Poster M16 - Development of Microfluidic Models for Sickle Cell Disease Mediated Carotid Artery Remodeling

Introduction: Affecting over 20 million people worldwide, sickle cell disease (SCD) is a hereditary blood disorder characterized by a misshapen beta-globin subunit, causing hemoglobin molecules to clump and red blood cells to sickle. Beyond hematological effects, SCD profoundly affects arterial structure and function. SCD-mediated arterial remodeling manifests as expanded arteries, marked by elastic lamina degradation and neointimal thickening. Arterial remodeling is linked to cathepsin K (catK), a robust elastase discharged by endothelial cells (ECs) in reaction to pro-inflammatory cytokines like tumor necrosis factor-alpha (TNFα), as well as low or oscillatory wall shear stress. Chronic inflammation from SCD leads to elevated plasma TNFα levels that may cause heightened proteolytic activity by cathepsin K shown by Keegan et al. (2012), exacerbating elastic lamina degradation and arterial dysfunction. There is a need to understand the interplay between these biomechanical and biochemical factors and their impact on cathepsin production and activity in the carotid artery to mitigate sickle cell disease-mediated artery damage. We are developing a microfluidic tissue model using fugitive ink and three-dimensional (3D) bioprinting to provide a controlled environment to isolate and study arterial pathology while offering flexibility in fabricating vessel geometries and stimulation with cytokines. Our goal with this microfluidic arterial system is to establish a modular model, enabling detailed examination of how cytokines and varied shear stress profiles influence catK activity, shedding light on the pathophysiology of arterial remodeling in SCD.

Materials and

Methods: To construct the 3D-engineered arterial model, a glass slide is coated in a thin layer of extracellular matrix (ECM) consisting of 25 mg/mL fibrinogen-7.5 wt. % gelatin mixture (gelbrin), and the layer is dehydrated. The coated slide is mounted in a custom-made aluminum chamber that aids device fabrication and imaging. Using an Allevi 3 bioprinter at 37°C, an artery template with inlet and outlet reservoirs is printed onto the gelbrin ECM with fugitive ink. The fugitive ink used is 25 wt. % Pluronic F127 and 1 wt. % high-molecular-weight polyethylene oxide. Following printing, the template is encased in ECM, sealed with a top glass slide, and the top plate of the aluminum chamber screwed down. The entire assembly is chilled to 4°C to liquefy the fugitive ink. The fugitive ink is flushed using a chilled phosphate-buffered saline solution, leaving open lumens encapsulated within the ECM (Fig. 1A). The fabricated vascular structure is warmed to 37°C and perfused with endothelial cell media overnight to prime with essential nutrients, growth factors, and signaling molecules. Endothelial cells are seeded in the device and incubated at 37°C under static conditions for 5 hours. Dynamic flow conditions are then introduced to the system, mimicking the hemodynamic forces experienced by the human common carotid artery. The device is perfused with media for 3-5 days to allow adherent cell clustering and growth until they reach a confluent monolayer lining the lumen.

Results, Conclusions, and Discussions: Diverse geometries were achieved, featuring straight, curved, and bifurcated configurations. Vessel fabrication encompassed a range of diameters from 360 to 3800 µm, including a bifurcated model mimicking mice carotid artery dimension at 688 µm. Customization to subject-specific geometries is foreseen, facilitating nuanced investigations into the impact of varied shear stress profiles on proteolytic activity. Following the seeding of ECs, physiological pulsatile flow simulation was achieved (~12 dyne/cm2) using a peristaltic pump calibrated to mimic flow and shear stress profiles in the carotid artery bifurcation. On nonbranching vessels, preliminary tests using immunofluorescence staining revealed the F-actin and nuclei of the cellular lumen monolayer (Fig. 1B, C). Pre-challenge with TNFα followed by imaging under flow revealed increased dextran permeability in these nonbranching vessels, suggesting enhanced vessel leakage and further revealing TNFα's role in modulating vessel integrity (Fig. 1D, E). We aim to deploy this imaging approach and carry out experiments involving TNFα on the bifurcated artery model, aiming to explore the impact of cytokines and shear stress profiles on protease activity.

Using a combination of 3D bioprinting and stimulation of ECs with TNFα, this model provides a controlled environment to study protease activity within straight and curved nonbranched vessels. Gelbrin ECM, characterized by a stiffness of 10.7 kPa, fostered EC growth and proliferation, with the potential for stiffness modulation to influence cell behavior. Using the tissue model developed, our aim is to use it as an adaptable approach in investigating the complex interplay between biomechanical forces and biochemical factors in sickle cell disease (SCD)-mediated arterial remodeling and other arterial pathologies. Our initial findings, using nonbranched vessels, underscore the role of TNFα in modulating vessel degradation and highlight the potential of this microfluidic vessel system as a modular model for dissecting the pathophysiology of arterial remodeling in SCD, offering insights for potential therapeutic interventions targeting artery dysfunction in this debilitating disease.

Acknowledgements (Optional): We would like to thank the Instrumentation Development and Engineering Application Solutions group at the National Institute of Biomedical Imaging and Bioengineering for their collaborative efforts.