Differentiation of skeletal muscle and integration of myotubes with silicon microstructures using serum-free medium and a synthetic silane substrate

Abstract

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.

Direct patterning of coplanar polyethylene glycol alkylsilane monolayers by deep-ultraviolet photolithography as a general method for high fidelity, long-term cell patterning and culture

“…Skeletal muscle was dissected from the hind limb thighs of rat pups at embryonic day 18 ͑Charles River Laboratories, Wilmington, MA͒ according to a previously published protocol, 18 with some modification. Tissue samples were collected in a sterile 15 ml centrifuge tube containing 1 ml of calcium and magnesium free phosphate buffered saline.…”

Mechanistic investigation of adult myotube response to exercise and drug treatment in vitro using a multiplexed functional assay system

“…Briefly, the automated system consisted of a class 2 photodiode laser positioned below the stage so that the beam was focused on the cantilever tip and reflected into the center of a four-quadrant photodetector (8,40). A pulse stimulator was used to generate pulses capable of eliciting contraction in the cultured myotubes.…”

“…Skeletal muscle is one tissue for which in vitro analogs have been developed that are capable of effectively recapitulating the functional maturation and performance of the native tissue through controlled stimulation and measurement of the contractile output of embryonic (8,39), neonatal/adult (2,3,16), and human (38) cultured cells. Such systems represent exciting possibilities for drug toxicity/efficacy studies, as well as for the modeling of human disease states in vitro.…”

Fetal bovine serum (FBS): Past – present – future

“…Also, quality and yield of cell culture-based biotechnology products, diagnostics and vaccines may have been affected (Gstraunthaler et al, 2014). As a very visible result of this turmoil in the serum market, the jan et al., 2006, 2011), hippocampal neurons (Varghese et al, 2009), motoneurons (MNs) (Das et al, 2003(Das et al, , 2007aGuo et al, 2010), sensory neurons (Guo et al, 2013;Rumsey et al, 2010), muscle (Das et al, 2006(Das et al, , 2007bVarghese et al, 2009), neuromuscular junction formation Guo et al, 2010;Smith et al, 2013), and a multi-organ system with functional readout (Oleaga et al, 2016).…”