Engineered functional skeletal muscle tissues for in vitro studies

Abstract

The skeletal muscle is the largest tissue of the human body. Its main function is to generate contractile forces, essential for locomotion, thermogenesis, and metabolism. Fundamental research on skeletal muscle in health and disease, and preclinical research for new therapies, are currently based on 2D in vitro cell cultures and in vivo animal models. However, these strategies have important shortcomings. For instance, conventional cell culture models cannot emulate the complex 3D architecture of native skeletal muscle, and the species-specific differences in animal models limit their relevance to humans. In contrast, engineered skeletal muscle tissues are emerging as in vitro 3D cell culture models that complement existing 2D strategies. These engineered tissues can offer an improved microenvironment resembling native muscle tissue, comprised of bundles of aligned, multinucleated fibers. Therefore, the main objective of this thesis was to develop 3D skeletal muscle tissues for in vitro studies of muscle metabolism and disease modeling. Skeletal muscle precursor cells were encapsulated in microfabricated hydrogel scaffolds, introducing the appropriate topographical and microenvironmental cues to guide muscle fiber formation. First, photocrosslinkable gelatin methacryloyl (GelMA)-based composite hydrogels were synthesized and evaluated as cell-laden bioinks for 3D bioprinting of murine skeletal muscle tissue. The fabrication conditions were optimized to ensure the biocompatibility of the process and promote in vitro myogenesis. Our results demonstrated that the composite hydrogels have a higher resistance to degradation than GelMA hydrogels. Thus, the bioprinted scaffolds maintained their 3D structure over a prolonged culture period. Furthermore, the shear stress during extrusion bioprinting combined with the appropriate scaffold geometry resulted in highly aligned myoblasts that correctly differentiated into multinucleated myotubes. Considering these results, GelMA-carboxymethylcellulose methacrylate (CMCMA) hydrogels were then used to generate skeletal muscle microtissues in long-lasting cell cultures. Photomold patterning of cell-laden GelMA-CMCMA filaments led to the formation of highly aligned 3D myotubes expressing sarcomeric proteins. Moreover, the presented protocols were highly biocompatible and reproducible. Murine skeletal muscle microtissues were fabricated in a microfluidic platform integrated with an electrical stimulation system and biosensors for monitoring muscle metabolism in situ. Here, we measured the contraction-induced release of muscle-secreted cytokines upon electrical or biological stimulation. The obtained results confirmed the endocrine function of the bioengineered tissues, obtaining in vivo-like responses upon exercise or endotoxin-induced inflammation. Then, the photomold patterning protocol was optimized for human cells to develop the first in vitro 3D model of myotonic dystrophy type 1 (DM1) human skeletal muscle. DM1 is the most prevalent hereditary myopathy in adults, and there is no effective treatment to date. We proved that 3D micropatterning enhances DM1 myotube formation compared to 2D cultures. Furthermore, we detected the reduced thickness of 3D DM1 myotubes compared to healthy controls, which was proposed as a new in vitro structural phenotype. Thus, as a proof-of-concept, we demonstrated that treatment with an antisense oligonucleotide, antagomiR-23b, could rescue both molecular and structural phenotypes in these bioengineered DM1 muscle tissues. Finally, animal-derived components were eliminated to develop in vitro functional tissues in xeno-free cell culture as a next step towards improving bioengineered human skeletal muscle tissues. Cell-laden nanocomposite hydrogels consisting of human platelet lysate and functionalized cellulose nanocrystals (HUgel) were fabricated in hydrogel casting platforms that implemented uniaxial tension during matrix remodeling. We modulated the content of cellulose nanocrystals to tune the mechanical and biological properties of HUgel and favor the formation of long, highly aligned myotube bundles. Additionally, we performed in situ force measurements of electrical stimulation-induced contractions. Altogether, the results presented in this thesis provide promising approaches to advanced cell culture models of skeletal muscle tissue that could be valuable tools for fundamental studies, disease modeling, and future personalized medicine.

El músculo esquelético tiene funciones esenciales para la salud que pueden verse afectadas por enfermedades neuromusculares o metabólicas. Actualmente, la investigación fundamental y preclínica se basa en cultivos celulares en 2D y modelos animales. Sin embargo, estos ensayos tienen relevancia limitada para la salud humana. En cambio, modelos in vitro de tejidos 3D que mimeticen la arquitectura y funcionalidad del músculo esquelético, podrían complementar las estrategias 2D tradicionales. Por lo tanto, el objetivo principal de esta tesis fue desarrollar tejidos de músculo esquelético en 3D para estudios sobre el metabolismo muscular y modelos de enfermedades in vitro. Los tejidos fueron desarrollados mediante diferentes técnicas de microfabricación de hidrogeles, en los que se encapsularon células precursoras del músculo esquelético introduciendo las señales topográficas adecuadas para guiar la formación de fibras musculares. Las propiedades de estos biomateriales fueron optimizadas para garantizar su biocompatibilidad y promover la miogénesis. Estos biomateriales mantienen su estructura durante periodos de cultivo prolongados, permitiendo la formación y diferenciación de miotubos 3D altamente alineados. La función endócrina de los tejidos fue evaluada utilizando un dispositivo músculo-en-un-chip, con el que fue posible medir la liberación de citoquinas secretadas tras estimulación eléctrica o biológica. Posteriormente, se desarrolló el primer modelo 3D de músculo esquelético humano para la distrofia miotónica tipo 1. Como prueba de concepto, demostramos que el tratamiento con un oligonucleótido antisentido, antagomiR-23b, podría rescatar fenotipos moleculares y estructurales en los tejidos fabricados a partir de células de pacientes. Finalmente, se desarrollaron tejidos funcionales en cultivos celulares xeno-free, con el objetivo de incrementar la relevancia de modelos humanos en los que fue posible medir las fuerzas generada por tejidos contráctiles. En conjunto, los resultados de esta tesis proporcionan enfoques prometedores para modelos avanzados de músculo esquelético que podrían ser herramientas valiosas para estudios fundamentales, modelos de enfermedades y medicina personalizada.