Extended 2D myotube culture recapitulates postnatal fibre type plasticity

Abstract

BackgroundThe 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.ResultsPrimary 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-015-0069-1) contains supplementary material, which is available to authorized users.

Anchored Cell Sheet Engineering: A Novel Scaffold‐Free Platform for in vitro Modeling

“…Maintaining such protracted cultures of various cell types, especially skeletal muscle cells, can pose challenges due to potential delamination post-differentiation and the resultant deleterious effects on cell viability. Nonetheless, numerous studies have successfully sustained viable and functional differentiated myoblasts over extended culture durations, both in 2D [52,53] and 3D [54,55] cultures. In this study, uncontrolled cell delamination was not observed, and sustained cell viability was validated through in vitro Toxicology Assay Kit at various intervals, coupled with live-dead staining at the end.…”

Mesenchymal stem cells and myoblast differentiation under HGF and IGF-1 stimulation for 3D skeletal muscle tissue engineering

“…Because myogenin is mostly expressed during terminal stages of myogenic differentiation [ 54 , 55 ], longer cultivation periods of at least up to 28 d would be one future goal. Although cell detachment after 28 d of cultivation made it impossible to analyse the gene expression during longer observation periods so far, coating with collagen type I or Maxgel™ (consisting of an undefined composition of human extracellular matrix components) may be a possibility to overcome cell detachment in future experiments [ 56 ]. Furthermore, myogenin is known to peak at some point of myoblast differentiation and then decline to lower expression afterwards.…”

Effects of a traditional Chinese medicine formula and its extraction on muscle fiber characteristics in finishing pigs, porcine cell proliferation and isoforms of myosin heavy chain gene expression in myocytes

“…Taken together, these results showed that highly pure SCs can be used as a cell model for the next experiment. As shown in the experiment, porcine myocytes were identified by proliferation and differentiation within 5 days with a nearly 80% differentiation fusion index manifested in an abundance of discrete MyHC [ 47 ]. We treated the myocytes with the TCMF extractions after 96 h of induction with 2% horse serum.…”