Differentiation of skeletal muscle and integration of myotubes with silicon microstructures using serum-free medium and a synthetic silane substrate.
This protocol describes a cell culture model to study the differentiation of fetal rat skeletal muscle cells. The model uses serum-free medium, a nonbiological substrate N-1[3(trimethoxysilyl)propyl] diethylenetriamine (DETA) and fabricated microcantilevers to promote the differentiation of dissociated rat myocytes into robust myotubes. In this protocol, we also describe how to characterize the myotubes on the basis of morphology, immunocytochemistry and electrophysiology. Here, four major techniques are employed: fabrication of cantilevers, surface modification of the glass and cantilever substrates with a DETA SAM, a serum-free medium and refined culture techniques. This culture system has potential applications in biocompatibility studies, bioartificial muscle engineering, skeletal muscle differentiation studies and for better understanding of myopathies and neuromuscular disorders. The model can be established in 26-33 d.
Das
Mainak
M
NanoScience Technology Center, University of Central Florida, Orlando, Florida 32826, USA.
Wilson
Kerry
K
Molnar
Peter
P
Hickman
James J
JJ
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002455:Cell Division; D016895:Culture Media, Serum-Free; D005333:Fetus; D008855:Microscopy, Electron, Scanning; D018485:Muscle Fibers, Skeletal / Q000166:cytology* / Q000648:ultrastructure; D018482:Muscle, Skeletal / Q000166:cytology* / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley; D012825:Silicon
D016895:Culture Media, Serum-Free; D012825:Silicon