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

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 non-biological, cell growth promoting substrate, N-1[3-(trimethoxysilyl) propyl] diethylenetriamine (DETA). 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 co-culture. Immunocytochemical evidence of nerve-muscle co-culture 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.

Functional myotube formation from adult rat satellite cells in a defined serum‐free system

“…The starting medium formulation, surface modification and plating technique was developed previously by our lab to create a system for the development of myotubes derived from embryonic E18 rat hind‐limbs . However, this system was insufficient to enable the development of myotubes from satellite cells isolated from the TA of adult rats.…”

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function <em>In Vitro</em>

“…Due to the undefined nature of animal sera, the effect of novel therapeutics may be confounded by the inclusion of such additives in the culture medium. Serum-free media formulations are available for both rodent and human skeletal muscle cultures 18–21 and provide a more controlled, well defined environment for performing compound assessment. Similarly, the use of biological coatings (collagen, laminin etc.)…”

Muscular dystrophy in a dish: engineered human skeletal muscle mimetics for disease modeling and drug discovery

“…Similarly, transforming growth factor (TGF)-β treatment has been shown to improve peak twitch and tetanic contractile activity, as well as sarcomere development and organization, in scaffold-free engineered skeletal muscle tissues [74]. Studies have also investigated the addition of a variety of neuronal growth factors to muscle cultures, and demonstrated that such supplementation can significantly enhance myotube differentiation and sarcomeric development in cultured skeletal muscle cells [75]. …”

“…Studies have shown that combining electrical stimulation with mechanical strain improves skeletal muscle maturation over and above what is achievable with either stimulus alone [29, 110]. Similarly, studies into the sequential addition of multiple growth factors describe the increased benefit of supplementation to cultures of skeletal muscle myotubes [75]. Likewise, the addition of neuronal factors has been shown to promote the spontaneous contractile activity of cultured human myotubes, and a lack of contraction is observed when these factors are omitted [111].…”