Extended 2D myotube culture recapitulates postnatal fibre type plasticity.pdf (2.7 MB)
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Extended 2D myotube culture recapitulates postnatal fibre type plasticity

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journal contribution
posted on 18.03.2016, 09:43 by Sujith Sebastian, Leah Goulding, Suresh V. Kuchipudi, Kin-Chow Chang
BACKGROUND. The traditional problems of performing skeletal muscle cell cultures derived from mammalian or avian species are limited myotube differentiation, and transient myotube persistence which greatly restricts the ability of myotubes to undergo phenotypic maturation. We report here on a major technical breakthrough in the establishment of a simple and effective method of extended porcine myotube cultures (beyond 50 days) in two-dimension (2D) that recapitulates key features of postnatal fibre types. RESULTS. Primary porcine muscle satellite cells (myoblasts) were isolated from the longissimus dorsi of 4 to 6 weeks old pigs for 2D cultures to optimise myotube formation, improve surface adherence and characterise myotube maturation. Over 95% of isolated cells were myoblasts as evidenced by the expression of Pax3 and Pax7. Our relatively simple approach, based on modifications of existing surface coating reagents (Maxgel), and of proliferation and differentiation (Ultroser G) media, typically achieved by 5 days of differentiation fusion index of around 80 % manifested in an abundance of discrete myosin heavy chain (MyHC) slow and fast myotubes. There was little deterioration in myotube viability over 50 days, and the efficiency of myotube formation was maintained over seven myoblast passages. Regular spontaneous contractions of myotubes were frequently observed throughout culture. Myotubes in extended cultures were able to undergo phenotypic adaptation in response to different culture media, including the adoption of a dominant postnatal phenotype of fast-glycolytic MyHC 2x and 2b expression by about day 20 of differentiation. Furthermore, fast-glycolytic myotubes coincided with enhanced expression of the putative porcine long intergenic non-coding RNA (linc-MYH), which has recently been shown to be a key coordinator of MyHC 2b expression in vivo. CONCLUSIONS. Our revised culture protocol allows the efficient differentiation and fusion of porcine myoblasts into myotubes and their prolonged adherence to the culture surface. Furthermore, we are able to recapitulate in 2D the maturation process of myotubes to resemble postnatal fibre types which represent a major technical advance in opening access to the in vitro study of coordinated postnatal muscle gene expression.

Funding

SS was supported by a PhD studentship jointly funded by University of Nottingham and Zoetis Inc. LG was supported by a Biotechnology and Biological Sciences Research Council-Zoetis CASE PhD studentship. We thank Zoe Redshaw and Paul Loughna for assistance in Pax immunocytochemistry and primary myoblasts isolation.

History

School

  • Mechanical, Electrical and Manufacturing Engineering

Published in

BMC Cell Biology

Volume

16

Issue

1

Citation

SEBASTIAN, S. ... et al., 2015. Extended 2D myotube culture recapitulates postnatal fibre type plasticity. BMC Cell Biology, 16 (23), DOI: 10.1186/s12860-015-0069-1.

Publisher

© Sebastian et al.

Version

VoR (Version of Record)

Publisher statement

This work is made available according to the conditions of the Creative Commons Attribution 4.0 International (CC BY 4.0) licence. Full details of this licence are available at: http://creativecommons.org/licenses/ by/4.0/

Publication date

2015

Notes

Issued under a CC-BY 4.0 licence (http://creativecommons.org/licenses/by/4.0/).

eISSN

1471-2121

Language

en