The high selectivity of the human blood-brain barrier (BBB) restricts delivery of many pharmaceuticals and therapeutic antibodies to the central nervous system. Here, we describe an in vitro microfluidic organ-on-a-chip BBB model lined by induced pluripotent stem cellderived human brain microvascular endothelium 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 and functional efflux pumps, and it displays selective transcytosis of peptides and antibodies previously observed in vivo. Increased 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.

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The highly specialized endothelial cells in brain capillaries are a key component of the blood-brain barrier, forming a network of tight junctions that almost completely block paracellular transport. In contrast to vascular endothelial cells in other organs, we show that brain microvascular endothelial cells resist elongation in response to curvature and shear stress. Since the tight junction network is defined by endothelial cell morphology, these results suggest that there may be an evolutionary advantage to resisting elongation by minimizing the total length of cell-cell junctions per unit length of vessel.

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 .