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

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.