Stable human brain microvascular endothelial cell line retaining its barrier‐specific nature independent of the passage number

Objectives: The breakdown of the blood–brain barrier (BBB) has been considered to be a key step in the disease process of a number of neuroimmunological disorders. Although several in vitro BBB models derived from human tissues have been established, no human conditionally immortalized in vitro BBB models using a temperature-sensitive SV40-T antigen (tsA58) and human telomerase reverse transcriptase (hTERT) have ever been reported. In the present study, we established a new human brain microvascular endothelial cell line harboring tsA58 and hTERT genes, and extensively characterized this new model.Methods: TY08 cells, derived from the human BBB and harboring tsA58, were infected with retroviruses possessing hTERT genes. We examined whether this new model retains its barrier-specific nature, independent of the passage number.Results: The obtained endothelial cell line, termed TY09, proliferated well under the permissive temperature and stopped growing under the nonpermissive temperature, despite the acquisition of hTERT as an additional immortalizing gene. Even with a high-passage number, the cells maintained a spindle-shaped morphology, the expression of the von Willebrand factor, tight junction proteins and transporters. Furthermore, we carried out a transendothelial transport study for TY09 cells and hCMEC/D3 cells, thereby proving that both cell lines have almost the same nature with respect to transcellular permeability of various hydrophilic and hydrophobic substances.Conclusions: The new stable conditionally immortalized TY09 cells, retaining the in vivo BBB functions, should facilitate the performance of future studies for determining the pathophysiology of various neuroimmunological diseases.