Culturing C2C12 myotubes on micromolded gelatin hydrogels accelerates myotube maturation
Abstract Background 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. However,...
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doaj-2638f220d5fa472eb7f0b24cea773c512020-11-25T03:19:51ZengBMCSkeletal Muscle2044-50402019-06-019111010.1186/s13395-019-0203-4Culturing C2C12 myotubes on micromolded gelatin hydrogels accelerates myotube maturationLance T. Denes0Lance A. Riley1Joseph R. Mijares2Juan D. Arboleda3Kendra McKee4Karyn A. Esser5Eric T. Wang6Department of Molecular Genetics and Microbiology, Center for Neurogenetics, Myology Institute, College of Medicine, University of FloridaDepartment of Physiology and Functional Genomics, Myology Institute, College of Medicine, University of FloridaDepartment of Physiology and Functional Genomics, Myology Institute, College of Medicine, University of FloridaDepartment of Molecular Genetics and Microbiology, Center for Neurogenetics, Myology Institute, College of Medicine, University of FloridaDepartment of Molecular Genetics and Microbiology, Center for Neurogenetics, Myology Institute, College of Medicine, University of FloridaDepartment of Physiology and Functional Genomics, Myology Institute, College of Medicine, University of FloridaDepartment of Molecular Genetics and Microbiology, Center for Neurogenetics, Myology Institute, College of Medicine, University of FloridaAbstract Background 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. 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. Methods We 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. Results C2C12 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. Conclusions Our 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.http://link.springer.com/article/10.1186/s13395-019-0203-4C2C12RNAseqMyotubesMicromoldingHydrogelsSarcomere |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Lance T. Denes Lance A. Riley Joseph R. Mijares Juan D. Arboleda Kendra McKee Karyn A. Esser Eric T. Wang |
spellingShingle |
Lance T. Denes Lance A. Riley Joseph R. Mijares Juan D. Arboleda Kendra McKee Karyn A. Esser Eric T. Wang Culturing C2C12 myotubes on micromolded gelatin hydrogels accelerates myotube maturation Skeletal Muscle C2C12 RNAseq Myotubes Micromolding Hydrogels Sarcomere |
author_facet |
Lance T. Denes Lance A. Riley Joseph R. Mijares Juan D. Arboleda Kendra McKee Karyn A. Esser Eric T. Wang |
author_sort |
Lance T. Denes |
title |
Culturing C2C12 myotubes on micromolded gelatin hydrogels accelerates myotube maturation |
title_short |
Culturing C2C12 myotubes on micromolded gelatin hydrogels accelerates myotube maturation |
title_full |
Culturing C2C12 myotubes on micromolded gelatin hydrogels accelerates myotube maturation |
title_fullStr |
Culturing C2C12 myotubes on micromolded gelatin hydrogels accelerates myotube maturation |
title_full_unstemmed |
Culturing C2C12 myotubes on micromolded gelatin hydrogels accelerates myotube maturation |
title_sort |
culturing c2c12 myotubes on micromolded gelatin hydrogels accelerates myotube maturation |
publisher |
BMC |
series |
Skeletal Muscle |
issn |
2044-5040 |
publishDate |
2019-06-01 |
description |
Abstract Background 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. 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. Methods We 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. Results C2C12 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. Conclusions Our 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. |
topic |
C2C12 RNAseq Myotubes Micromolding Hydrogels Sarcomere |
url |
http://link.springer.com/article/10.1186/s13395-019-0203-4 |
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