Comparison of the rate of dedifferentiation with increasing passages among cell sources for an in vitro model of the blood-brain barrier.

Cell culture-based blood-brain barrier (BBB) models are useful experimental tools for developing central nervous system drugs. Several endothelial cell sources exist for BBB models, including primary cultured brain endothelial cells and immortalized cell lines. Among them, primary cell-based models are considered suitable for the functional analysis of the BBB; however, little is known about the utility of low-passage brain endothelial cells for this purpose. In this study, we investigated the effect of passage on brain endothelial cells from human, mouse and rat brain tissue as BBB models. We established in vitro BBB models using primary brain endothelial cells (Passage 1-Passage 4) from humans, mice, and rats. To analyze the effect of cell type on BBB function, we evaluated transendothelial electrical resistance (TEER) and performed immunofluorescence staining of tight junction proteins. Among the brain endothelial cell models, TEER was highest in the Passage 1 (P1) cell-based BBB model. There was no adequate increase in TEER in other low-passage cultures (P2-P4). A confluent, non-overlapping, uniform monolayer of cells in all P1 cell-based models was visible on immunostaining of tight junction proteins, whereas it was weak or undetectable in more passaged cultures. Increasing passages cultured of brain endothelial cells did not exhibit restrictive BBB function regardless of the cell source and despite culturing with pericytes and astrocytes. Among the tested culture models, only the lowest cultured cell-based models are suitable for functional analysis of the BBB.