Use of an NVU-mimetic hydrogel within the transwell insert allows the NVU cells to be grown in 3D beneath a BMEC monolayer without having separate compartments that physically separate the different cell types. (A) Monoculture: BMECs grown on a hydrogel that mimics the physical properties of the NVU ECM with a basement membrane coating to facilitate BMEC monolayer development. (B) Co-culture: BMECs are seeded on top of a basement membrane-coated hydrogel layer that is laden with another cell type, e.g. neurons, thereby enabling physical and biochemical interactions between the two cell types. (C) Tri-culture: neurons and astrocytes, for example, are both encapsulated within the hydrogel and cultured in 3D allowing both cell types to interact with each other within the hydrogel and with the BMEC monolayer. (D) Quad-culture: neurons, astrocytes and pericytes, for example, are grown within the 3D hydrogel layer with BMECs on the surface.