Comparison of a Rat Primary Cell-Based Blood-Brain Barrier Model With Epithelial and Brain Endothelial Cell Lines: Gene Expression and Drug Transport

Abstract

Cell culture-based blood-brain barrier (BBB) models are useful tools for screening of CNS drug candidates. Cell sources for BBB models include primary brain endothelial cells or immortalized brain endothelial cell lines. Despite their well-known differences, epithelial cell lines are also used as surrogate models for testing neuropharmaceuticals. The aim of the present study was to compare the expression of selected BBB related genes including tight junction proteins, solute carriers (SLC), ABC transporters, metabolic enzymes and to describe the paracellular properties of nine different culture models. To establish a primary BBB model rat brain capillary endothelial cells were co-cultured with rat pericytes and astrocytes (EPA). As other BBB and surrogate models four brain endothelial cells lines, rat GP8 and RBE4 cells, and human hCMEC/D3 cells with or without lithium treatment (D3 and D3L), and four epithelial cell lines, native human intestinal Caco-2 and high P-glycoprotein expressing vinblastine-selected VB-Caco-2 cells, native MDCK and MDR1 transfected MDCK canine kidney cells were used. To test transporter functionality, the permeability of 12 molecules, glucopyranose, valproate, baclofen, gabapentin, probenecid, salicylate, rosuvastatin, pravastatin, atorvastatin, tacrine, donepezil, was also measured in the EPA and epithelial models. Among the junctional protein genes, the expression level of occludin was high in all models except the GP8 and RBE4 cells, and each model expressed a unique claudin pattern. Major BBB efflux (P-glycoprotein or ABCB1) and influx transporters (GLUT-1, LAT-1) were present in all models at mRNA levels. The transcript of BCRP (ABCG2) was not expressed in MDCK, GP8 and RBE4 cells. The absence of gene expression of important BBB efflux and influx transporters BCRP, MRP6, -9, MCT6, -8, PHT2, OATPs in one or both types of epithelial models suggests that Caco-2 or MDCK models are not suitable to test drug candidates which are substrates of these transporters. Brain endothelial cell lines GP8, RBE4, D3 and D3L did not form a restrictive paracellular barrier necessary for screening small molecular weight pharmacons. Therefore, among the tested culture models, the primary cell-based EPA model is suitable for the functional analysis of the BBB.

Simvastatin, edaravone and dexamethasone protect against kainate-induced brain endothelial cell damage

“…The tightness of the BBB model was tested by the measurement of P app for marker molecules fluorescein (4.72 × 10 −6 cm/s) and albumin (0.31 × 10 −6 cm/s) which were in the range we measured in previous studies [29,41]. Kainate treatment (100 μM) significantly increased the permeability of the BBB model for both markers at the 1 and 24 h time points (Fig.…”

Cell-based, animal and H1 receptor binding studies relative to the sedative effects of ketotifen and norketotifen atropisomers

“…Although RBE4 cells express both P-gp [28] and BCRP, [29] the latter is reported to be expressed at a very low level. [52] In addition, MRP 1, 3 and 5 have been found at much lower levels in RBE4 cells than in Caco-2 cells. Moreover, P-gp is reported to be overexpressed in Caco-2 cells [38] making their monolayers suitable for screening of efflux transporter substrates.…”

“…The contrast between the predominance of influx in RBE4 cell uptake and efflux in Caco‐2 monolayer permeability may be due to differences between endothelial and epithelial cells in transporter expression. Although RBE4 cells express both P‐gp and BCRP, the latter is reported to be expressed at a very low level . In addition, MRP 1, 3 and 5 have been found at much lower levels in RBE4 cells than in Caco‐2 cells.…”

A pump‐free tricellular blood–brain barrier on‐a‐chip model to understand barrier property and evaluate drug response

“…We used primary cells rather than immortalized cell lines that are commonly used to construct in vitro BBB models because most microvascular cell lines only exhibit limited facets of the endothelial phenotype and have limited responses to proinflammatory stimuli (Unger, Krump‐Konvalinkova, Peters, & Kirkpatrick, 2002). Primary cells have a more significant response toward cytokine stimulation as well as the formation of vessel‐like structures in vitro (Unger et al, 2002; Veszelka et al, 2018).…”

“…In vitro coculture of primary ECs, pericytes, and astrocytes have been established as a useful tool for BBB model. (Fazakas et al, 2011; Thomsen, Burkhart, & Moos, 2015; Veszelka et al, 2018). Various pathological conditions in the CNS, including cerebral ischemia and neuroinflammation, can compromise the tightness of the BBB, allowing large proteins to leak out and blood cells to enter into the brain tissue (Abbott, 2013; Daneman & Prat, 2015).…”