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Developing a novel serum-free cell culture model of skeletal muscle differentiation by systematically studying the role of different growth factors in myotube formation

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Abstract

This work describes the step-by-step development of a novel, serum-free, in vitro cell culture system resulting in the formation of robust, contracting, multinucleate myotubes from dissociated skeletal muscle cells obtained from the hind limbs of fetal rats. This defined system consisted of a serum-free medium formulation developed by the systematic addition of different growth factors as well as a nonbiological cell growth promoting substrate, N-1[3-(trimethoxysilyl) propyl] diethylenetriamine. Each growth factor in the medium was experimentally evaluated for its effect on myotube formation. The resulting myotubes were evaluated immunocytochemically using embryonic skeletal muscle, specifically the myosin heavy chain antibody. Based upon this analysis, we propose a new skeletal muscle differentiation protocol that reflects the roles of the various growth factors which promote robust myotube formation. Further observation noted that the proposed skeletal muscle differentiation technique also supported muscle–nerve coculture. Immunocytochemical evidence of nerve–muscle coculture has also been documented. Applications for this novel culture system include biocompatibility and skeletal muscle differentiation studies, understanding myopathies, neuromuscular disorders, and skeletal muscle tissue engineering.

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Acknowledgments

The F1.652 monoclonal antibody developed by Helan Blau was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA. This work was supported by DARPA grant DARPA-ITO N65236-01-1-7400 and NIH grant number 5R01 NS 050452. The initial experiments for this work were performed in the Bioengineering Department at Clemson University, Clemson, SC.

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Authors and Affiliations

  1. NanoScience Technology Center, University of Central Florida, 12424 Research Parkway, Suite 400, Orlando, FL, 32826, USA

    Mainak Das, John W. Rumsey, Neelima Bhargava, Lisa Riedel, Jung Fong Kang & James J. Hickman

  2. Department of Bioengineering, Clemson University, Clemson, SC, 29634, USA

    Mainak Das, Cassie Gregory, Lisa Riedel, Jung Fong Kang & James J. Hickman

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  1. Mainak Das
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  2. John W. Rumsey
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Correspondence to James J. Hickman.

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Editor: J. Denry Sato

Mainak Das and John W Rumsey contributed equally to this work.

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Das, M., Rumsey, J.W., Bhargava, N. et al. Developing a novel serum-free cell culture model of skeletal muscle differentiation by systematically studying the role of different growth factors in myotube formation. In Vitro Cell.Dev.Biol.-Animal 45, 378–387 (2009). https://doi.org/10.1007/s11626-009-9192-7

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