Bioengineered in vitro skeletal muscles as new tools for muscular dystrophies preclinical studies

Muscular dystrophies are a group of highly disabling disorders that share degenerative muscle weakness and wasting as common symptoms. To date, there is not an effective cure for these diseases. In the last years, bioengineered tissues have emerged as powerful tools for preclinical studies. In this review, we summarize the recent technological advances in skeletal muscle tissue engineering. We identify several ground-breaking techniques to fabricate in vitro bioartificial muscles. Accumulating evidence shows that scaffold-based tissue engineering provides topographical cues that enhance the viability and maturation of skeletal muscle. Functional bioartificial muscles have been developed using human myoblasts. These tissues accurately responded to electrical and biological stimulation. Moreover, advanced drug screening tools can be fabricated integrating these tissues in electrical stimulation platforms. However, more work introducing patient-derived cells and integrating these tissues in microdevices is needed to promote the clinical translation of bioengineered skeletal muscle as preclinical tools for muscular dystrophies

Xeno-free bioengineered human skeletal muscle tissue using human platelet lysate-based hydrogels

Bioengineered human skeletal muscle tissues have emerged in the last years as new in vitro systems for disease modeling. These bioartificial muscles are classically fabricated by encapsulating human myogenic precursor cells in a hydrogel scaffold that resembles the extracellular matrix. However, most of these hydrogels are derived from xenogenic sources, and the culture media is supplemented with animal serum, which could interfere in drug testing assays. On the contrary, xeno-free biomaterials and culture conditions in tissue engineering offer increased relevance for developing human disease models. In this work, we used human platelet lysate-based nanocomposite hydrogels (HUgel) as scaffolds for human skeletal muscle tissue engineering. These hydrogels consist of human platelet lysate reinforced with cellulose nanocrystals (a-CNC) that allow tunable mechanical, structural, and biochemical properties for the 3D culture of stem cells. Here, we developed hydrogel casting platforms to encapsulate human muscle satellite stem cells in HUgel. The a-CNC content was modulated to enhance matrix remodeling, uniaxial tension, and self-organization of the cells, resulting in the formation of highly aligned, long myotubes expressing sarcomeric proteins. Moreover, the bioengineered human muscles were subjected to electrical stimulation, and the exerted contractile forces were measured in a non-invasive manner. Overall, our results demonstrated that the bioengineered human skeletal muscles could be built in xeno-free cell culture platforms to assess tissue functionality, which is promising for drug development applications

Bioengineered in vitro 3D model of myotonic dystrophy type 1 human skeletal muscle

Myotonic dystrophy type 1 (DM1) is the most common hereditary myopathy in the adult population. The disease is characterized by progressive skeletal muscle degeneration that produces severe disability. At present, there is still no effective treatment for DM1 patients, but the breakthroughs in understanding the molecular pathogenic mechanisms in DM1 have allowed the testing of new therapeutic strategies. Animal models and in vitro two-dimensional cell cultures have been essential for these advances. However, serious concerns exist regarding how faithfully these models reproduce the biological complexity of the disease. Biofabrication tools can be applied to engineer human three-dimensional (3D) culture systems that complement current preclinical research models. Here, we describe the development of the first in vitro 3D model of DM1 human skeletal muscle. Transdifferentiated myoblasts from patient-derived fibroblasts were encapsulated in micromolded gelatin methacryloyl-carboxymethyl cellulose methacrylate hydrogels through photomold patterning on functionalized glass coverslips. These hydrogels present a microstructured topography that promotes myoblasts alignment and differentiation resulting in highly aligned myotubes from both healthy and DM1 cells in a long-lasting cell culture. The DM1 3D microtissues recapitulate the molecular alterations detected in patient biopsies. Importantly, fusion index analyses demonstrate that 3D micropatterning significantly improved DM1 cell differentiation into multinucleated myotubes compared to standard cell cultures. Moreover, the characterization of the 3D cultures of DM1 myotubes detects phenotypes as the reduced thickness of myotubes that can be used for drug testing. Finally, we evaluated the therapeutic effect of antagomiR-23b administration on bioengineered DM1 skeletal muscle microtissues. AntagomiR-23b treatment rescues both molecular DM1 hallmarks and structural phenotype, restoring myotube diameter to healthy control sizes. Overall, these new microtissues represent an improvement over conventional cell culture models and can be used as biomimetic platforms to establish preclinical studies for myotonic dystrophy

