骨骼肌是由收缩性肌纤维组成的有丝分裂后组织,所述收缩性肌纤维定向并连接到结缔组织的不同层。然而,成年肌纤维保留再生能力,以响应损伤,激活经典的肌肉干细胞室,即,卫星细胞(SC),它们是有丝分裂的静止细胞,直到需要生长或修复为止,并且位于肌纤维的基底层和肌膜之间。SC从静止状态向活化的转变,承诺,分化涉及对新生物学功能的遗传和表观遗传适应,需要蛋白质表达谱的动态变化。有趣的是,一些活动和信号调节增殖,承诺,分化,和卫星细胞的存活/凋亡也在体外部分概述,利用强大的标记,可靠的技术,和可重复的协议。多年来,已经设计了不同的肌肉细胞培养技术,包括胚胎或出生后肌肉的原代培养,生肌细胞系,和三维(3D)骨骼肌结构。典型的二维(2D)肌肉细胞培养不能完全概括活肌肉组织的复杂性,限制了它们对生理研究的有用性。功能3D培养模型的开发代表了克服现有体外模型局限性的有效替代方法。增加我们对各种细胞类型所扮演的角色以及它们如何相互作用的理解。在这一章中,已经描述了二维和三维细胞培养的发展,改进卫星小区隔离的技术方面,肌肉细胞生长和分化的最佳培养条件,以及开发三维骨骼肌结构所需的程序。
Skeletal muscle is a postmitotic tissue composed of contractile myofibers that are oriented and connected to different layers of connective tissue. Nevertheless, adult muscle fibers retain the capacity to regenerate in response to damage, activating the classical muscle stem cell compartment, namely, satellite cells (SCs), which are mitotically quiescent cells until required for growth or repair and are localized between the basal lamina and sarcolemma of myofibers. The transition of SCs from the quiescent state toward activation, commitment, and differentiation involves the genetic and epigenetic adaptation to novel biological functions, entailing dynamic changes in the protein expression profile. Interestingly, some of the activities and signaling regulating proliferation, commitment, differentiation, and survival/apoptosis of satellite cells have been also partially recapitulated in vitro, taking advantage of robust markers, reliable techniques, and reproducible protocols. Over the years, different techniques of muscular cell culture have been designed including primary cultures from embryonic or postnatal muscle, myogenic cell line, and three-dimensional (3D) skeletal muscle construct. Typical two-dimensional (2D) muscle cell culture cannot fully recapitulate the complexity of living muscle tissues, restricting their usefulness for physiological studies. The development of functional 3D culture models represents a valid alternative to overcome the limitations of already available in vitro model, increasing our understanding of the roles played by the various cell types and how they interact. In this chapter, the development of bidimensional and three-dimensional cell cultures have been described, improving the technical aspect of satellite cell isolation, the best culture-based conditions for muscle cell growth and differentiation, and the procedures required to develop a three-dimensional skeletal muscle construct.