A face-to-face comparison of claudin-5 transduced human brain endothelial (hCMEC/D3) cells with porcine brain endothelia
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Fluids and Barriers of the CNS Open Access
RESEARCH
A face‑to‑face comparison of claudin‑5 transduced human brain endothelial (hCMEC/ D3) cells with porcine brain endothelial cells as blood–brain barrier models for drug transport studies Birthe Gericke1†, Kerstin Römermann1†, Andreas Noack1†, Sandra Noack2, Jessica Kronenberg1, Ingolf Ernst Blasig3† and Wolfgang Löscher1,4*†
Abstract Background: Predictive in vitro models of the human blood–brain barrier (BBB) are essential in early drug discovery and development. Among available immortalized human brain capillary endothelial cell lines (BCECs), the hCMEC/ D3 cell line has become the most widely used in vitro BBB model. However, monolayers of hCMEC/D3 cells form only moderately restrictive barriers, most likely because the major tight junction protein, claudin-5, is markedly downregulated. Thus, hCMEC/D3 monolayers cannot be used for vectorial drug transport experiments, which is a major disadvantage of this model. Methods: Here we transduced hCMEC/D3 cells with a claudin-5 plasmid and compared the characteristics of these cells with those of hCMEC/D3 wildtype cells and primary cultured porcine BCECs. Results: The claudin-5 transduced hCMEC/D3 exhibited expression levels (and junctional localization) of claudin-5 similar to those of primary cultured porcine BCECs. The transduced cells exhibited increased TEER values (211 Ω c m2) and reduced paracellular mannitol permeability (8.06%/h), indicating improved BBB properties; however, the barrier properties of porcine BCECs (TEER 1650 Ω cm2; mannitol permeability 3.95%/h) were not reached. Hence, vectorial transport of a selective P-glycoprotein substrate (N-desmethyl-loperamide) was not observed in claudin-5 transduced hCMEC/D3 (or wildtype) cells, whereas such drug transport occurred in porcine BCECs. Conclusions: The claudin-5 transduced hCMEC/D3 cells provide a tool to studying the contribution of claudin-5 to barrier tightness and how this can be further enhanced by additional transfections or other manipulations of this widely used in vitro model of the BBB. Keywords: P-glycoprotein, Transwell, Porcine brain endothelial cells, Primary culture
*Correspondence: wolfgang.loescher@tiho‑hannover.de † Birthe Gericke, Kerstin Römermann and Andreas Noack contributed equally to this study † Ingolf Ernst Blasig and Wolfgang Löscher share equal senior authorship 1 Department of Pharmacology, Toxicology, and Pharmacy, University of Veterinary Medicine Hannover, Hannover, Germany Full list of author information is available at the end of the article
Background The blood–brain barrier (BBB) protects the brain against numerous potentially toxic compounds, but also restricts passage of most medically used drugs [1–3]. The barrier function of the BBB is primarily a result of tight junctions (TJs) between adjacent brain
© The Author(s) 2020. 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 form
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