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

  • Mainak Das
  • John W. Rumsey
  • Neelima Bhargava
  • Cassie Gregory
  • Lisa Riedel
  • Jung Fong Kang
  • James J. Hickman


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.


Muscle differentiation Muscle-nerve coculture Myotube Serum-free medium Synthetic substrate 



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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Copyright information

© The Society for In Vitro Biology 2009

Authors and Affiliations

  • Mainak Das
    • 1
    • 2
  • John W. Rumsey
    • 1
  • Neelima Bhargava
    • 1
  • Cassie Gregory
    • 2
  • Lisa Riedel
    • 1
    • 2
  • Jung Fong Kang
    • 1
    • 2
  • James J. Hickman
    • 1
    • 2
  1. 1.NanoScience Technology CenterUniversity of Central FloridaOrlandoUSA
  2. 2.Department of BioengineeringClemson UniversityClemsonUSA

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