Poster T16 - Development of a microfluidic model of infective endocarditis

Introduction: A heart valve infection, also called infective endocarditis (IE), develops when there is a bacterial infection that forms on the native heart or prosthetic valve. These infections are very difficult to treat, and clinical data suggest that IE is a biofilm-associated infection. IE is relatively rare, but the incidence is rising with increased use of intracardiac and vascular devices. Staphylococcus aureus (S. aureus) accounts for the most infections on native heart valves. Animal models of IE, particularly the rabbit and rat, are well-established and commonly used to evaluate the efficacy of novel therapeutics for bacterial IE but are limited in their ability to generate predictable and reproducible biofilms. Static human cell cultures lack the physiological stimuli and tissue architecture found in vivo. Organs on a chip can overcome some of these animal model and in vitro cell-based shortcomings. We have created an organ on a chip model of the aortic valve that can provide a dynamic, 3D culture environment with viable porcine aortic valve endothelial and interstitial cells for up to 21 days. The goal of this project is to create an in vitro model of IE to understand IE development and provide a testbed for new therapeutic strategies.

Materials and

Methods: Porcine aortic valve endothelial cells (PAVEC) and interstitial cells (PAVIC) were used to model the aortic valve cell populations. PAVEC were seeded onto 3D collagen I gels at 95,000 cells/cm2, and PAVIC seeded within the 1.5 mg/mL collagen I hydrogel at 1x106 cells/mL. PAVEC were allowed 4 hours to attach to the hydrogel at 37°C before steady flow was initiated at 1 dyne/cm2 or 10 dyne/cm2. Cells were grown in DMEM+ 10% heat-inactivated FBS and 0.4% L-arginine, diluted with PBS for 2 days. Staphylococcus aureus was cultured for a period of 16 hours in 5 mL of reinforced clostridial medium in orbital shaker at 37¬oC. Bacteria were introduced into the device on day 2 at a concentration of 103 CFU/mL, allowed to attach for 1 hour, and then flow was resumed for an additional 3 days. Mammalian and bacterial cell viability and biofilm thickness were determined at 24 hours, 48 hours, and 72 hours.

Results, Conclusions, and Discussions: Figure 1 shows the viability of PAVIC and PAVEC exposed to 10 dyne/cm2 steady shear stress and DMEM +/- L-arginine or DMEM diluted with PBS +/- L-arginine. The viability of cells cultured in 70% DMEM diluted with PBS with 0.4% L-arginine showed no statistical difference when compared with 100% DMEM controls, therefore this condition was used for subsequent experiments. With an increase of L-arginine to 1% there was a statistically significant decrease in mammalian cell viability. S. aureus maintained high viability within the microfluidic device with 70% DMEM diluted with PBS and 0.4% L-arginine after 72 hours both with and without the mammalian cells. There was a significant decrease in viability of PAVEC/PAVIC at 72 hours post-infection.
PAVIC and PAVEC in a 3D collagen I scaffold remain viable with biofilm inhibiting medium for 3 days. L-arginine, an amino acid, is a precursor for the synthesis of proteins and inside the body is converted to nitric oxide (NO). Excessive amounts of NO production can cause oxidative stress and damage to mitochondrial function. Higher concentrations of L-arginine lead to cell death by apoptosis, which is shown in Figure 1 when comparing cell viability at 1% and 0.4%. L-arginine also acts to slow biofilm growth, as it disrupts biofilm formation through the destabilization of the bacterial extracellular matrix.
: A microfluidic model of IE was developed by incorporating S. aureus biofilms into an existing model of the aortic valve. Mammalian aortic valve cells and S. aureus remain viable in the device for up to 72 hours. This in vitro model provides a platform to understand the early stages of IE development.

Acknowledgements (Optional): We acknowledge funding from the Clifford D. Clark Diversity Fellowship and NSF CMMI 1919438.