Abstract WP347: Evaluating Brain Microvascular Endothelial Cell Dysfunction to Stenosis in a Tissue-Engineered Model of the Blood-Brain Barrier

Introduction: Highly specialized brain microvascular endothelial cells (BMECs) form the first defense of the blood-brain barrier (BBB), regulating the exchange of molecules, cells, and pathogens in and out of the brain. Ischemic stroke, a heterogeneous disease with various etiological subtypes and risk factors, places stress on the BBB. While macroscopic BBB changes in ischemic stroke are observable in animal models, individual cell phenotypical and functional changes remain largely unknown due to poor spatiotemporal resolution. Our objective is to use human-based, tissue-engineered microvessel models to capture these details in response to stenosis, a hallmark of ischemic stroke. Methods: Pluripotent stem cells were differentiated into BMECs (iBMECs), validated for BBB phenotype, and seeded into 150 μm channels in collagen I/Matrigel hydrogels. Microvessels were matured for two days under physiological flow (~5 dyne cm -2 ). To model stenosis, a microactuator was mounted on a 3D printed platform and connected to a capillary tube (~1 mm diameter). Barrier function was probed by perfusing microvessels with Lucifer yellow + 70 kDa dextran after 4 h of varied stenosis %, followed by thresholding in ImageJ to track focal leaks. Reactive oxygen species (ROS) generation was assessed using a live cell ROS stain and imaging on the microvessel polar planes with an epifluorescent microscope. The zonula occludens-1 (ZO-1) fluorescent tag on the iBMECs outlined cells for single-cell analyses in CellProfiler using CellPose. Results and Conclusions: A tissue-engineered BBB model from the Searson Lab was modified (Fig. 1A) to include a microactuator for inducing adjustable (0-100%) and reversible stenosis during the perfusion of iBMEC&HUVEC microvessels within a hydrogel ECM (Fig. 1B, C). Preliminary results show focal leaks forming immediately after stenosis (Fig. 2A, B), with distribution increasing along the microvessel length as stenosis intensifies (Fig. 2C). Using ZO-1 labeled iBMECs for cell segmentation from epifluorescence images, we found that ROS significantly increases with greater stenosis (Fig. 3A, B), particularly in the center and upstream of microvessels (Fig. 3C). In conclusion, our findings suggest that stenosis disrupts barrier function, with the disruption not always being regionally dependent. We will further investigate whether these changes are due to physical or chemical factors, such as altered wall shear stress or oxygen/glucose bottlenecks.