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. 2020 Aug 26;17(1):53.
doi: 10.1186/s12987-020-00212-5.

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 Gericke  1 , Kerstin Römermann  1 , Andreas Noack  1 , Sandra Noack  2 , Jessica Kronenberg  1 , Ingolf Ernst Blasig  3 , Wolfgang Löscher  4   5
Affiliations

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 Gericke et al. Fluids Barriers CNS. .

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 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.

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; Porcine brain endothelial cells; Primary culture; Transwell.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Schematic illustration of the experimental workflow. hCMEC/D3 cells stably expressing Cldn5-YFP were generated by lentiviral transduction. HEK293T cells were used as packing cell line to produce lentiviral particles for transduction of hCMEC/D3-WT cells. At day 0, prior to experiments, pBCECs were freshly isolated from porcine brain. On the same day, pBCECs, hCMEC/D3-WT and -Cldn5-YFP cells were seeded on semipermeable membranes of two chamber devices, each in triplicates. During experiments and cultivation, medium of hCMEC/D3-Cldn5-YFP cells was supplemented with doxycycline (1 µg/mL). After seeding cells attach, proliferate and differentiate to form a continuous monolayer with tight junctions sealing the paracellular cleft between adjacent cells. Transendothelial resistance (TEER) was measured once daily until 7 days after seeding of the cells, using an EVOM Volt-Ohm meter equipped with a STX2/chopstick, consisting of a fixed pair of electrodes. For comparison, TEER of hCMEC/D3 cells was also measured using an EndOhm-6 chamber (not illustrated). At day 7 after seeding, the cells were used for measuring paracellular permeability of mannitol and transcellular drug transport using the Pgp substrate N-desmethyl-loperamide (dLop). Pgp functionality was assessed by rhodamine 123 (Rho123) uptake assay. Another set of cells was grown to analyze Cldn5 and Pgp expression by Western blot (WB) and Cldn5 localization by immunofluorescent staining (IF) 7 days after seeding. Cldn5 expression in hCMEC/D3-Cldn5-YFP cells was also analyzed 21 days after seeding. Drawings are not to scale
Fig. 2
Fig. 2
Comparison of cell morphology and localization and expression of claudin-5 and Pgp in hCMEC/D3-WT, hCMEC/D3-Cldn5-YFP and pBCECs. a Claudin-5 was indirectly stained (hCMEC/D3-WT and pBCECs) or visualized by YFP tag in the stably transduced hCMEC/D3-Cldn5-YFP cell line (for better visualization both claudin-5 and Cldn5-YFP are depicted in green). F-Actin is shown in red and cell nuclei are counterstained in blue by DAPI. b An xz scan of the cell layer revealed interendothelial (junctional) localization of claudin-5 (green) in the two cell lines and pBCECs. Scale bars: 10 µm. c Phase contrast micrographs of hCMEC/D3-WT, hCMEC/D3-Cldn5-YFP and primary pBCEC cultures. d Purity of pBCEC isolation, evaluated by fluorescent staining for the endothelial cell marker CD31 (green). Cell nuclei were counterstained with DAPI (blue). e Length and width of hCMEC/D3 and pBCECs. f Representative Western blot showing claudin-5 (Cldn5) expression in hCMEC/D3-WT (7 days after seeding), hCMEC/D3-Cldn5-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. Data are represented as mean ± SEM of n = 3 independent experiments. 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. Data are represented as mean ± SEM of n = 3 independent experiments. Significant intergroup differences are indicated by asterisk (*P < 0.05)
Fig. 3
Fig. 3
Transendothelial electrical resistance in hCMEC/D3-WT, hCMEC/D3-Cldn5-YFP and pBCECs. TEER values are shown as Ω cm2 and results are expressed as mean ± SEM of 3 replicates. Values in a and c were determined with chopstick electrodes, whereas values in b and d were determined with an EndOhm chamber. *P < 0.0001 as determined by two-way ANOVA using Bonferroni posthoc analysis
Fig. 4
Fig. 4
Pgp is functional in hCMEC/D3 cells as indicated by the effect of the Pgp inhibitor tariquidar (TQ; 0.5 µM) in the Rho123 uptake assay, in which alterations in Pgp efflux are indirectly measured by determining intracellular concentrations of the Pgp substrate Rho123. Data are shown as mean ± SEM of six experiments. Significant differences between treatments are indicated by asterisk (P < 0.0001). a Shows data from the Rho123 uptake assay in nontransduced (WT) hCMEC/D3 cells in the absence or presence of doxycycline (Dox). Doxycycline (1 µg/mL) did not alter the functionality of Pgp. Tariquidar significantly increased the uptake of Rho123 in WT cells both in the absence and presence of doxycycline to the same extent. b Shows data from the Rho123 uptake assay in transduced hCMEC/D3-Cldn5-YFP cells in the absence or presence of Dox. Again, Dox (1 µg/mL) did not alter the functionality of Pgp. Tariquidar significantly increased the uptake of Rho123 in the transduced cells both in the absence and presence of Dox to the same extent. Consistent with the similar expression of Pgp in the two cell lines (see Fig. 2i), the functionality of Pgp was comparable in hCMEC/D3-WT and hCMEC/D3-Cldn5-YFP cells
Fig. 5
Fig. 5
Concentration equilibrium transport assay (CETA) of Pgp substrate N-desmethyl-loperamide (dLop) in hCMEC/D3-WT, hCMEC/D3-Cldn5-YFP and pBCECs. Data are shown as percentage of the initial drug concentration (= 100%) in the apical and basolateral chamber of the Thincert system over time. a An increase of the drug concentration in the apical chamber and a simultaneous reduction in the basolateral chamber indicates drug transport by Pgp across the primary pBCEC monolayer. Almost no drug transport was measurable when the experiment was repeated in the presence of the Pgp inhibitor verapamil (50 µM) as a control. b, c A Pgp-mediated ([3H]dLop) transport across the hCMEC/D3 monolayer could not be observed
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