Positioning Microtissues on the Differentiation Continuum Reveals their Utility for Studying Skeletal Myopathies and Metabolic Disease

Insulin resistance in skeletal muscle is the primary defect in type 2 diabetes (T2D). While current in vitro models utilize two-dimensional (2D) myotubes, these cultures express low levels of insulin-responsive GLUT4 and elevated levels of insulin-independent GLUT1, limiting their relevance for modeling insulin resistance. Human skeletal muscle microtissues, differentiated under uniaxial tension across two microposts, exhibit greater insulin responsiveness than 2D myotubes and are amenable to contractile stimulation. However, a microtissue-based model of T2D has not been developed. The motivation of this thesis was the establishment of a microtissue model to investigate T2D-associated impairments in force generation, myokine signaling, and protein synthesis, thereby establishing a multiplex platform for the study of dysregulation in T2D. First, to ensure accurate contractile assessments, we developed protocols to identify the electrical field stimulation parameters eliciting peak twitch and tetanus force from microtissues. The generalizability of these approaches was demonstrated across protocol users, micropost culture devices, and distinct myoblast cell line types. Our results revealed cell type-specific contractile properties, with all microtissues contracting and relaxing more slowly than native muscle. Next, to ensure accurate assessment of protein synthesis, we implemented stable isotope amino acid tracer to measure fraction-specific muscle protein synthesis rates in microtissues, demonstrating improved accuracy over puromycin-based methods. We uncovered that the myofibrillar protein fraction of microtissues is more stable than the sarcoplasmic fraction, a property of native human muscle absent in 2D myotubes. Finally, to evaluate the relevance of microtissues for modeling insulin resistance, we characterized their glucose transporter profile. Microtissues exhibited significantly higher glucose uptake potential than 2D myotubes, with a ~60-fold higher GLUT4:GLUT1 ratio. However, they required supraphysiological insulin concentrations to maintain functional integrity under conventional insulin-based differentiation protocols. Through this work, we demonstrated the need for an insulin-free differentiation media formulation to enable T2D modeling. In parallel, we benchmarked the functional and metabolic properties of microtissues relative to 2D myotubes and native muscle. These findings identify a hurdle for the development of a multiplex microtissue-based T2D model while also delivering a comparative framework to guide platform selection for the study of skeletal myopathies and metabolic disease.

Department dc:contributor.department

Biomedical Engineering

Year dc:date.issued

2025

Author dc:creator

• Tiper, Yekaterina

Advisor dc:contributor.advisor

• Gilbert, Penney M.

• Contractile force
• Engineered skeletal muscle
• GLUT4
• Myotubes
• Protein synthesis
• Type 2 diabetes

Statement dc:rights

• Attribution 4.0 International

Licence dc:rights.uri

• http://creativecommons.org/licenses/by/4.0/

Handle dc:identifier.uri

https://hdl.handle.net/1807/144835

OAI identifier oai:identifier

oai:utoronto.scholaris.ca:1807/144835