Building μSiM hVasc-SkM as a drug development tool for Juvenile Dermatomyositis (JDM)

Introduction: : Juvenile dermatomyositis (JDM) is a rare idiopathic autoimmune disease that predominately affects female with an average onset at age of 4-9. It has a systemic effect but typically affects muscle and skin, causing muscle weakness, rash and loss of capillaries. Patients experience infiltration of immune cells, including monocyte, macrophages, and T-cells, into muscle, and an increased type I interferon score. Current treatment uses corticosteroids, which are broadly immunosuppressive with significant toxicity. However, 2/3 of children under treatment still experience chronic active disease, causing muscle damage including atrophy and calcification. There is a need to improve the treatment, yet there is no suitable model to test therapeutics. Our lab previously modeled JDM by treating engineered myobundles made of primary human skeletal muscle cells with IFNβ and observed a dose dependent force reduction. To further elaborate this model for better representation of JDM, we will incorporate components representing the capillaries and immune cells through the new µSiM Vasc-SkM system (Figure 1). A confluent layer of endothelial cells is cultured on the porous silicon membrane to form the perfusable Vasc component, developed by our collaborator. The bottom flow channel hosts a skeletal muscle component with and without IFNβ pretreatment to model JDM muscle. Monocytes are perfused through the Vasc flow channel to model immune cell interactions in JDM. Once the system setup is mature, we will introduce myobundles made of myoblasts from JDM patients. Our goal is to establish this µSiM Vasc-SkM system for accurate JDM modeling and drug development.

Materials and

Methods: : The µSiM Vasc-SkM system contains a perfusable vascular component (Vasc) and Skeletal muscle component (SkM) (Figure 1). The Vasc component consists of a porous silicon membrane that hosts an endothelium at the bottom of the flow channel. The SkM component consists of a flow channel with two deflectable PDMS beams near the ends and two parallel electrodes underneath the flow channel on a piece of glass. An engineered skeletal muscle myobundle was made between the beams. Each component was fabricated and matured separately and assembled into the µSiM Vasc-SkM system for perfusion. After integration, each channel was perfused separately with either the predetermined integrated system media, or with the culture media for the corresponding tissue type. This way, the tissues cultured in the system influence each other only through the membrane below the endothelium. The wellbeing of the endothelium was determined with phase contrast images and immunofluorescence for CD31. The myobundle was analyzed for their contractile force generated under electrical stimulation. The beam deflection caused by contracting myobundles was videotaped and analyzed through MATLAB to produce force tracings. For control nylon myobundles, they were fabricated and analyzed following our standard protocol, with their forces measured using our force transducer setup. To model JDM, myobundles underwent pre-treatment of IFNβ before integration and CellTracker stained monocytes were perfused through the Vasc and visualized through immunofluorescence imaging. Different perfusion rates will be tested for optimal representation of JDM monocyte infiltration from the endothelium into the SkM and the subsequent monocyte attachment on myobundles.

Results, Conclusions, and Discussions:: We first ensured that the µSiM Vasc-SkM setup and the flow culture did not damage the myobundles (Figure 2A-D). We observed similar forces between the traditional control myobundles and hMoC myobundles with and without perfusion during integration, indicating no damage from the chip on myobundles. Perfusion during the force test was found optional as it did not cause significant change in force.

We then compared the influence of different perfusion culture conditions on the myobundles (Figure 2E-H). We observed no significant reduction in force before and after perfusion in the µSiM Vasc-SkM with and without the endothelium and with the regular culture media for the endothelium and myobundles in their corresponding channels or with the integrated media in both channels. The force reduction in hMoC cultured on rocker was likely due to the hindered media exchange by hMoC structure. However, the differentiation media flowing through the SkM channel may influence the maturation of the endothelium. Hence the integrated system media was used in the subsequent experiments.

Using the µSiM Vasc-SkM chip, we modeled JDM by treating myobundles with 1000 IU/mL IFNβ (Figure 3&2EGH). We observed increased monocyte adhesion in the endothelium integrated with IFNβ pretreated myobundles and the myobundles themselves, compared to the untreated control. This increased monocyte adhesion indicated an increased inflammation in the muscle and endothelium. It was also a sign of increased monocyte migration underneath the endothelium into the SkM channel.

In conclusion, we developed the µSiM Vasc-SkM system and applied the system for JDM modeling. We determined the desirable perfusion culture conditions for the endothelium and the myobundles integrated in this µSiM Vasc-SkM system. We modeled JDM by integrating endothelium with IFNβ pretreated myobundles and observed increased inflammation through increased monocyte compared to untreated control chips. We are currently testing the monocyte adhesion and migration under a lower and more JDM-relevant shear stress, modeling the activity of immune cells at the capillary. Our next step is to streamline the protocol and determine a more stable trend in endothelium inflammation and myobundles force. We will further reduce the system media volume for reliable analysis of secreted cytokines.

Acknowledgements and/or References (Optional):: This study is funded by NIH NCATS, AHA, and IFER. We would like to thank our collaborators at University of Rochester as a member of the TraCe-bMPS group.