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

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 cm 2) and reduced paracellular mannitol permeability (8.06%/h), indicating improved BBB properties; however, the barrier properties of porcine BCECs (TEER 1650 Ω cm 2 ; 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.

Similarities and differences in the localization, trafficking, and function of P-glycoprotein in MDR1-EGFP-transduced rat versus human brain capillary endothelial cell lines

“…TEER values were comparable in all cell lines, reaching maximal values of ~ 13–16 Ω cm 2 when measured with an EndOhm chamber. For hCMEC/D3 cells, these low TEER values were comparable to values reported in the literature when using an epithelial Volt-Ohm meter (EVOM) coupled to an EndOhm chamber for TEER measurements [ 43 , 47 ], whereas TEER measurements with chopstick electrodes result in significantly higher values [ 21 , 43 , 48 ]. When TEER was measured with an EndOhm chamber in primary rBCECs, values were in the range of 80–140 Ω cm 2 (Fig.…”

“…In the present study, the CETA assay was used to determine the flux of the radiolabeled Pgp substrate [ N -methyl- 3 H]N-desmethyl loperamide ([ 3 H]dLop) across hCMEC/D3- MDR1 -EGFP and RBE4- MDR1 -EGFP monolayers. Although Kannan et al [ 42 ] have shown that [ 3 H]dLop is trapped in lysosomes, this does not restrict its use for studying Pgp-mediated vectorial drug transport in BCECs, as demonstrated recently by us in primary cultured porcine BCECs [ 43 ]. Cells were grown on permeable ThinCert® filter supports (12-well, 0.4 µm pore size, PET, Greiner Bio-One).…”

Endothelial genetic deletion of CD147 induces changes in the dual function of the blood‐brain barrier and is implicated in Alzheimer’s disease

“…Abcb1 , which encodes MDR1/P‐gp, is involved in BBB transporter function and Aβ clearance in the brain 31,32 . As brain uptake is often restricted by the active efflux transporter ABCB1, we evaluated the effect of CD147 ablation on ABCB1 by Rho123 uptake assay 33 . We found a reduction in Rho123 accumulation in BECs isolated from EC‐KO mice (Figure 5E,F), confirming that endothelial CD147 deficiency resulted in an increase in ABCB1 expression.…”

Development of Real-Time Transendothelial Electrical Resistance Monitoring for an In Vitro Blood-Brain Barrier System

“…Moreover, the TEER values measured in vivo have been reported to be as high as 5900 Ω·cm 2 [ 33 ]. Even the human brain endothelial cell lines (hCMEC/D3) used in co-culture with astrocytes have yielded relatively low TEER value and less than 500 Ω·cm 2 TEER value is measured in currently available in vitro models [ 34 , 35 , 36 , 37 , 38 , 39 , 40 ]. In this study, we demonstrated that a microfluidic platform can allow the exposure of cells to dynamic conditions that provide 5 dyne/cm 2 shear stress simulation.…”