Establishment of an In Vitro Neurovascular Unit Model With Blood and Brain Components and Investigation of Its Blood–Brain Barrier

The neurovascular unit (NVU), including the blood-brain barrier (BBB), governs the interaction between neural tissue and blood vessels. The BBB is a highly selective interface that regulates molecular exchange between the bloodstream and the central nervous system. This study aimed to develop a structurally relevant, multicellular in vitro NVU model integrating both vascular and brain microenvironments to evaluate BBB function. A fibrous membrane mimicking the basement membrane was fabricated via electrospinning, while a methacrylated hyaluronic acid (MeHA)-collagen hydrogel was used on the brain side. Endothelial cells (ECs) were cultured on the vascular side, and astrocytes, pericytes, and neuronal model cells were embedded within the hydrogel. The model was optimized for cell viability and endothelial monolayer formation. Cell behavior was assessed via immunocytochemistry, and barrier function was evaluated using TEER measurements and permeability assays with fluorescein, 0.4 and 20 kDa dextran, ceftriaxone, and amikacin. CD31 expression was elevated in the multicellular model, indicating improved endothelial integrity. The model achieved a TEER of 166.86 ± 5.75 Ω versus 121.70 ± 13.58 Ω cm² in monoculture. Permeability to tracers was significantly reduced in the multicellular model, and ceftriaxone showed higher transport than amikacin, reflecting human BBB selectivity. This model provides a physiologically relevant platform for neurovascular research and drug screening.