herx
authors.forename: R
authors.initials: R
authors.lastname: Monette
authors.forename: J P
authors.initials: JP
authors.lastname: Durkin
authors.forename: D B
authors.initials: DB
authors.lastname: Stanimirovic
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D000957:Antigens, Viral, Tumor / Q000096:biosynthesis; D001812:Blood-Brain Barrier / Q000502:physiology*; D002196:Capillaries; D002448:Cell Adhesion; D002455:Cell Division; D002460:Cell Line; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic; D002560:Cerebrovascular Circulation / Q000502:physiology*; D002999:Clone Cells; D004553:Electric Conductivity; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology*; D006801:Humans; D007444:Inulin / Q000493:pharmacokinetics; D007621:Karyotyping; D008564:Membrane Potentials; D008833:Microcirculation / Q000502:physiology*; D008954:Models, Biological; D051381:Rats; D013539:Simian virus 40 / Q000235:genetics; D013395:Sucrose / Q000493:pharmacokinetics; D014162:Transfection; D005723:gamma-Glutamyltransferase / Q000378:metabolism
chemical_list: D000957:Antigens, Viral, Tumor; D013395:Sucrose; D007444:Inulin; D005723:gamma-Glutamyltransferase; D000469:Alkaline Phosphatase