Three-dimensional (3D) in vitro models of human skeletal muscle mimic aspects of native tissue structure and function, thereby providing a promising system for disease modeling, drug discovery or pre-clinical validation, and toxicity testing. Widespread adoption of this research approach is hindered by the lack of easy-to-use platforms that are simple to fabricate and that yield arrays of human skeletal muscle micro-tissues (hMMTs) in culture with reproducible physiological responses that can be assayed non-invasively. Here, we describe a design and methods to generate a reusable mold to fabricate a 96-well platform, referred to as MyoTACTIC, that enables bulk production of 3D hMMTs. All 96-wells and all well features are cast in a single step from the reusable mold. Non-invasive calcium transient and contractile force measurements are performed on hMMTs directly in MyoTACTIC, and unbiased force analysis occurs by a custom automated algorithm, allowing for longitudinal studies of function. Characterizations of MyoTACTIC and resulting hMMTs confirms the capability of the device to support formation of hMMTs that recapitulate biological responses. We show that hMMT contractile force mirrors expected responses to compounds shown by others to decrease (dexamethasone, cerivastatin) or increase (IGF-1) skeletal muscle strength. Since MyoTACTIC supports hMMT long-term culture, we evaluated direct influences of pancreatic cancer chemotherapeutics agents on contraction competent human skeletal muscle myotubes. A single application of a clinically relevant dose of Irinotecan decreased hMMT contractile force generation, while clear effects on myotube atrophy were observed histologically only at a higher dose. This suggests an off-target effect that may contribute to cancer associated muscle wasting, and highlights the value of the MyoTACTIC platform to non-invasively predict modulators of human skeletal muscle function. Skeletal muscle is one of the most abundant tissues in the human body and it enables critical physiological and functional activities, such as thermogenesis 1 and mobility 2. There are many degenerative and fatal diseases of skeletal muscle that remain untreated and the underlying pathology of some muscle related diseases is not fully understood. The use of animal models to study skeletal muscle diseases has improved our understanding of in vivo drug response and disease pathology 3,4. However, in some cases animal models fail to accurately predict drug response in humans, in part due to species specific differences leading to inaccurate disease symptoms 5,6. Furthermore, animal models are expensive and time consuming making them less desirable for drug testing 7. As a result, a push to establish in vitro models of human skeletal muscle with reliable phenotypic readouts for drug testing is underway with the goal of improving therapeutic outcomes in humans.

Functional assessment of stem cell-mediated endogenous repair relies on animal studies. Here an in vitro assay is described that recapitulates important early steps of the in vivo skeletal muscle endogenous repair (MEndR) process. The assay is integrated with a custom semi-automated image analysis pipeline to enable high-content data analysis of donor-derived muscle fiber content and morphology. Myotube sheets, generated by infiltrating a cellulose scaffold with myoblasts, are engrafted with muscle stem cells (MuSCs), injured to induce a regenerative microenvironment, and muscle repair is assessed. Significantly, the spatiotemporal dynamics of in vitro repair closely matched those observed in vivo, when both stem cells and injury are present. By exploiting the easy imaging geometry of the engineered tissue, cellular mechanisms of action driving the MuSC response to the regenerative template are explored. In vivo outcomes of two known modulators of MuSC-mediated repair, measured by donor fiber production, MuSC niche repopulation, and response to a secondary injury, are phenocopied in the platform only when both the stem cells and injured 3D template are present. The MEndR platform represents a powerful opportunity to explore MuSC-mediated repair and potentially compress the discovery pipeline by combining drug screening and validation in one step.

Adult skeletal muscle tissue harbors the capacity for self-repair due to the presence of tissue resident muscle stem cells (MuSCs). Advances in the area of prospective MuSC isolation demonstrated the potential of cell transplantation therapy as a regenerative medicine strategy to restore strength and long-term regenerative capacity to aged, injured, or diseased skeletal muscle tissue. However, cell loss during ejection, limits to post-injection proliferation, and poor donor cell dispersion distal to the injection site are amongst hurdles to overcome to maximize MuSC transplant impact. Here, we assess a physical blend of hyaluronan and methylcellulose (HAMC) as a bioactive, shear thinning hydrogel cell delivery system to improve MuSC transplantation efficiency. Using in vivo transplantation studies, we found that the HAMC delivery system results in a >45% increase in the number of donor-derived fibers as compared to saline delivery. Furthermore, we observed a significant improvement in donor fiber dispersion when transplanted MuSCs were delivered in the HAMC hydrogel. Studies to assess primary myoblast and MuSC viability in HAMC culture revealed no differences compared to the media control even when the cells were first ejected through a syringe and needle or exposed to regenerating skeletal muscle extract to mimic the transplantation procedure. However, when we quantified absolute numbers, we found that more cells pass through the syringe and needle when delivered in HAMC. Culture in HAMC also increased the proportion of MuSCs in cell cycle, via a CD44-independent mechanism. An effect on myoblast proliferation was not observed, suggesting a hierarchical effect. Finally, a series of transplant studies indicated that HAMC delivery does not influence passive cell clearance or alter the host immune response, but instead may serve to support in vivo expansion by delaying differentiation following transplant. Therefore, we conclude that MuSC engraftment efficacy is improved by delivering the therapeutic cell population within HAMC.