Abstract
BackgroundPredictive 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.MethodsHere 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.ResultsThe 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 cm2) 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.ConclusionsThe 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.
Highlights
Predictive in vitro models of the human blood–brain barrier (BBB) are essential in early drug discovery and development
Cell nuclei were counterstained with DAPI. e Length and width of hCMEC/D3 and porcine BCECs (pBCECs). f Representative Western blot showing claudin-5 (Cldn5) expression in hCMEC/D3-WT (7 days after seeding), hCMEC/ D3-Cldn5-Yellow fluorescent protein (YFP) (7 and 21 days after seeding) and pBCEC cultures (7 days after seeding). β-actin was used as a loading control. g Quantification of Western blot bands and normalization of claudin-5 expression to β-actin
Significant intergroup differences are indicated by asterisk (*P < 0.05). h Representative Western blot showing Pgp expression in hCMEC/D3-WT, hCMEC/D3-Cldn5-YFP and pBCEC cultures. β-actin was used as a loading control. i Quantification of Western blot bands and normalization of Pgp expression to β-actin
Summary
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. Numerous in vitro models of the BBB, based on primary BCECs or immortalized BCEC lines, have been developed for studying drug transport [8]. Validation of drug transport data using BCECs in more sophisticated models is usually necessary [11] In this respect, primary cultures of bovine or porcine BCECs may be used, the sequence of proteins expressed by these models differ from their human homologues, which may result in differences in drug affinities and transport rates [8]. Immortalized human BCECs can be used as an alternative [9]
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