A Novel Neonatal Blood-Brain Barrier on a Chip Closely Approximates the In Vivo Microenvironment

Understanding the role of the blood-brain barrier (BBB) in the development and progression of neonatal neural pathologies has been hampered by a lack of physiologically relevant in vitro models of BBB. This also hampers the discovery and testing of pharmacological properties of CNS-targeting therapeutics. To address these challenges, we have developed a novel dynamic neonatal blood-brain barrier on a chip (B3C) that mimics the three dimensional morphology, size and flow characteristics of microvessels in vivo. The microfluidics based model comprises of a side-by-side architecture of vascular channels and the tissue compartment allowing simultaneous real-time visualization of both compartments. The porous interface between the vascular channels and the tissue compartment allows for biochemical and cellular communication between the vascular and tissue cells. Primary brain endothelial cells (RBEC) isolated from neonatal rats were seeded in the vascular channels of B3C and maintained under shear flow conditions, while neonatal rat astrocytes were cultured under static conditions in the tissue compartment of the B3C. RBEC formed continuous endothelial lining with a central lumen along the length of the vascular channels of B3C and exhibited tight junction formation, as measured by the expression of zonula occludens-1 (ZO-1). The barrier formed by RBEC in B3C under flow was strengthened by the presence of ACM or astrocytes as assessed by the permeability of 40kDa dextran, the expression of tight junction molecule (e.g. ZO-1) and transendothelial electrical resistance (TEER). Consistent with the observed features of the in vivo BBB, B3C allowed astrocyte endfeet-endothelial cellular interactions. Moreover, we showed that B3C exhibits significantly improved barrier characteristics compared to the classical transwell model with B3C permeability matching the in vivo BBB permeability in neonatal rats. This fully developed BBB model is the first ever dynamic in vitro neonatal BBB on a chip (B3C) that closely models the in vivo microenvironment of the neonatal brain, offers the flexibility of real time analysis, and provides a platform for studies of BBB function as well as screening of novel therapeutics in a realistic setting. Support or Funding Information This study is supported by a grant from Shriners Hospitals for Children. Neonatal B3C (A) assembled on a microscope slide (B) allows RBEC culture under flow in vascular channel (C) which forms a 3D culture (D) exhibits increased ZO-1 (green) expression in the presence of ACM (E), B3C allows co-culture of RBEC (green: ZO-1) and astrocytes (red: GFAP) (F), presence of ACM and astrocytes causes significant reduction in permeability in B3C (G), and B3C approaches in vivo BBB permeability compared to transwell. Nuclei are blue. *significant difference, p<0.01. Neonatal B3C (A) assembled on a microscope slide (B) allows RBEC culture under flow in vascular channel (C) which forms a 3D culture (D) exhibits increased ZO-1 (green) expression in the presence of ACM (E), B3C allows co-culture of RBEC (green: ZO-1) and astrocytes (red: GFAP) (F), presence of ACM and astrocytes causes significant reduction in permeability in B3C (G), and B3C approaches in vivo BBB permeability compared to transwell. Nuclei are blue. *significant difference, p<0.01.