Microenvironment-Driven 3-Dimensional Biofabrication Enables Sequential Maturation of the Blood-Brain Barrier

Three-dimensional (3D) in vitro blood-brain barrier (BBB) models are essential for studying neurovascular development, drug permeability, and disease mechanisms, yet most fail to recapitulate the temporal sequence of cellular maturation. Here, we present a spatiotemporally organized 3D BBB model engineered via dynamic microenvironmental regulation. Temporally controlled enzymatic modulation of an alginate/collagen dual-network hydrogel enabled transition in matrix properties from strong to weak mechanical confinement, thereby triggering the embedded cells to shift from a quiescent to an active state and further enabling the construction of a BBB model with sequential endothelial and supporting cell development. The resulting 3D BBB model exhibits improved barrier integrity, reduced macromolecular leakage, and physiologically relevant drug-selective permeability. This strategy demonstrates that temporally controlled microenvironmental cues can guide multicellular tissue development, providing a versatile platform for neurovascular research and central-nervous-system-targeted drug evaluation.