Microfluidic blood-brain barrier model provides in vivo-like barrier properties for drug permeability screening

Abstract

Efficient delivery of therapeutics across the neuroprotective blood-brain barrier (BBB) remains a formidable challenge for central nervous system drug development. High-fidelity in vitro models of the BBB could facilitate effective early screening of drug candidates targeting the brain. In this study, we developed a microfluidic BBB model that is capable of mimicking in vivo BBB characteristics for a prolonged period and allows for reliable in vitro drug permeability studies under recirculating perfusion. We derived brain microvascular endothelial cells (BMECs) from human induced pluripotent stem cells (hiPSCs) and cocultured them with rat primary astrocytes on the two sides of a porous membrane on a pumpless microfluidic platform for up to 10 days. The microfluidic system was designed based on the blood residence time in human brain tissues, allowing for medium recirculation at physiologically relevant perfusion rates with no pumps or external tubing, meanwhile minimizing wall shear stress to test whether shear stress is required for in vivo-like barrier properties in a microfluidic BBB model. This BBB-on-a-chip model achieved significant barrier integrity as evident by continuous tight junction formation and in vivo-like values of trans-endothelial electrical resistance (TEER). The TEER levels peaked above 4000 Ω · cm² on day 3 on chip and were sustained above 2000 Ω · cm² up to 10 days, which are the highest sustained TEER values reported in a microfluidic model. We evaluated the capacity of our microfluidic BBB model to be used for drug permeability studies using large molecules (FITC-dextrans) and model drugs (caffeine, cimetidine, and doxorubicin). Our analyses demonstrated that the permeability coefficients measured using our model were comparable to in vivo values. Our BBB-on-a-chip model closely mimics physiological BBB barrier functions and will be a valuable tool for screening of drug candidates. The residence time-based design of a microfluidic platform will enable integration with other organ modules to simulate multi-organ interactions on drug response. Biotechnol. Bioeng. 2017;114: 184-194. © 2016 Wiley Periodicals, Inc.

Keywords: TEER; blood-brain barrier; human iPS cells; organ on a chip; permeability.

Figure 1.

Design of the BBB-on-a-Chip. (A) Schematic exploded view of the microfluidic platform. The device consists of a cell insert and three 3D printed plastic layers: i) a bottom perfusion layer with microchannels and bottom electrodes, ii) a middle layer that forms reservoirs and the neuronal chamber, and iii) a top lid layer with top electrodes that covers the neuronal chamber and the reservoirs minimizing evaporation. The cell insert was made from two silicone sheets and a sandwiched porous polycarbonate membrane, and was assembled between the bottom and the middle layers. (B) The assembled device, with or without the lid. A red dye was used for visualization of microchannels, the neuronal chamber and reservoirs. (C) Side view showing the fluid pathway, electrode wiring connected to a Millicell-ERS Volt-Ohm Meter and the BBB co-cultural orientation. The zoom-in panel showing the cross section was drawn to scale except for cells and the porous membrane. A step chamber with a height of h_sc was introduced to minimize shear stress on the BMEC surface.

Figure 2.

Simulation results for the shear stress on BMEC surface. The maximum wall shear stress occurred near the entrance/exit to the microchannels, while relatively lower shear stress levels were uniformly present near the center (A). Both the average and the maximum levels of shear stress on BMEC surface decreased dramatically when the step chamber height increased from 0 to 0.5 mm (A-B). The average shear stress levels showed an inversely proportional relationship with the step chamber height squared (C).

Figure 3.

BBB characteristic barrier integrity established and maintained on chip. (A) Cocultures of hiPSC-derived BMECs and astrocytes were maintained on chip and examined by immunostaining for the tight junction proteins, ZO-1 and claudin-5. Representative fluorescence images reveal continuous networks of claudin-5 (red) and ZO-1 (green) on both day 3 and day 10. Nuclear staining with DAPI in blue. Scale bar, 50 μm. (B) TEER values from BBBoC increased remarkably within 2–3 days on chip and sustained at high levels up to 10 days. Controls include BMEC (green) and astrocyte (red) monocultures. Values are means ± SEM; n = 10 – 16.

Figure 4.

In vivo-like permeability to fluorescence tracers and drugs. (A) BBB permeability to FITC-dextrans inversely correlates with Stokes-Einstein radius. (B) TEER values, an index of BBB integrity, decreased by doxorubicin treatment in a dose-dependent manner, while (C) the apparent permeability to doxorubicin remained the same for different doses. (D) Permeability to FITC-dextrans and small molecule drugs measured using our BBB-on-a-Chip model correlates with in vivo data (Avdeef, 2012; Shi et al., 2014). All data presented as mean (± standard error of the mean). n = 3. *p<0.05, #p<0.005 compared to pre-treatment; $ compared to 2.5μM group. NS, non-significant.