The blood-brain barrier (BBB) is unique because it has selective permeability (which also changes in some diseased conditions). The BBB is a complex system made up of a structurally distinct, continuous endothelial cell layer separating the blood from the extracellular fluid of the brain. The luminal plasma membrane of the endothelial cells is directed towards the blood, while the abluminal plasma membrane faces the brain. The presence of adhesion molecules and tight junctions between endothelial cells and the low density of pinocytes are some of the key structural features that make the BBB selectively permeable.
Fig.1 The multicellular structure of the blood-brain barrier (BBB)/neurovascular unit (NVU). (Daneman, 2015)
The BBB is responsible for physiologically protecting the brain from exposure to toxins and ill effects. However, this physiological protective function of the BBB presents a key challenge to the pharmaceutical fraternity, with a need to circumvent it to deliver drugs to the brain in various CNS disorders. While actual or potential routes across the BBB are under rigorous control to maintain the homeostatic functions of the barrier, many of them are altered or disturbed in pathology. Hence therapeutic delivery strategies need to consider both the possible routes for the transendothelial movement of therapeutics and how these routes are modified pathologies. CNS drugs should possess specific physicochemical properties, which allow them to penetrate the blood-brain barrier.
Endothelial cells plated on synthetic materials have long been crafted for simulation of the BBB. Simple in vitro models have utilized endothelial cells plated in a monolayer to evaluate junctional integrity. Yet more sophisticated studies have utilized neurons, stem cells, microglia, lymphocytes, astrocytes, and/or pericytes, in addition to endothelial cells, to enhance paracellular BBB integrity for various analysis platforms.
Co-cultured cells in extracellular matrix (ECM) protein solutions, such as collagen I, laminin, fibronectin, and/or matrigel have assisted in evaluating the complexity of BBB architecture for three-dimensional modeling. While the brain consists mainly of neurons and glial cells, 10 to 25% of the total volume is ECM, which includes hyaluronic acid, proteoglycans, tenascins, link proteins, and laminin. The combination of these ECM components allows for ample support of neurons, glia, and endothelial-pericytes among a loose meshwork of soft material that is suitable for growth, migration, and extension. The use of these rich EC matrices enables observations of the adjacent interaction among BBB cells pre- and post-therapy in a three-dimensional culture setting, often with the formation of capillary-like tubes formed by adjacent endothelial cells.
Dynamic microfluidic systems, such as BBB-on-a-chip, enhance the co-culture models mentioned above by incorporating realistic geometries and physiological fluid flow. Like normal physiologic state, these dynamic systems allow for intraluminal pressure and shear stress to produce capillary-like models with low permeability, high TEER, and expression of relevant transporters and efflux proteins. These microfluidic models consist of a cylindrical chamber made of a porous cell culture substrate with a micropump, which allows media to flow through and around the chamber(s). Endothelial cells are seeded on the luminal surface and other neurovascular unit cells can be seeded on the abluminal surface.
In recent years, three-dimensional printing has emerged as an alternative manufacturing process. The 3D printing approach allows faster and cheaper fabrication, built-in assay technology, and greater flexibility in design. These microfluidic devices enable dynamic evaluation of BBB integrity, permeability, and cell invasion through real-time measurements of TEER, dye permeability, and fluorescent cell imaging. These real-time readouts are advantageous for studying drug permeability across the BBB, dye extravasation in response to drug screening, and cell invasion by immune cells or tumor cells. When coupled with the co-culture of iPSC-derived endothelial cells, pericytes, astrocytes, and neurons, these microfluidic devices closely resemble physiologic BBB.
|BBB model||Model subtype||Applications/ biological relevance||Advantages||Limitations|
|Polycarbonate or polyethylene terephthalate membrane, varied pore size and pore density||Cell permeability/extravasation assays, junctional protein fluorescence expression, TEER||Short time assay for permeability assay, low-cost, co-cultured cells, high-throughput screening||Static system, lack of luminal shear stress, poor endothelial cell differentiation, and adaptation to microenvironmental cells|
|Matrigel, hyaluronic acid, laminin, collagen I, fibronectin, scaffold matrix||Evaluation of close interactions between cells, cross-signaling, angiogenesis, gene expression||Low cost, co-cultured cells||Static system, unable to perfuse vasculature, unable to utilize for high-throughput screening, contains major cell-derived matrices but lacks minor ECM proteins/factors|
|Microfluidic devices||Physiologic representation of cell invasion, dye extravasation, drug sensitivity, cell growth, drug permeability, gene expression||Dynamic model of cell-cell interaction, high TEER values, co-cultured cells, cell imaging, and permeability evaluable in real-time, shear stress||Prolonged time for assay development and permeability assessment, expensive, time-consuming, technical expertise necessary, not suitable for high-throughput screens|
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