Developing a novel serum-free cell culture model of skeletal muscle differentiation by systematically studying the role of different growth factors in myotube formation.
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.
Das
Mainak
M
NanoScience Technology Center, University of Central Florida, Orlando, FL 32826, USA.|Department of Bioengineering, Clemson University, Clemson, SC 29634, USA.
Rumsey
John W
JW
NanoScience Technology Center, University of Central Florida, Orlando, FL 32826, USA.
Bhargava
Neelima
N
NanoScience Technology Center, University of Central Florida, Orlando, FL 32826, USA.
Gregory
Cassie
C
Department of Bioengineering, Clemson University, Clemson, SC 29634, USA.
Reidel
Lisa
L
NanoScience Technology Center, University of Central Florida, Orlando, FL 32826, USA.|Department of Bioengineering, Clemson University, Clemson, SC 29634, USA.
Kang
Jung Fong
JF
NanoScience Technology Center, University of Central Florida, Orlando, FL 32826, USA.|Department of Bioengineering, Clemson University, Clemson, SC 29634, USA.
Hickman
James J
JJ
NanoScience Technology Center, University of Central Florida, Orlando, FL 32826, USA.|Department of Bioengineering, Clemson University, Clemson, SC 29634, USA.
D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects; D048430:Cell Shape; D018920:Coculture Techniques; D016895:Culture Media, Serum-Free; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology* / Q000502:physiology; D008954:Models, Biological; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011073:Polyamines / Q000494:pharmacology; D051381:Rats; D013499:Surface Properties
D016895:Culture Media, Serum-Free; D036341:Intercellular Signaling Peptides and Proteins; D011073:Polyamines; C005391:diethylenetriamine