C2C12 co-culture on a fibroblast substratum enables sustained survival of contractile, highly differentiated myotubes with peripheral nuclei and adult fast myosin expression
C2C12 co-culture on a fibroblast substratum enables sustained survival of contractile, highly differentiated myotubes with peripheral nuclei and adult fast myosin expression
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DOI：
10.1002/cm.20010
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发表时间：
2004-07-01
影响因子：
--
通讯作者：
North, KN
中科院分区：
文献类型：
--
作者：
Cooper, ST;Maxwell, AL;North, KN
我们描述了一种简单的培养方法，用于使用汇合成纤维细胞的饲养层获得高度分化的克隆 C2C12 肌管，并使用该系统记录收缩蛋白表达和肌纤维形态方面的表达。使用胶原蛋白或层粘连蛋白包被的组织培养塑料的传统培养方法通常会导致肌管脱离和重组的循环模式，很少产生成熟成体表型的肌管。成纤维细胞培养基上的 C2C12 共培养有助于收缩肌管的持续培养，从而产生成熟的肌节记录，并具有外周迁移细胞核的证据。免疫印迹分析表明，结蛋白、原肌球蛋白、肌动蛋白、α-肌动蛋白-2 和慢肌球蛋白在整个生肌分化过程中均可检测到，而成体快肌球蛋白重链亚型、肌营养不良蛋白相关复合物的成员和 α-肌动蛋白-3 直到分化 > 6 天（与收缩活动开始同时）才以显着水平表达。成熟肌管的电刺激揭示了典型且可重复的钙瞬变，证明了钙处理蛋白的功能成熟。免疫细胞化学染色显示大部分肌管 (70-80%) 具有明确的肌节区，并且观察到结蛋白的条纹染色模式，表明中间丝网络与肌节区的对齐。我们报告说，培养体积影响发育中的肌管的融合指数和肌节发育速率，并提出成纤维细胞饲养层提供弹性基质以支持收缩活动，并可能分泌有助于肌管发育的生长因子和细胞外基质蛋白。 (C) 2004 Wiley-Liss, Inc.
We describe a simple culture method for obtaining highly differentiated clonal C2C12 myotubes using a feeder layer of confluent fibroblasts, and document the expression of contractile protein expression and aspects of myofibre morphology using this system. Traditional culture methods using collagen- or laminin-coated tissue-culture plastic typically results in a cyclic pattern of detachment and reformation of myotubes, rarely producing myotubes of a mature adult phenotype. C2C12 co-culture on a fibroblast substratum facilitates the sustained culture of contractile myotubes, resulting in a mature sarcomeric register with evidence for peripherally migrating nuclei. Immunoblot analysis demonstrates that desmin, tropomyosin, sarcomeric actin, alpha-actinin-2 and slow myosin are detected throughout myogenic differentiation, whereas adult fast myosin heavy chain isoforms, members of the dystrophin-associated complex, and alpha-actinin-3 are not expressed at significant levels until >6 days of differentiation, coincident with the onset of contractile activity. Electrical stimulation of mature myotubes reveals typical and reproducible calcium transients, demonstrating functional maturation with respect to calcium handling proteins. Immunocytochemical staining demonstrates a well-defined sarcomeric register throughout the majority of myotubes (70-80%) and a striated staining pattern is observed for desmin, indicating alignment of the intermediate filament network with the sarcomeric register. We report that culture volume affects the fusion index and rate of sarcomeric development in developing myotubes and propose that a fibroblast feeder layer provides an elastic substratum to support contractile activity and likely secretes growth factors and extracellular matrix proteins that assist myotube development. (C) 2004 Wiley-Liss, Inc.