Composite biomaterials as long-lasting scaffolds for 3D bioprinting of highly aligned muscle tissue

New biocompatible materials have enabled the direct 3D printing of complex functional living tissues, such as skeletal and cardiac muscle. Gelatinmethacryloyl (GelMA) is a photopolymerizable hydrogel composed of natural gelatin functionalized with methacrylic anhydride. However, it is difficult to obtain a single hydrogel that meets all the desirable properties for tissue engineering. In particular, GelMA hydrogels lack versatility in their mechanical properties and lasting 3D structures. In this work, a library of composite biomaterials to obtain versatile, lasting, and mechanically tunable scaffolds are presented. Two polysaccharides, alginate and carboxymethyl cellulose chemically functionalized with methacrylic anhydride, and a synthetic material, such as poly(ethylene glycol) diacrylate are combined with GelMA to obtain photopolymerizable hydrogel blends. Physical properties of the obtained composite hydrogels are screened and optimized for the growth and development of skeletal muscle fibers from C2C12 murine cells, and compared with pristine GelMA. All these composites show high resistance to degradation maintaining the 3D structure with high fidelity over several weeks. Altogether, in this study a library of biocompatible novel and totally versatile composite biomaterials are developed and characterized, with tunable mechanical properties that give structure and support myotube formation and alignment

Muscle-on-a-chip with an on-site multiplexed biosensing system for in situ monitoring of secreted IL-6 and TNF-α

Despite the increasing number of organs-on-a-chip that have been developed in the past decade, limited efforts have been made to integrate a sensing system for in situ continual measurements of biomarkers from three-dimensional (3D) tissues. Here, we present a custom-made integrated platform for muscle cell stimulation under fluidic conditions connected with a multiplexed high-sensitivity electrochemical sensing system for in situ monitoring. To demonstrate this, we use our system to measure the release levels and release time of interleukin 6 and tumor necrosis factor alpha in vitro by 3D muscle microtissue under electrical and biological stimulations. Our experimental design has enabled us to perform multiple time point measurements using functionalized screen-printed gold electrodes with sensitivity in the ng mL−1 range. This affordable setup is uniquely suited for monitoring factors released by 3D single cell types upon external stimulation for metabolic studies

Microphysiological sensing platform for an in-situ detection of tissue-secreted cytokines

Understanding the protein-secretion dynamics from single, specific tissues is critical toward the advancement of disease detection and treatments. However, such secretion dynamics remain difficult to measure in vivo due to the uncontrolled contributions from other tissue populations. Here, we describe an integrated platform designed for the reliable, near real-time measurements of cytokines secreted from an in vitro single-tissue model. In our setup, we grow 3D biomimetic tissues to discretize cytokine source, and we separate them from a magnetic microbead-based biosensing system using a Transwell insert. This design integrates physiochemically controlled biological activity, high-sensitivity protein detection (LOD < 20 pg mL−1), and rapid protein diffusion to enable non-invasive, near real-time measurements. To showcase the specificity and sensitivity of the system, we use our setup to probe the inflammatory process related to the protein Interleukine 6 (IL-6) and to the Tumor Necrosis Factor (TNF-α). We show that our setup can monitor the time-dependence profile of IL-6 and TNF-α secretion that results from the electrical and chemical stimulation of 3D skeletal muscle tissues. We demonstrate a novel and affordable methodology for discretizing the secretion kinetics of specific tissues for advancing metabolic-disorder studies and drug-screening applications

