Interfacial Morphology Shapes Endothelial Organization and Function in a Blood-Brain Barrier-on-a-Chip

Organ-on-a-chip technologies have advanced rapidly as platforms for modeling human physiology and disease. In particular, the integration of hydrogels has enabled closer mimicry of tissue microenvironments and facilitated coupling with organoids and other three-dimensional cellular systems. Despite these advances, the mechanical stability of hydrogels and the integrity of the barrier interface remain poorly controlled, limiting reproducibility and functional fidelity in barrier-on-a-chip models such as the blood-brain barrier (BBB). Herein, we present a three-channel BBB-on-a-chip (μBBB) that incorporates an in situ photo-cross-linkable hydrogel system to precisely regulate interfacial stability during device operation. Computational fluid dynamics analysis revealed that subtle variations in hydrogel interface morphology markedly alter local shear stress distribution, with slightly protruded interfaces providing more favorable conditions for endothelial organization. Guided by these insights, we introduced a biocompatible photo-cross-linkable agent that reinforces the stability of the hydrogel interface, yielding a robust and stable barrier-forming surface. Stabilization of the hydrogel interface significantly improved endothelial cell viability, reduced cell invasion into the matrix, and enhanced BBB-associated functional readouts. Transcriptomic analysis further revealed lower inflammation- and sprouting-related pathways, consistent with a more quiescent and physiologically relevant endothelial phenotype. Together, this work identifies hydrogel-cell interfacial stability as a critical yet underappreciated determinant of μBBB performance and offers a simple, adaptable strategy to improve the reliability of hydrogel-integrated organ-on-a-chip systems.