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. 2017 Sep 4;7:10396. doi: 10.1038/s41598-017-11220-1

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Setup and characterization of the in vitro model of the BBB based on primary rat BCECs and astrocytes. (A) Primary BCECs derived from young rats were setup in Transwell co-culture with astrocytes, hereby yielding a polarized layer of BCECs to be used for uptake and transcytosis experiments. (B) TEER was measured continuously to evaluate the tightness of the in vitro BBB model. The TEER values were approximately 400 Ω*cm² at the time of the experiments, and this value was stabile throughout the duration of any experiment. (C) After reaching high TEER values, the resulting tight BCEC monolayers expressed both transferrin receptors (upper panel) and the TJ-related protein, ZO-1 (lower panel). The positive staining for the transferrin receptors was found associated with the luminal membrane as well as in the cell cytoplasm, whereas the ZO-1 staining presented as a homogenous lining of the intercellular junctions, suggesting the presence of functional tight junctions. Scale bar depicts 10 µm. BBB: Blood-brain barrier. BCEC: Brain capillary endothelial cells. TEER: Transendothelial electrical resistance. DAPI: Diamino-phenylindole. TJ: Tight junction. ZO-1: Zonula occludens 1.