RT Journal Article SR Electronic T1 Establishment of murine in vitro blood-brain barrier models using immortalized cell lines: co-cultures of brain endothelial cells, astrocytes, and neurons JF bioRxiv FD Cold Spring Harbor Laboratory SP 435990 DO 10.1101/435990 A1 Fakhriedzwan Idris A1 Siti Hanna Muharram A1 Zainun Zaini A1 Suwarni Diah YR 2018 UL http://biorxiv.org/content/early/2018/10/05/435990.abstract AB Blood-brain barrier (BBB) is a selective barrier formed by the endothelial cells that line cerebral microvessels. It serves as a physical barrier due to the presence of complex tight junctions between adjacent endothelial cells which limits the paracellular movement of most molecules across the BBB. Many in vitro models of the BBB have been established to mimic these in vivo properties with limited success. In this study, we described the properties of a cell-based murine in vitro BBB model in five configurations constructed using immortalized cell lines in a 12-well format Transwell system: murine brain endothelial cells (bEnd.3) grown in a monoculture, or as co-culture in contact with astrocytes, or without contact with astrocytes or neurons, and triple co-culture combining the three cell lines. We found that only contact and triple co-culture model closely mimic the in vivo BBB tightness as evaluated by apparent permeability (Papp) of sucrose and albumin producing the lowest Papp values of 0.56 ± 0.16 × 10−6 cms−1 and 3.30 ± 0.51 × 10−6 cms−1, respectively, obtained in triple co-culture model. Co-culturing of bEnd.3 with astrocytes increased the expression of occludin as shown by western blot analysis, and immunohistochemistry showed an increase in peripheral localization of occludin and claudin-5. In addition, we found conditioned media were able to increase in vitro BBB model tightness through the modulation of tight junction proteins localization. We conclude that the presence of astrocytes and neurons in close proximity to brain endothelial cells is essential to produce a tight in vitro BBB model.