Skeletal muscle is increasingly considered an endocrine organ secreting myokines and extracellular vesicles (exosomes and microvesicles), which can affect physiological changes with an impact on different pathological conditions, including regenerative processes, aging, and myopathies. Primary human myoblasts are an essential tool to study the muscle vesicle secretome. Since their differentiation in conditioned media does not induce any signs of cell death or cell stress, artefactual effects from those processes are unlikely. However, adult human primary myoblasts senesce in long-term tissue culture, so a major technical challenge is posed by the need to avoid artefactual effects resulting from pre-senescent changes. Since these cells should be studied within a strictly controlled pre-senescent division count (<21 divisions), and yields of myoblasts per muscle biopsy are low, it is difficult or impossible to amplify sufficiently large cell numbers (some 250 × 106 myoblasts) to obtain sufficient conditioned medium for the standard ultracentrifugation approach to exosome isolation. Thus, an optimized strategy to extract and study secretory muscle vesicles is needed. In this study, conditions are optimized for the in vitro cultivation of human myoblasts, and the quality and yield of exosomes extracted using an ultracentrifugation protocol are compared with a modified polymer-based precipitation strategy combined with extra washing steps. Both vesicle extraction methods successfully enriched exosomes, as vesicles were positive for CD63, CD82, CD81, floated at identical density (1.15-1.27 g.ml−1), and exhibited similar size and cup-shape using electron microscopy and NanoSight tracking. However, the modified polymer-based precipitation was a more efficient strategy to extract exosomes, allowing their extraction in sufficient quantities to explore their content or to isolate a specific subpopulation, while requiring >30 times fewer differentiated myoblasts than what is required for the ultracentrifugation method. In addition, exosomes could still be integrated into recipient cells such as human myotubes or iPSC-derived motor neurons. Modified polymer-based precipitation combined with extra washing steps optimizes exosome yield from a lower number of differentiated myoblasts and less conditioned medium, avoiding senescence and allowing the execution of multiple experiments without exhausting the proliferative capacity of the myoblasts.

中文翻译：

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从人原代肌细胞提取外泌体的优化方法。

骨骼肌越来越被认为是分泌肌动蛋白和细胞外囊泡（外泌体和微囊泡）的内分泌器官，它们可以影响生理变化，并影响不同的病理状况，包括再生过程，衰老和肌病。原代人成肌细胞是研究肌肉囊泡分泌组的必不可少的工具。由于它们在条件培养基中的分化不会引起细胞死亡或细胞应激的任何迹象，因此不太可能产生来自这些过程的人工影响。然而，成年人类原代成肌细胞在长期组织培养中会衰老，因此需要避免由衰老前变化引起的假象效应构成了主要的技术挑战。由于应在严格控制的衰老前分裂计数（<21个分裂）内研究这些细胞，而且每次肌肉活检中成肌细胞的产率很低，很难或不可能扩增足够大的细胞数量（约250×106个成肌细胞）以获得足够的条件培养基，以用于标准的超速离心方法来分离外泌体。因此，需要一种优化的策略来提取和研究分泌性肌肉小泡。在这项研究中，为体外培养人类成肌细胞优化了条件，并将使用超速离心方案提取的外泌体的质量和产量与改良的基于聚合物的沉淀策略以及额外的洗涤步骤进行了比较。两种囊泡提取方法均成功富集了外泌体，因为囊泡以相同的密度（1.15-1.27 g.ml-1）漂浮于CD63，CD82，CD81阳性，并通过电子显微镜和NanoSight跟踪显示出相似的大小和杯形。然而，基于聚合物的改性沉淀是提取外泌体的更有效策略，允许提取足够多的提取物以探索其内容或分离特定的亚群，同时所需的分化成肌细胞比超速离心方法少30倍以上。 。此外，外泌体仍可以整合到受体细胞中，例如人肌管或iPSC衍生的运动神经元。改性的基于聚合物的沉淀与额外的洗涤步骤相结合，可从较少数量的分化成肌细胞和较少条件培养基中优化外泌体产量，避免了衰老，并允许执行多个实验而不会耗尽成肌细胞的增殖能力。