Development of a 3D printed perfusable in vitro blood-brain barrier model for use as a scalable screening tool.

ABSTRACT

Despite recent technological advances in drug discovery, the success rate for neurotherapeutics remains alarmingly low compared to treatments for other areas of the body. One of the biggest challenges for delivering therapeutics to the central nervous system (CNS) is the presence of the blood-brain barrier (BBB). In vitro blood-brain barrier models with high predictability are essential to aid in designing parameters for new therapeutics, assess their ability to cross the BBB, and investigate therapeutic strategies that can be employed to enhance transport. Here, we demonstrate the development of a 3D printable hydrogel blood-brain barrier model that mimics the cellular composition and structure of the blood-brain barrier with human brain endothelial cells lining the surface, pericytes in direct contact with the endothelial cells on the abluminal side of the endothelium, and astrocytes in the surrounding printed bulk matrix. We introduce a simple, static printed hemi-cylinder model to determine design parameters such as media selection, co-culture ratios, and cell incorporation timing in a resource-conservative and high-throughput manner. Presence of cellular adhesion junction, VE-Cadherin, efflux transporters, P-glycoprotein (P-gp) and Breast cancer resistance protein (BCRP), and receptor-mediated transporters, Transferrin receptor (TfR) and low-density lipoprotein receptor-related protein 1 (LRP1) were confirmed via immunostaining demonstrating the ability of this model for screening in therapeutic strategies that rely on these transport systems. Design parameters determined in the hemi-cylinder model were translated to a more complex, perfusable vessel model to demonstrate its utility for determining barrier function and assessing permeability to model therapeutic compounds. This 3D-printed blood-brain barrier model represents one of the first uses of projection stereolithography to fabricate a perfusable blood-brain barrier model, enabling the patterning of complex vessel geometries and precise arrangement of cell populations. This model demonstrates potential as a new platform to investigate the delivery of neurotherapeutic compounds and drug delivery strategies through the blood-brain barrier, providing a useful in vitro screening tool in central nervous system drug discovery and development.