A Microfluidic In Vitro Three-Dimensional Dynamic Model of the Blood-Brain Barrier to Study the Transmigration of Immune Cells

Abstract

To study the biodistribution of new chemical and biological entities, an in vitro model of the blood-brain barrier (BBB) may become an essential tool during early phases of drug discovery. Here, we present a proof-of-concept of an in-house designed three-dimensional BBB biochip designed by us. This three-dimensional dynamic BBB model consists of endothelial cells and astrocytes, co-cultured on opposing sides of a polymer-coated membrane under flow mimicking blood flow. Our results demonstrate a highly effective BBB as evidenced by (i) a 30-fold increase in transendothelial electrical resistance (TEER), (ii) a significantly higher expression of tight junction proteins, and (iii) the low FITC-dextran permeability of our technical solution as compared to a static in vitro BBB model. Importantly, our three-dimensional BBB model effectively expresses P-glycoprotein (Pg-p), a hallmark characteristic for brain-derived endothelial cells. In conclusion, we provide here a complete holistic approach and insight to the whole BBB system, potentially delivering translational significance in the clinical and pharmaceutical arenas.

Keywords: FITC-dextran permeability; P-glycoprotein; TripleB slides; blood–brain barrier; central nervous system; endothelial cells; microfluidic device; transendothelial electrical resistance.

Figure 1

The fabrication process of the TripleB fluidic chip, beginning with the sub-assembly in (a) which shows the formation of the upper fluid channel (for endothial cells) comprising two rigid bodies (upper channel part 1 and upper channel 2), which are 3D printed using biocompatible MED610 resin, and are then sealed along the edges using uncured MED610 resin, which is then separately cured with a UV lamp; (b) then, the assembly of the removeable membrane is performed, which consists of a porous membrane with 3.0 µm pore size (Oxyphen) and spacer tape to support the delicate porous membrane structure, which are then sandwiched between an upper and lower layer of silicone rubber (Shore hardness 30 A) to ensure a tight seal between the rigid bodies of the upper and lower channels—the shapes of these components were fabricated using mechanical die-cutting and punching and exposed a surface area of 4 × 8 mm or 0.32 cm² on either side of the porous membrane for the growth of the endothelial and astrocyte cells; (c) the formation of the lower fluid channel (for astrocyte cells) comprising two rigid bodies (lower channel part 1 and part 2). These parts are fabricated and sealed using the same method as the upper channel. These three sub-assemblies are combined together, as shown with (d) a render and (e) a photograph of the fabricated TripleB fluidic chip. A photograph of the novel transwell insert is shown in (f) which comprises the two rigid bodies (the flanged carrier and the cover with bayonet-type connection) and the removable membrane assembly.

Figure 2

Demonstrations of the flexibility of prototyping devices with unconventional geometries by 3D-printing using a Polyjet printer, including (a) an exploded view of a 3D, vascular capillary-shaped BBB model based on Polyjet-printed rigid upper and lower housings to form two fluidic channels, containing a 1-mm diameter hollow cylinder which was micromachined as a scaffolding structure for the membrane layer, which is affixed to the scaffold with spacer tape. Mechanically punched silicone o-rings were used to seal the inner and outer channels from each other; (b) a render of the device, and (c) a photograph of the final prototyped device; (d) a CAD model of a novel transwell insert based on a dimensions of an insert for a standard 24-well transfer plate, where the yellow section is a removable 2 × 2 mm membrane with a 3D-printed frame, which was inserted into a 3D-printed flanged carrier; (e) a photograph of the prototyped novel 24-well transwell plate insert with removable membrane.

Figure 3

The clinical user flow of the TripleB model. Firstly (a) the red-colored removable membrane is seeded using pipettes with poly-L-lysine and endothelial cells on the upper half of the membrane and collogen and astrocyte cells on the lower half. The seeded membrane is then positioned inside the novel 6-well transwell insert, in this case the flanged carrier component, which is then secured in place with a 3D-printed cover with bayonet-type connections to keep the membrane barrier sealed. Once assembled (b), this can be placed in a standard 6-well transwell plate with the required nutrients in the well and on the inside of the insert for the initial incubation. After the initial incubation, (c) the membrane can be removed from the novel transwell insert and placed in the 3D-printed lower channel housing of the TripleB, which is then covered with the upper channel housing. The assembled fluidic chip is then placed into a 3D-printed clamping device (d) to ensure the fluidic channels do not leak, and then growth media can be added via pipettes to Luer-locks inputs of the upper and lower channels, to support the growth of endothelial cells and astrocyte cells, respectfully. From there, (e) fluidic flow tubes from the Ibidi pump are connected to the input and output Luer-locks of the upper channel to induce a strain on the endothelial cells due to the flow circulating through the system (which is indicated in the direction of the arrow), shown in (f) where three TripleB models are connected in parallel and further incubated under flow conditions.

Figure 4

The 3D TripleB slides demonstrate the presence of continuous laminar flow. To model the flow inside of the microfluidic slide, the CFDs for the 3D designed slide were generated, which shows (A) the range of velocities as measured from 3 different positions in the bottom chamber of the slide (B) and the presence of unidirectional laminar flow in the TripleB slides.

Figure 5

Endothelial cells grow in the direction of the laminar flow in dynamic BBB model. BBB was cultured under static conditions (A) or under a laminar flow of 4–15 dyn/cm² (B) for 8 days Confocal images obtained following staining of the hCMEC/D3 cells for DAPI (blue), and ZO-1 (green) merged when grown on collagen-coated microporous membranes. Arrows indicate the direction of the flow. Images were taken using an UltraVIEW confocal microscope.

Figure 6

Highly rigorous blood–brain barrier formation takes place when the cells are cultured in the presence of flow. TEER was measured following the growth of hCMEC/D3 and astrocytes on permeable membranes (A) under static and dynamic (4–15 dyn/cm²) conditions. The TEERs of hCMEC/D3 were measured when grown on permeable inserts in the absence and presence of shear stress. Steady-state TEER values were typically reached in the static BBB by day 4 of culture and were attained by the TripleB slides by day 5. (n = 6). Permeability to the tracer molecule FITC–dextran; (B) a significant decrease in the apparent permeability to FITC–dextran was induced in the BBB cultured in dynamic flow conditions as compared to the static BBBs (n = 4) (C) The ability of immune cells to cross the BBB was significantly downregulated under dynamic flow conditions. Migratory capacity of PBMCs was evaluated across a static and a dynamic BBB. Significantly lower numbers of PBMCs were recovered from the TripleB culture as compared to the amount of cells harvested from a static culture of BBB. (n = 4, *** p ≤ 0.001).

Figure 7

TripleB cultured barriers demonstrate a much higher expression of P-glycoprotein along with other endothelial-cell-specific markers when compared to transwell-cultured barriers. Phenotypic differences in the static and dynamic cultured BBB (A) BBB cultured in the TripleB slides showed a significantly higher expression of EC-specific markers compared with the static in vitro BBB. (B) mRNA encoding the tight junction proteins SLC2A1, TJP1, OCLDN1 and CLDN5 was found to be significantly upregulated in TripleB cultures as compared to the static BBB cultures (n = 4, * p ≤ 0.05; ** p ≤ 0.01).