Abstract
BackgroundSkeletal muscle contributes to roughly 40% of lean body mass, and its loss contributes to morbidity and mortality in a variety of pathogenic conditions. Significant insights into muscle function have been made using cultured cells, in particular, the C2C12 myoblast line. However, differentiation of these cells in vitro typically yields immature myotubes relative to skeletal muscles in vivo. While many efforts have attempted to improve the maturity of cultured myotubes, including the use of bioengineered substrates, lack of molecular characterization has precluded their widespread implementation. This study characterizes morphological, molecular, and transcriptional features of C2C12 myotubes cultured on crosslinked, micropatterned gelatin substrates fabricated using previously established methods and compares them to myotubes grown on unpatterned gelatin or traditional plasticware.MethodsWe used immunocytochemistry, SDS-PAGE, and RNAseq to characterize C2C12 myotubes grown on micropatterned gelatin hydrogels, unpatterned gelatin hydrogels, and typical cell culture substrates (i.e., plastic or collagen-coated glass) across a differentiation time course. The ability to form aligned sarcomeres and myofilament protein concentration was assessed. Additionally, the transcriptome was analyzed across the differentiation time course.ResultsC2C12 myotubes grown on micropatterned gelatin hydrogels display an increased ability to form aligned sarcomeres as well as increased contractile protein content relative to myotubes cultured on unpatterned gelatin and plastic. Additionally, genes related to sarcomere formation and in vivo muscle maturation are upregulated in myotubes grown on micropatterned gelatin hydrogels relative to control myotubes.ConclusionsOur results suggest that growing C2C12 myotubes on micropatterned gelatin hydrogels accelerates sarcomere formation and yields a more fully matured myotube culture. Thus, the use of micropatterned hydrogels is a viable and simple approach to better model skeletal muscle biology in vitro.
Highlights
Protein expression, and transcriptomics, we found that micropatterned gelatin hydrogels accelerate and advance myogenic maturation and sarcomere formation in C2C12 myotubes when compared to traditional culture methods
Micropatterned C2C12 myotubes develop more sarcomeres than those grown on other substrates In this study, we compared C2C12 myotubes cultured on three different substrates
Because one defining property of skeletal muscle in vivo is the presence of sarcomeres yet most C2C12 myotube cultures do not yield robust sarcomeres, we examined sarcomere formation in vitro
Summary
Skeletal muscle contributes to roughly 40% of lean body mass, and its loss contributes to morbidity and mortality in a variety of pathogenic conditions. Significant insights into muscle function have been made using cultured cells, in particular, the C2C12 myoblast line. Differentiation of these cells in vitro typically yields immature myotubes relative to skeletal muscles in vivo. Loss of skeletal muscle mass during aging and pathogenesis is a known contributor to morbidity and mortality, a large contingent of research aims to prolong health by maintaining muscle quality [3,4,5,6,7]. While there is no replacement for studies of muscle in vivo, muscle cell culture models allow for more rapid and facile manipulation to address mechanistic questions and perform drug screening experiments. The C2C12 subclone has become widely used in the skeletal muscle field as a cell culture model [9,10,11,12]
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