Poster BB7 - Building the μSiM Vasc-SkM containing engineered vessel mimic and muscle for studying the relation between Rheumatoid Arthritis and Atherosclerosis

Introduction: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that primarily targets the joints, but also systemically promotes inflammation. Patients with RA experience muscle weakness and elevated inflammatory cytokines in their circulation. RA patients also have 1.5-2 fold higher cardiovascular disease (CVD) risk than their age-and-gender-matched general population. Whereas CVD is the leading cause of death globally, with atherosclerosis being its major cause. The increased inflammation might be the cause of the increased atherosclerosis in RA patients, but the specific mechanism is yet unknown. There are a variety of animal models available for either atherosclerosis or rheumatoid arthritis, but each has their own limitations. Animal models can have quite different physiology and pathology than human, and the generation of animal models can be complicated technique wise. There is also a limited number of animal models that recapitulate both diseases. One example is a mouse model with both the RA and atherosclerosis phenotype, but it’s complicated to produce and the disease phenotype takes a relatively long time to develop. Previous experiments with integrated systems of myobundles and tissue engineered blood vessels (TEBVs) in our lab demonstrated increased inflammation in TEBVs cultured with myobundles made with muscle cells donated by RA patients. Though the integrated system has advantages, its fabrication is lengthy and meticulous, and the system is cumbersome with large media volume and dead spaces. Hence, we aim to make a substitute for the integrated system that removes these limitations and can be made more easily in larger quantities.

Materials and

Methods: The modified system (μSiM Vasc-SkM) contains a vascular component (Vasc) made of a blood vessel mimic and a skeletal muscle component (SkM) with a myobundle (Figure 1). The modification is based on the results obtained from our previous integrated system of tissue engineered blood vessels (TEBVs) made of fibroblasts and endothelial cells and myobundles, and the modular microphysiological system enabled by a silicon membrane(μSiM) capable of hosting a layer of barrier tissue, developed by McGrath lab. The Vasc composes of iPSC derived endothelial cells (viEC) and iPSC derived smooth muscle cells (viSMC) on a porous silicon membrane and matured under flow to form a barrier tissue. The SkM contains a tissue engineered myobundle made of fibrin based hydrogel with skeletal muscle cells isolated form rheumatoid arthritis (RA) patients. The media formulations tested contain a combination of the myobundle differentiation media (Diff) and endothelial cell culture media (EC) or TEBV culture media (EC2) or viSMC culture media (viSMC), with or without the addition of 10ng/mL VEGF. Phase contrast images are taken to analyze cell coverage, and viSMC is stained with calponin for maturation. For myobundle contractile profile analysis for media test, the myobundles undergo different electrical stimulation for twitch (1Hz, 1s), tetanus (20Hz, 1s), and fatigue (20Hz, 30s). ANSYS Mechanical is used to simulate beam deformation when force was applied. PDMS beams are coated with overnight incubation of 2mg/mL polydopamine in 10mM Tris buffer. Videos are taken for myobundles cultured on PDMS beams during electrical stimulation.

Results, Conclusions, and Discussions: The integrated culture media for the Vasc and SkM component were first determined. We found that a mixture of 75% differentiation media for myobundles with 25% of either viSMC culture media or DMEM based TEBV culture media with VEGF had least influence on cell and tissue function. There was significant force reduction in RA myobundles for of all test media groups compared to the Diff media control (Figure 2). However, for Diff:EC2_75:25+VEGF and Diff:viSMC_75:25+VEGF, the force reductions were still within an acceptable range. The Diff:EC2_75:25+VEGF and Diff:viSMC_75:25+VEGF were then tested in 2D viSMC culture for 4 days with viSMC media as control case. There was no significant change in viSMC coverage. Mean fluorescent intensity of calponin in viSMC did not show significant difference between media conditions (Figure3).
Meanwhile, the SkM component of the µSiM was setup for myobundle culture and contractile force analysis. As myobundles will be attached to deflectable PDMS beams for culture and force analysis, ANSYS was used to model the deflection of deformed beam to ensure feasibility of the system and estimate calibration factor for conversion of the beam deflection to myobundle contractile force (Figure 4). 0.4mN is predicted to cause a 15μm deflection, which is larger than the resolution we can achieve, proving the system feasible. In addition, we coated the PDMS beams with polydopamine to increase its hydrophilicity for improved myobundle attachment. In our preliminary test with a small set of myobundles, they all remained attached to the beams after 18 days of culture, and video taken for myobundle Tetanus showed beam deflection upon stimulation.
In conclusion, we found two promising integrated culture media candidates (the Diff:EC2_75:25+VEGF and Diff:viSMC_75:25+VEGF), and proved feasibility of culture and functionality analysis of myobundles on PDMS beams in the SkM compartment. Our next steps are to validate viEC survival and function of the media candidates, calibrating the beam provided by vendor and compare to results from ANSYS, setting up the flow with the Vasc and SkM, and improving electrode for myobundle stimulation when they are integrated into the μSiM Vasc-SkM.

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