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.

Basement Membrane Proteins Influence Brain Capillary Endothelial Barrier Function In Vitro

“…Most of them are based on primary culture of BCECs, although immortalized cell lines of BCECs have also been used (Roux et al, 1994;Muruganandam et al, 1997). One of the major problems of in vitro studies is the loss of blood-brain barrier properties such as low ion permeability and the activity of certain enzymes in culture (Mischeck et al, 1989;Laterra and Goldstein, 1993).…”

Glioma tropism of lentivirally transduced hematopoietic progenitor cells

“…The primary human glioma cultures were established from samples with high tumor content (20). The source and the generation of the astrocytic cell line SV-FHAS have been described (21). SN of glioma cells (SN-G) were generated in serum-free medium (SFM): 1.5x10 6 glioma cells were first seeded in serum-containing medium in T75 cell culture flasks.…”

Blood-Brain Barrier and Effectiveness of Therapy Against Brain Tumors

“…It was used to study the cytoadherence of Plasmodium falciparum-infected erythrocytes (Prudhomme et al, 1996). Human cerebral microvascular endothelial cells SV-HCEC, transfected with the plasmid pSV3-neo coding for the SV40 large T antigen, was utilized to serve as a human BBB in vitro model showing the expression of factor VIII-related antigen, the uptake of acetylated lowdensity lipoprotein, the binding of fluorescently labeled lectins, the expression of transferrin receptor, and high activities of the ALP and -GTP (Muruganandam et al, 1997). Immortalized brain capillary endothelial cell lines (TM-BBB1-5) were established from 3 transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene displaying the expression of Glut-1 and P-gp.…”