Professor Rutgers University New York, New York, United States
Introduction:: Chronic neurovascular disorders (CNDs) encompassing the brain, spinal cord, and the retina are increasing at a faster rate than the production of therapeutics for these conditions. The implementation of microfluidic systems has emerged as a promising technology to streamline the testing of therapeutics for CNDs due to their high-throughput, cost-effective, and reproducible capacity to test scientific hypotheses. Our group has developed a novel microfluidic system, the HμB (High-efficiency μ-fluidic Barrier), which is able to assess impedance-based cell barrier integrity under physiological flow conditions. The HμB is a cost-effective platform manufactured using laser-cutting of acrylic sheets. This device features a porous membrane for cell co-culture, built-in electrodes to record changes in cell resistance and capacitance in real time, and two separate flow rates along the membrane to model changes in barrier transport. In this work, we have used the HμB to model the inner blood retinal barrier (iBRB) under diabetic conditions to simulate proliferative diabetic retinopathy (PDR) at the neurovascular interface between retinal capillaries and neuroretina. . The HμB enabled us to study the individual and collective contribution of the cells that comprise the iBRB, demonstrating that Muller glia (MG), the predominant glial cell of the retina, are fundamental to maintaining the integrity of the iBRB in diabetic conditions and accelerate the development of CNDs treatments.
Materials and Methods:: Materials: The HμB’s geometry was designed in SolidWorks and printed on polymethyl methacrylate (PMMA) via laser-cutting machining. This device has 2 independent microchannels (top and bottom) divided by a porous polycarbonate membrane of 0.4μm pore diameter, where cells can adhere on each side and interface via paracrine signaling. The HμB features 2 clear built-in electrodes to assess cell impedance across the cell barriers seeded on the membrane. Methods: ECs and MG were harvested from diabetic rats and cultured in high glucose media with advanced glycation end products. Cell types were seeded either individually or in co-culture in the HμB and allowed to adhere and form a confluent monolayer. Cells were subjected to two constant independent flow rates at relevant physiological conditions (capillary blood flow velocity and interstitial flow velocity) for 4-5 days . Likewise, antiangiogenic drugs were introduced on the top channel, while barrier integrity was quantified using an impedance analyzer configured to work with The HμB. Permeability across the cell barriers was assessed by measuring dextran concentrations that crossed the cell barriers over time.
Results, Conclusions, and Discussions:: Results: Experiments in the HμB took place in a microscope stage chamber at 37C and 5%CO2, connected to two syringe pumps and an impedance analyzer that recorded cell-impedance every hour for 4-5 days (Figure 1A-B). Cells formed confluent monolayers on each side of the porous membrane with viability over 95%. The impedance profile of each cell type individually or in co-culture changed over time, elucidating the individual contribution of each cell type to the barrier’s integrity. In the presence of diabetic conditions, cell impedance decreased with respect to control groups. MG monolayers have greater impedance values than those of ECs. Hence, significantly contributing to the integrity of the iBRB when in co-culture with ECs under diabetic conditions. (Figure 1C-D). Immunocytochemistry of tight junctions and gap junctions in both ECs and MG in the HμB provided further insight in the barrier’s integrity when exposed to diabetic conditions and antiangiogenic drugs (Figure 1E). Conclusion: In this work we have developed a novel microfluidic system that can be utilized to study a variety of neurovascular barriers. We modeled the iBRB under induced PDR conditions in the HμB to study changes in barrier integrity in real time. Further, we tested a common therapeutic (antiangiogenic drug) used to treat PDR and assessed its effects in regulating the barrier integrity. Results show that antiangiogenic drugs are insufficient to have significant restorative effect and require combinatory therapeutics. The HμB will serve as a new drug testing platform to streamline dose-dependent therapies under physiological flow conditions, expediting the transition of safe and effective drug products for CNDs into the market.