In-vitro blood-brain barrier models for drug screening and permeation studies: an overview

Abstract

The blood-brain barrier (BBB) is comprised of brain microvascular endothelial central nervous system (CNS) cells, which communicate with other CNS cells (astrocytes, pericytes) and behave according to the state of the CNS, by responding against pathological environments and modulating disease progression. The BBB plays a crucial role in maintaining homeostasis in the CNS by maintaining restricted transport of toxic or harmful molecules, transport of nutrients, and removal of metabolites from the brain. Neurological disorders, such as NeuroHIV, cerebral stroke, brain tumors, and other neurodegenerative diseases increase the permeability of the BBB. While on the other hand, semipermeable nature of BBB restricts the movement of bigger molecules i.e. drugs or proteins (>500 kDa) across it, leading to minimal bioavailability of drugs in the CNS. This poses the most significant shortcoming in the development of therapeutics for CNS neurodegenerative disorders. Although the complexity of the BBB (dynamic and adaptable barrier) affects approaches of CNS drug delivery and promotes disease progression, understanding the composition and functions of BBB provides a platform for novel innovative approaches towards drug delivery to CNS. The methodical and scientific interests in the physiology and pathology of the BBB led to the development and the advancement of numerous in vitro models of the BBB. This review discusses the fundamentals of BBB structure, permeation mechanisms, an overview of all the different in-vitro BBB models with their advantages and disadvantages, and rationale of selecting penetration prediction methods towards the critical role in the development of the CNS therapeutics.

Keywords: BBB; BMECs; CNS; TJs; blood-brain barrier; brain microvascular endothelial cells; central nervous system; iPSCs; in-silico prediction methods; induced pluripotent cells; proteins; tight junctions.

Figure 1

Structure and functionality of the Blood-Brain Barrier (BBB): (A) Brain structure- The brain has several barriers, including the BBB, the outer blood-cerebrospinal fluid (CSF)–brain barrier, and the blood–CSF barrier; (B) BBB structure- The BBB is formed by endothelial cells (ECs) that are in close association with astrocyte end feet and pericytes, forming a physical barrier; (C) BBB transport- Routes for molecular traffic across the BBB are shown. Some transporters are energy-dependent (P-glycoprotein, P-gp) and act as efflux transporters; (D) Tight junctions- Tight junctions are typically located on the apical region of ECs. The tight junctions form complex networks that result in multiple barriers that restrict the penetration of polar drugs into the brain.,

Figure 2

Schematic representation of mechanisms available for drugs transport across the BBB: Schematic shows the main mechanism behind the drugs or small molecule transport across the BBB ie receptor-mediated transcytosis; adsorptive transcytosis (passive transport), diffusion or active transport. Notes: Reprinted from Adv Drug Deliv Rev, 103, Nair M, Jayant RD, Kaushik A, Sagar V., Getting into the brain: potential of nanotechnology in the management of NeuroAIDS, 202–217, Copyright 2016, with permission from Elsevier.

Figure 3

Schematic representation of different in vitro BBB models: (A) Configurations for in vitro static BBB Models using brain capillary endothelial cells (BCECs) (i) Monolayer models: are constructed using BCECs on the upper side of microporous semipermeable membrane (transwell), (ii) Non-contact co-culture: Astrocytes seeded at the bottom of the culture wells with BCECs; (iii) 2D co-culture contact models: endothelial cells are grown on porous cell culture inserts and co-cultured with primary astrocytes. Reprinted from J Pharm Sci, 105(2), Tornabene E, Brodin B, Stroke and drug delivery—in vitro models of the ischemic blood-brain barrier, Page Nos.398–405, Copyright 2016, with permission from Elsevier. (B) Cone and Plate viscometer apparatus. (C) Dynamic in vitro blood–brain barrier (DIV-BBB) model: The endothelial cells (ECs) are cultured inside the fibronectin-coated surface of hollow fibers made up of polypropylene. This system allows co-culture because astrocytes can be cultured on the outer surface of the hollow fibers. (B) and (C) adapted from J Pharm Sci, 101(4), Naik P, Cucullo L, In vitro blood-brain barrier models: current and perspective technologies, Page Nos.1337–1354, Copyright 2012, with permission from Elsevier. (D) Microfluidic-based in vitro BBB models: layered PDMS channels sandwiching a polyester membrane and the organization of b.End3 endothelial cells, pericyte, and astrocytes in the co-culture model. Reprinted with permission from Wang JD, Khafagy E-S, Khanafer K, Takayama S, ElSayed MEH. Organization of endothelial cells, pericytes, and astrocytes into a 3D microfluidic in vitro model of the blood–brain barrier. Mol Pharm. 2016;13(3):895–906. Copyright © 2016 American Chemical Society. (E) Stem cell-derived in-vitro BBB model: Undifferentiated iPSCs were differentiated simultaneously into ECs and neural cells, and then brain like ECs were purified on a selective matrix and co-cultured with astrocytes, the ECs exhibited a high TEER and formed networks of tight junctions. Abbreviations: ACM-Astrocyte-conditioned medium; BMEC- Brain microvascular endothelial cell; and TEER- Transendothelial electric resistance; iPSCs- Induced pluripotent stem cells; UM- Unconditioned medium; E6-Essential medium; EC-Endothelial cell medium supplemented with bFGF (Basic fibroblast growth factor); RA-Retinoic acid.

Figure 4

Applications of BBB models in drug discovery and development.