Abstract
To identify suitable cell lines for a mimetic system of in vivo blood-brain barrier (BBB) for drug permeability assessment, we characterized two immortalized cell lines, ECV304 and bEnd3 in the respect of the tightness, tight junction proteins, P-glycoprotein (P-gp) function and discriminative brain penetration. The ECV304 monoculture achieved higher transendothelial electrical resistance (TEER) and lower permeability to Lucifer yellow than bEnd3. However, co-culture with rat glioma C6 cells impaired the integrity of ECV304 and bEnd3 cell layers perhaps due to the heterogeneity among C6 cells in inducing BBB characteristics. The immunostaining of ZO-1 delivered distinct bands along cell borders on both cell lines while those of occludin and claudin-5 were diffused and weak. P-gp functionality was only proved in bEnd3 by Rhodamine 123 (R123) uptake assay. A permeability test of reference compounds displayed a similar rank order (digoxin < R123 < quinidine, verapamil < propranolol) in ECV304 and bEnd3 cells. In comparison with bEnd3, ECV304 developed tighter barrier for the passage of reference compounds and higher discrimination between transcellular and paracellular transport. However, the monoculture models of ECV304 and bEnd3 fail to achieve the sufficient tightness of in vitro BBB permeability models with high TEER and evident immunostaining of tight junction proteins. Further strategies to enhance the paracellular tightness of both cell lines to mimic in vivo BBB tight barrier deserve to be conducted.
Highlights
The blood-brain barrier (BBB), mainly composed of endothelial cells that line brain capillaries, is characterized by the presence of tight junctions and efflux transporter systems
Primary culture of endothelial cells isolated from porcine, bovine, rodent and human can closely reproduce the in vivo BBB characteristics of tight junction and expression of efflux transporters
ECV304, C6 and bEnd3 cell lines were obtained from American Type Culture Collection (ATCC), Medium 199 (M199), Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), trypsin (0.25%)-EDTA (0.02%) solution, penicillin-streptomycin solution and Hank’s balanced salt solution (HBSS) were purchased from Hyclone (Logan, UT, USA), Lucifer yellow, quinidine, digoxin and Rhodamine 123 (R123) were purchased from Sigma Aldrich
Summary
The blood-brain barrier (BBB), mainly composed of endothelial cells that line brain capillaries, is characterized by the presence of tight junctions and efflux transporter systems. Primary culture of endothelial cells isolated from porcine, bovine, rodent and human can closely reproduce the in vivo BBB characteristics of tight junction and expression of efflux transporters. Brain capillary endothelial cell lines such as mouse bEnd, porcine PBMEC/C1-2 and human hCMEC/D3, and non-cerebral cell lines like CaCo-2, MDCK-MDR1 and ECV304 could form the tight paracellular barrier and represent popular cell lines for BBB studies [3,4,5]. Flow-based hollow-fiber models, microfluidic models and human pluripotent stem cells-derived models have been established for BBB studies. They require sophisticated expertises and are not in wide usage at present [6]
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