# Building the blood-brain barrier: a scalable self-assembling 3D model of the brain microvasculature under unidirectional flow

## TL;DR

A new 3D model of the blood-brain barrier allows researchers to study its function and test therapies for neurological diseases.

## Contribution

A scalable, self-assembling 3D BBB model with unidirectional flow and high-throughput capabilities is introduced.

## Key findings

- Human BBB vascular networks remain viable for at least 14 days with direct cell-cell interactions.
- Tri-cultures of endothelial cells, pericytes, and astrocytes show improved barrier function and vessel alignment.
- The model enables unidirectional perfusion without pumps, suitable for studying BBB function and therapies.

## Abstract

The blood vessels of the central nervous (CNS) system form a tight, protective blood-brain barrier (BBB). This barrier is essential for healthy CNS function but also poses a hurdle in the treatment of increasingly common neurological disorders. Additionally, BBB dysfunction is a hallmark of many neurological diseases, further emphasizing a need for a better understanding of BBB function in health and disease. We present a human self-assembling 3D model of the BBB in a microfluidic cell culture platform that allows culture of 48 models in parallel on one tissue culture plate. Human brain microvascular endothelial cells, pericytes, and astrocytes form highly reproducible BBB vascular networks under unidirectional perfusion and remain viable for a minimum of 14 days. Immunostaining reveals close cell-cell interactions with pericytes and astrocyte endfeet in direct contact with the brain microvasculature. Compared to endothelial monocultures, co-cultures with astrocytes or pericytes result in improved barrier function, lower vessel diameters, increased branching, and alignment of the vessels in the direction of fluid flow. These results were most pronounced in tri-cultures containing all three cell types. Unlike similar models previously reported, this brain microvasculature model allows for unidirectional perfusion without the need for pumps and syringes. Combined with its high-throughput nature, this feature renders the model suitable for studies of BBB function in health and disease, and assessment of potential BBB restorative therapies.

The online version contains supplementary material available at 10.1186/s12987-026-00765-x.