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-a-chip (Organ Chip) model of the BBB lined by induced pluripotent stem cell-derived 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.\n\nThe 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 function1. The brain microvascular endothelium differs from that found in peripheral capillaries based on its complex tight junctions, which restrict paracellular transit and instead, require that transcytosis be used to transport molecules from the blood through the endothelium and into the CNS2. BMVECs also express multiple broad-spectrum efflux pumps on their luminal surface that inhibit uptake of lipophilic molecules, including many drugs, into the brain3,4. The astrocytes and pericytes provide signals that are required for differentiation of the BMVECs5,6, and all three cell types are needed to maintain BBB integrity in vivo as well as in vitro7-9. 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 BBB10. 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 humans11-14. 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 research8,9.\n\nDevelopment of human induced pluripotent stem (iPS) cell technology has enabled differentiation of brain-like microvascular endothelial cells (iPS-BMVECs) that exhibit many properties of the human BBB, including well-organized tight junctions, expression of nutrient transporters and polarized efflux transporter activity15,16. The trans-endothelial electrical resistance (TEER) values exhibited by the permeability barrier generated by these human iPS-BMVECs reach physiological levels ([~]3000-5000 {Omega}{middle dot}cm2) within 24-48 h when cultured in Transwell inserts or within a microfluidic organ-on-a-chip (Organ Chip) device15,17-19, a level that is more than an order of magnitude higher than TEER values previously reported in other in vitro human BBB models6,17,20.\n\nHowever, the usefulness of these iPS-BMVEC models for studies on targeted delivery to the CNS is limited because they can only maintain these high TEER levels for [~]2 days, and the expression of efflux pumps in these iPS-BMVECs does not fully mimic those of human brain endothelium in vivo21. Here, we describe the development of an enhanced human BBB model created with microfluidic Organ Chip culture technology22,23 that contains human iPS-BMVECs interfaced with primary human pericytes and astrocytes, and that uses a developmentally-inspired differentiation protocol24-26. The resulting human BBB Chip exhibits physiologically relevant levels of human BBB function for at least one week in vitro, including low barrier permeability and expression of multiple efflux pumps and transporter functions that are required for analysis of drug and therapeutic antibody transport.