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. 2009 Jul-Aug;45(7):378-387.
doi: 10.1007/s11626-009-9192-7. Epub 2009 May 9.

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 #  1   2 , John W Rumsey #  1 , Neelima Bhargava  1 , Cassie Gregory  2 , Lisa Reidel  1   2 , Jung Fong Kang  1   2 , James J Hickman  1   2
Affiliations

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 et al. In Vitro Cell Dev Biol Anim. 2009 Jul-Aug.

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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Figures

Figure 1
Figure 1
Flow chart showing the technique utilized to grow robust myotubes and muscle-nerve cocultures. The Formulation IX and the medium change protocol, which is the most optimal for myotube and coculture growth.
Figure 2
Figure 2
Different morphologies of myotubes stained with embryonic, myosin heavy chain antibodies (Red) and clustering of acetylcholine receptors (Green) on the membrane surface of myotubes. Scale bar is 50 μm. A. Chain like morphology of the myotubes. B. Branched morphology of the myotubes. C. Spindle shaped morphology of a single myotube. B. Cylinder shaped morphology of a single myotube. E–H. Different morphologies of myotubes indicating the clustering of acetylcholine receptors (Green) on the membrane surface of different myotubes.
Figure 3
Figure 3
Coculture of skeletal muscle* sensory neurons (DRG)* spinal cord neurons. All neurons were stained with the antibody against neurofilament-M (150 KD) (Red) and the myotubes were stained with the antibody against embryonic myosin heavy chain (F 1.652) (Green). Scale bar is 75 μm. A. The large sensory neurons (DRG) were stained with NF 150 (Red). The neurons are shown with white arrows. The myotubes stained with F1.652 (Green) were seen in the same field. B. The neuron process in red are noted running parallel to the myotubes (Green). C. The multi-polar motoneuron was noted in close proximity with the myotubes. In the frame multiple large sensory neurons were observed. All the neurons are pointed out by white arrows. D. A bundle of multiple processes of neurons crossed the striated, branched myotube. E. A single multi-polar motoneuron was observed to wrap around the mytotube. F. Neuron processes wrapping on the myotubes.
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