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Establishment of a simplified in vitro porcine blood-brain barrier model with high transendothelial electrical resistance.

ABSTRACT: Good in vitro blood-brain barrier (BBB) models that mimic the in vivo BBB phenotype are essential for studies on BBB functionality and for initial screening in drug discovery programmes, as many potential therapeutic drug candidates have poor BBB permeation. Difficulties associated with the availability of human brain tissue, coupled with the time and cost associated with using animals for this kind of research have led to the development of non-human cell culture models. However, most BBB models display a low transendothelial electrical resistance (TEER), which is a measure of the tightness of the BBB. To address these issues we have established and optimised a robust, simple to use in vitro BBB model using porcine brain endothelial cells (PBECs). The PBEC model gives high TEER without the need for co-culture with astrocytes (up to 1300 O cm(2) with a mean TEER of ~800 O cm(2)) with well organised tight junctions as shown by immunostaining for occludin and claudin-5. Functional assays confirmed the presence of high levels of alkaline phosphatase (ALP), and presence of the efflux transporter, P-glycoprotein (P-gp, ABCB1). Presence of the breast cancer resistance protein (BCRP, ABCG2) was confirmed by TaqMan real-time RT-PCR assay. Real-time RT-PCR assays for BCRP, occludin and claudin-5 demonstrated no significant differences between batches of PBECs, and also between primary and passage 1 PBECs. A permeability screen of 10 compounds demonstrated the usefulness of the model as a tool for drug permeability studies. Qualitative and quantitative results from this study confirm that this in vitro porcine BBB model is reliable and robust; it is also simpler to generate than most other BBB models. This article is part of a Special Issue entitled Electrical Synapses.

SUBMITTER: Patabendige A

PROVIDER: S-EPMC3694297 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Establishment of a simplified in vitro porcine blood-brain barrier model with high transendothelial electrical resistance.

Patabendige Adjanie A Skinner Robert A RA Abbott N Joan NJ

Brain research 20120710

Good in vitro blood-brain barrier (BBB) models that mimic the in vivo BBB phenotype are essential for studies on BBB functionality and for initial screening in drug discovery programmes, as many potential therapeutic drug candidates have poor BBB permeation. Difficulties associated with the availability of human brain tissue, coupled with the time and cost associated with using animals for this kind of research have led to the development of non-human cell culture models. However, most BBB model ...[more]

PMID: 22789905

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Project description:The blood-brain barrier (BBB) is an active and complex diffusion barrier that separates the circulating blood from the brain and extracellular fluid, regulates nutrient transportation, and provides protection against various toxic compounds and pathogens. Creating an in vitro microphysiological BBB system, particularly with relevant human cell types, will significantly facilitate the research of neuropharmaceutical drug delivery, screening, and transport, as well as improve our understanding of pathologies that are due to BBB damage. Currently, most of the in vitro BBB models are generated by culturing rodent astrocytes and endothelial cells, using commercially available transwell membranes. Those membranes are made of plastic biopolymers that are nonbiodegradable, porous, and stiff. In addition, distinct from rodent astrocytes, human astrocytes possess unique cell complexity and physiology, which are among the few characteristics that differentiate human brains from rodent brains. In this study, we established a novel human BBB microphysiologocal system, consisting of a three-dimensionally printed holder with a electrospun poly(lactic- co-glycolic) acid (PLGA) nanofibrous mesh, a bilayer coculture of human astrocytes, and endothelial cells, derived from human induced pluripotent stem cells (hiPSCs), on the electrospun PLGA mesh. This human BBB model achieved significant barrier integrity with tight junction protein expression, an effective permeability to sodium fluorescein, and higher transendothelial electrical resistance (TEER) comparing to electrospun mesh-based counterparts. Moreover, the coculture of hiPSC-derived astrocytes and endothielial cells promoted the tight junction protein expression and the TEER value. We further verified the barrier functions of our BBB model with antibrain tumor drugs (paclitaxel and bortezomib) and a neurotoxic peptide (amyloid ? 1-42). The human microphysiological system generated in this study will potentially provide a new, powerful tool for research on human BBB physiology and pathology.

Dataset Information

Establishment of a simplified in vitro porcine blood-brain barrier model with high transendothelial electrical resistance.

Publications

Establishment of a simplified in vitro porcine blood-brain barrier model with high transendothelial electrical resistance.

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