3D brain angiogenesis model to reconstitute maturation of functional human blood-brain barrier in vitro

0. Human central nervous system (CNS) vasculature in brain expresses a distinctive barrier phenotype, the blood–brain barrier (BBB), which protects the brain against harmful pathogens. Since the BBB contributes to low success rate in CNS pharmacotherapy by restricting drug transportation, the development of an in vitro human BBB model has been in demand. Previous models were unable to fully represent the complex threedimensional (3D) anatomical structure or specific barrier phenotypes of the matured BBB. In this study, we present a physiological 3D microfluidic model of the human BBB that mimics its developmental process including CNS angiogenesis and subsequent maturation in concert with perivascular cells. We used microfluidic hydrogel patterning to precisely and sequentially load perivascular cells into the model, investigate the role of each cell type on BBB phenotypes. We confirmed the necessity of the tri-culture system (brain endothelium with pericytes and astrocytes) to attain the characteristic BBB vascular morphology such as minimized diameter and maximized junction expression. In addition, endothelial-perivascular cell interaction was also critical in reconstituting p-glycoprotein (p-gp), efflux transporter in our model that works as metabolic barrier of BBB and blocks drug to enter CNS. The 3D hydrogel matrix was tuned with hyaluronic acid (HA) to optimize the interaction between endothelial cells and astrocytes. Our in vitro BBB system mimics CNS angiogenesis and characteristic features of BBB. We expect the model will contribute to deeper understanding of neurodegenerative diseases and cost-efficient development of effective CNS medications.