Establishing a microfluidic co-culture blood–brain barrier model using human brain endothelial cells and astrocytes

The blood–brain barrier (BBB) is a highly selective physiological interface that regulates molecular transport between the bloodstream and the central nervous system. In vitro BBB models are valuable tools for studying endothelial barrier formation, cell–cell interactions, and responses to dynamic flow conditions. This protocol describes the establishment of a three-dimensional microfluidic co-culture BBB model using human brain endothelial cells and primary human astrocytes cultured in separate but interconnected compartments of a microfluidic chip. The protocol includes chip preparation, extracellular matrix coating, endothelial and astrocyte seeding, introduction of programmable shear flow, transendothelial electrical resistance (TEER) measurement, fixation, immunofluorescent staining, and confocal imaging. Shear flow is applied to the endothelial compartment using programmable syringe pumps to support endothelial alignment and tight junction formation under dynamic culture conditions. Barrier formation is evaluated using TEER measurements and immunofluorescent characterization of endothelial and astrocytic markers, including cluster of differentiation 31, zonula occludens-1, and glial fibrillary acidic protein. This methodology provides a reproducible workflow for establishing a microfluidic endothelial–astrocyte co-culture model suitable for studying BBB-associated cellular interactions and barrier formation under controlled flow conditions. The protocol is intended to support reproducible implementation of BBB chip workflows by providing detailed guidance for device preparation, cell culture, flow setup, imaging, and data acquisition.