Hypoxia-enhanced Blood-Brain Barrier Chip recapitulates human barrier function, drug penetration, and antibody shuttling properties

Abstract

The highly specialized human brain microvascular endothelium forms a selective blood-brain barrier (BBB) with adjacent pericytes and astrocytes that restricts delivery of many pharmaceuticals and therapeutic antibodies to the central nervous system. Here, we describe an in vitro microfluidic 'organ-on-achip' (Organ Chip) model of the BBB lined by induced pluripotent stem cellderived human brain microvascular endothelium (iPS-BMVEC) interfaced with primary human brain astrocytes and pericytes that recapitulates the high level of barrier function of the in vivo human BBB for at least one week in culture. The endothelium expresses high levels of tight junction proteins, multiple functional efflux pumps, and displays selective transcytosis of peptides and anti-transferrin receptor antibodies previously observed in vivo. This increased level of barrier functionality was accomplished using a developmentally-inspired induction protocol that includes a period of differentiation under hypoxic conditions. This enhanced BBB Chip may therefore represent a new in vitro tool for development and validation of delivery systems that transport drugs and therapeutic antibodies across the human BBB.The human blood-brain barrier (BBB) is a unique and selective physiological barrier that controls transport between the blood and the central nervous system (CNS) to maintain homeostasis for optimal brain function. The BBB is composed of brain microvascular endothelial cells (BMVECs) that line the capillaries as well as surrounding extracellular matrix (ECM), pericytes, and astrocytes, which create a microenvironment that is crucial to BBB function 1 . The brain microvascular endothelium differs from that found in peripheral capillaries based on its complex tight junctions, which restrict 3 paracellular transit and instead, require that transcytosis be used to transport molecules from the blood through the endothelium and into the CNS 2 . BMVECs also express multiple broad-spectrum efflux pumps on their luminal surface that inhibit uptake of lipophilic molecules, including many drugs, into the brain 3,4 . The astrocytes and pericytes provide signals that are required for differentiation of the BMVECs 5,6 , and all three cell types are needed to maintain BBB integrity in vivo as well as in vitro 7-9 . The BBB is also of major clinical relevance because dysfunction of the BBB associated is observed in many neurological diseases, and the efficacy of drugs designed to treat neurological disorders is often limited by their inability to cross the BBB 10 . Unfortunately, neither animal models of the BBB nor in vitro cultures of primary or immortalized human BMVECs alone effectively mimic the barrier and transporter functions of the BBB observed in humans [11][12][13][14] . Thus, there is a great need for a human BBB model that could be used to develop new and more effective CNS-targeting therapeutics and delivery technologies as well as advance fundamental and translational research 8,9 .

Lab-On-A-Chip for the Development of Pro-/Anti-Angiogenic Nanomedicines to Treat Brain Diseases

“…More importantly, they can be specifically functionalized to target particular pathways and sites on the BMECs. They are designed to have greater BBB permeability [2,6,9,11,17,19], stability [5,9], half-life [11,18,82,83] and shelf-life [5], high-site specific targeting [10,14,84], and a controlled load-release of drugs [18,19,21,84,85].…”

“…In addition, the cellular uptake, transport via the transcellular/paracellular routes and drug-release kinetics, needs to be evaluated. Various device geometries have been reported for brain vasculature models (Figures 2 and 3); parallel channels (PCs), with interconnecting micro-gaps, [42][43][44][45]51,56,90], hollow-microtubes (HTs) [2,47,49,50,52], and others, concentric-ring (CR) channels [54], multi-layered channels [53], hybrid-microchambers [57], and microwells [58]. Each of the above designs has its own advantage in terms of angiogenesis drug screening application.…”

“…The human brain is the primary center for our cognitive activities and is commonly recognized as the most complex organ of the human body. Due to its important role in human survival, the brain is guarded by a boney skull (elastic modulus, 2.4 ± 1.5 GPa) [1] and a robust blood-brain barrier (BBB) (Transendothelial Electrical Resistance (TEER),~5000 Ω cm 2 ) [2]. While excellent for protecting the brain, these natural barriers have also made it difficult to study and treat brain disorders, in particular, diseases that require rapid treatment.…”

“…Some of the above devices were already evaluated for drug screening [32,34,36,38,40], and a few studied the effect of NPs [35,41] and NMs [39].Microfluidic technology has also been used to model brain vasculogenesis/angiogenesis [42][43][44][45][46], BBB [2,42,[47][48][49][50][51][52][53], brain tissues [54,55], and brain angiogenesis-related cellular events such as inflammation [47,50], cell migration [56], cell-cell interactions [57], etc., see Table 2. In addition, specific conditions, involving pathological-angiogenesis, such as brain tumors [46,[54][55][56]58], ischemic strokes [59], and neurodegenerative disorders including Alzheimer's disease (AD) [60], Parkinson's disease (PD) [61], and Huntington's disease (HD) [62], could also be created on-chip.In addition to this, LOCs have been developed for studying the biocompatibility, cellular uptake and transport of NMs [2,27], many of them focusing on brain angiogenesis [2,45,63]. Further, LOCs are also used to synthesis NMs [64].…”

“…In addition to this, LOCs have been developed for studying the biocompatibility, cellular uptake and transport of NMs [2,27], many of them focusing on brain angiogenesis [2,45,63]. Further, LOCs are also used to synthesis NMs [64].…”