Extended 2D myotube culture recapitulates postnatal fibre type plasticity.
Extended 2D myotube culture recapitulates postnatal fibre type plasticity.
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DOI：
10.1186/s12860-015-0069-1
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发表时间：
2015-09-17
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影响因子：
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通讯作者：
Chang KC
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作者：
Sebastian S;Goulding L;Kuchipudi SV;Chang KC
进行源自哺乳动物或禽类物种的骨骼肌细胞培养的传统问题是有限的肌管分化和短暂的肌管持续性，这极大地限制了肌管经历表型成熟的能力。我们在这里报告了一个重大的技术突破，建立了一个简单有效的方法，延长猪肌管培养（超过50天）在二维（2D），概括了出生后的纤维类型的关键特征。从4至6周龄猪的背最长肌分离原代猪肌肉卫星细胞（成肌细胞）用于2D培养以优化肌管形成、改善表面粘附和肌管成熟。超过95%的分离细胞是成肌细胞，如Pax 3和Pax 7的表达所证明的。我们相对简单的方法，基于现有表面包被试剂（Maxgel）和增殖和分化（Ultroser G）培养基的修改，通常通过5天的分化融合指数达到约80%，表现为大量的离散肌球蛋白重链（MyHC）慢肌管和快肌管。在50天内，肌管活力几乎没有恶化，并且肌管形成的效率维持在7个成肌细胞传代。在整个培养过程中，经常观察到肌管有规律的自发收缩。扩展培养中的肌管能够在不同的培养基中进行表型适应，包括在分化的第20天左右采用显性出生后表型的快速糖酵解MyHC 2x和2b表达。此外，快速糖酵解肌管与增强表达的假定的猪长基因间非编码RNA（linc-MYH），最近已被证明是一个关键的协调员的MyHC 2b在体内的表达。我们修改后的培养方案允许猪成肌细胞有效分化和融合成肌管，并延长其粘附到培养表面。此外，我们能够在2D中概括肌管的成熟过程，以类似于出生后的纤维类型，这代表了开放获得协调的出生后肌肉基因表达的体外研究的重大技术进步。本文的在线版本（doi：10.1186/s12860-015-0069-1）包含补充材料，可供授权用户使用。
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. 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. 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. The online version of this article (doi:10.1186/s12860-015-0069-1) contains supplementary material, which is available to authorized users.