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  • Engineering a 3D in vitro model of human skeletal muscle at the single fiber scale.

Engineering a 3D in vitro model of human skeletal muscle at the single fiber scale.

PloS one (2020-05-07)
Anna Urciuolo, Elena Serena, Rusha Ghua, Susi Zatti, Monica Giomo, Nicolò Mattei, Massimo Vetralla, Giulia Selmin, Camilla Luni, Nicola Vitulo, Giorgio Valle, Libero Vitiello, Nicola Elvassore
ABSTRACT

The reproduction of reliable in vitro models of human skeletal muscle is made harder by the intrinsic 3D structural complexity of this tissue. Here we coupled engineered hydrogel with 3D structural cues and specific mechanical properties to derive human 3D muscle constructs ("myobundles") at the scale of single fibers, by using primary myoblasts or myoblasts derived from embryonic stem cells. To this aim, cell culture was performed in confined, laminin-coated micrometric channels obtained inside a 3D hydrogel characterized by the optimal stiffness for skeletal muscle myogenesis. Primary myoblasts cultured in our 3D culture system were able to undergo myotube differentiation and maturation, as demonstrated by the proper expression and localization of key components of the sarcomere and sarcolemma. Such approach allowed the generation of human myobundles of ~10 mm in length and ~120 μm in diameter, showing spontaneous contraction 7 days after cell seeding. Transcriptome analyses showed higher similarity between 3D myobundles and skeletal signature, compared to that found between 2D myotubes and skeletal muscle, mainly resulting from expression in 3D myobundles of categories of genes involved in skeletal muscle maturation, including extracellular matrix organization. Moreover, imaging analyses confirmed that structured 3D culture system was conducive to differentiation/maturation also when using myoblasts derived from embryonic stem cells. In conclusion, our structured 3D model is a promising tool for modelling human skeletal muscle in healthy and diseases conditions.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
N-Methylethylenediamine, 95%
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Poly(dimethylsiloxane), viscosity 1.0 cSt (25 °C)
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Anti-Laminin antibody produced in rabbit, 0.5 mg/mL, affinity isolated antibody, buffered aqueous solution
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Monoclonal Anti-α-Actinin (Sarcomeric) antibody produced in mouse, clone EA-53, ascites fluid
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Anti-Myosin (Skeletal, Fast) antibody, Mouse monoclonal, clone MY-32, purified from hybridoma cell culture
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Laminin from Engelbreth-Holm-Swarm murine sarcoma basement membrane, 1-2 mg/mL in Tris-buffered saline, 0.2 μm filtered, BioReagent, suitable for cell culture
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Anti-Desmin antibody produced in rabbit, whole antiserum