A Novel Dynamic Neonatal Blood-Brain Barrier on a Chip.

Studies of neonatal neural pathologies and development of appropriate therapeutics are hampered by a lack of relevant in vitro models of neonatal blood-brain barrier (BBB). To establish such a model, we have developed a novel blood-brain barrier on a chip (B3C) that comprises a tissue compartment and vascular channels placed side-by-side mimicking the three-dimensional morphology, size and flow characteristics of microvessels in vivo. Rat 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). ZO-1 expression significantly increased with shear flow in the vascular channels and with the presence of astrocyte conditioned medium (ACM) or astrocytes cultured in the tissue compartment. Consistent with in vivo BBB, B3C allowed endfeet-like astrocyte-endothelial cell interactions through a porous interface that separates the tissue compartment containing cultured astrocytes from the cultured RBEC in the vascular channels. The permeability of fluorescent 40 kDa dextran from vascular channel to the tissue compartment significantly decreased when RBEC were cultured in the presence of astrocytes or ACM (from 41.0 ± 0.9 x 10-6 cm/s to 2.9 ± 1.0 x 10-6 cm/s or 1.1±0.4 x 10-6 cm/s, respectively). Measurement of electrical resistance in B3C further supports that the addition of ACM significantly improves the barrier function in neonatal RBEC. Moreover, B3C exhibits significantly improved barrier characteristics compared to the transwell model and B3C permeability was not significantly different from the in vivo BBB permeability in neonatal rats. In summary, we developed a first dynamic in vitro neonatal BBB on a chip (B3C) that closely mimics the in vivo microenvironment, offers the flexibility of real time analysis, and is suitable for studies of BBB function as well as screening of novel therapeutics

B³C exhibits significantly improved barrier function compared to the transwell model and closely approximates the permeability of neonatal <i>in vivo</i> BBB.

<p>B³C and Transwell BBB were constructed with neonatal RBEC in the presence of ACM. Permeability of 40 kDa dextran in B³C is significantly lower than transwell but not significantly different from that of <i>in vivo</i> BBB in neonatal rats. (** indicates significant difference p<0.01, *** indicates significant difference p<0.001, one-way ANOVA with Tukey’s multiple comparison test; n = 3–4 experiments per group).</p

Neonatal RBEC exhibit distinct barrier structure and function compared to adult RBEC in B³C.

<p>Neonatal RBEC alone (A) and neonatal RBEC + ACM (B) exhibit distinctly weaker and less organized ZO-1 staining (arrows) compared to adult RBEC alone (C) and adult RBEC + ACM (D). In the presence of ACM, neonatal RBEC exhibit discontinuous bands of ZO-1 staining (B) compared to neonatal RBEC in the absence of ACM where ZO-1 staining is discontinuous and granular (A). Presence of ACM has a significantly larger impact on both permeability (E) and electrical resistance (F) in neonatal RBEC compared to adult RBEC. Inset panels show higher magnification of white squared regions. (** indicates significant difference p<0.01, student’s t-test; n = 3–4 experiments per group).</p

Passage of fluorescent dextran from the vascular channel to tissue compartment of B³C under shear flow.

<p>Permeability of Texas Red 40 kDa dextran from vascular channel to the tissue compartment in a cell-free B³C after 5 min (A), 15 min (B), 30 min (C), 60 min (D) and 120 min (E) from the initiation of flow in vascular channel. Normalized tissue intensity in a cell-free B³C increases linearly with time in the tissue compartment (F).</p

Neonatal RBEC cultured under flow in the vascular channel of B³C form a complete lumen.

<p>Full view of B³C device showing RBEC cultured in the two vascular channels (A). Magnified view of inset from panel (A) showing a section of the vascular channel with cultured RBEC (B). 3D reconstruction of confocal images of neonatal RBEC cultured in B³C stained with f-actin (green) and Draq5 (red) after 5 days in culture maintained under flow of 0.01 μl/min in RBEC medium (C)—(F); images are shown with a Y-axis rotation of 0, 140, 210 and 330 degrees in (C), (D), (E) and (F) respectively. All images were acquired after 5 days of 0.01 μl/min of flow of RBEC medium over cultured RBEC in the vascular channel of B³C.</p

ACM enhances the barrier properties of neonatal RBEC more significantly in B³C as compared to transwell.

<p>Presence of ACM increases electrical resistance of neonatal RBEC in both B³C (A) and transwell (B), the electrical resistance measurements are from day 5. Presence of ACM increases resistance more significantly in B³C as compared to the transwell model (C). Please note that the units of electrical resistance for B³C and transwell are different as noted in the results section. [* indicates significant difference p<0.05, ** indicates significant difference p<0.01, *** indicates significant difference p<0.001, one-way ANOVA (panels A and B) or two-way ANOVA (panel C) with Tukey’s multiple comparison test; n = 3 experiments per group].</p

Tight junction formation by neonatal RBEC under static and flow conditions as indicated by immunofluorescence staining of ZO-1.

<p>RBEC cultured for 5 days under static conditions were stained with ZO-1 (red) and Hoechst 33342 (blue) (A). RBEC cultured for 5 days under flow of RBEC medium in B³C were stained with ZO-1 (red) and Hoechst 33342 (blue) (B). RBEC cultured under flow of ACM for 5 days in B³C were stained with ZO-1 (green) and Hoechst 33342 (blue) (C). Bright field image of B³C showing RBEC in the vascular channels after 5 days of co-culture with rat astrocytes in the tissue compartment of B³C (D). Immunofluorescence staining of RBEC in vascular channel for ZO-1 (green) and astrocytes in tissue compartment for GFAP (red) (E). Magnified view of interface with pores from panel E showing staining of cells inside the pores, ZO-1 (green), GFAP (red) and nuclei (blue) (F).</p