Engineered functional skeletal muscle tissues for in vitro studies

[eng] 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.[spa] 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

Microphysiological sensing platform for an in-situ detection of tissue-secreted cytokines

Understanding the protein-secretion dynamics from single, specific tissues is critical toward the advancement of disease detection and treatments. However, such secretion dynamics remain difficult to measure in vivo due to the uncontrolled contributions from other tissue populations. Here, we describe an integrated platform designed for the reliable, near real-time measurements of cytokines secreted from an in vitro single-tissue model. In our setup, we grow 3D biomimetic tissues to discretize cytokine source, and we separate them from a magnetic microbead-based biosensing system using a Transwell insert. This design integrates physiochemically controlled biological activity, high-sensitivity protein detection (LOD < 20 pg mL−1), and rapid protein diffusion to enable non-invasive, near real-time measurements. To showcase the specificity and sensitivity of the system, we use our setup to probe the inflammatory process related to the protein Interleukine 6 (IL-6) and to the Tumor Necrosis Factor (TNF-α). We show that our setup can monitor the time-dependence profile of IL-6 and TNF-α secretion that results from the electrical and chemical stimulation of 3D skeletal muscle tissues. We demonstrate a novel and affordable methodology for discretizing the secretion kinetics of specific tissues for advancing metabolic-disorder studies and drug-screening applications

Composite biomaterials as long-lasting scaffolds for 3D bioprinting of highly aligned muscle tissue

New biocompatible materials have enabled the direct 3D printing of complex functional living tissues, such as skeletal and cardiac muscle. Gelatinmethacryloyl (GelMA) is a photopolymerizable hydrogel composed of natural gelatin functionalized with methacrylic anhydride. However, it is difficult to obtain a single hydrogel that meets all the desirable properties for tissue engineering. In particular, GelMA hydrogels lack versatility in their mechanical properties and lasting 3D structures. In this work, a library of composite biomaterials to obtain versatile, lasting, and mechanically tunable scaffolds are presented. Two polysaccharides, alginate and carboxymethyl cellulose chemically functionalized with methacrylic anhydride, and a synthetic material, such as poly(ethylene glycol) diacrylate are combined with GelMA to obtain photopolymerizable hydrogel blends. Physical properties of the obtained composite hydrogels are screened and optimized for the growth and development of skeletal muscle fibers from C2C12 murine cells, and compared with pristine GelMA. All these composites show high resistance to degradation maintaining the 3D structure with high fidelity over several weeks. Altogether, in this study a library of biocompatible novel and totally versatile composite biomaterials are developed and characterized, with tunable mechanical properties that give structure and support myotube formation and alignment

Composite biomaterials as long-lasting scaffolds for 3D bioprinting of highly aligned muscle tissue

New biocompatible materials have enabled the direct 3D printing of complex functional living tissues, such as skeletal and cardiac muscle. Gelatinmethacryloyl (GelMA) is a photopolymerizable hydrogel composed of natural gelatin functionalized with methacrylic anhydride. However, it is difficult to obtain a single hydrogel that meets all the desirable properties for tissue engineering. In particular, GelMA hydrogels lack versatility in their mechanical properties and lasting 3D structures. In this work, a library of composite biomaterials to obtain versatile, lasting, and mechanically tunable scaffolds are presented. Two polysaccharides, alginate and carboxymethyl cellulose chemically functionalized with methacrylic anhydride, and a synthetic material, such as poly(ethylene glycol) diacrylate are combined with GelMA to obtain photopolymerizable hydrogel blends. Physical properties of the obtained composite hydrogels are screened and optimized for the growth and development of skeletal muscle fibers from C2C12 murine cells, and compared with pristine GelMA. All these composites show high resistance to degradation maintaining the 3D structure with high fidelity over several weeks. Altogether, in this study a library of biocompatible novel and totally versatile composite biomaterials are developed and characterized, with tunable mechanical properties that give structure and support myotube formation and alignment