Gameiro Dos Santos Pedro, Soares Ana Rita, Thorsteinsdóttir Sólveig, Rodrigues Gabriela
Centre for Ecology, Evolution and Environmental Changes and Global Change & Sustainability Institute, Department of Animal Biology, Faculty of Sciences, University of Lisbon;
Centre for Ecology, Evolution and Environmental Changes and Global Change & Sustainability Institute, Department of Animal Biology, Faculty of Sciences, University of Lisbon.
J Vis Exp. 2023 Mar 3(193). doi: 10.3791/65069.
The extracellular matrix (ECM) plays a crucial role in providing structural support for cells and conveying signals that are important for various cellular processes. Two-dimensional (2D) cell culture models oversimplify the complex interactions between cells and the ECM, as the lack of a complete three-dimensional (3D) support can alter cell behavior, making them inadequate for understanding in vivo processes. Deficiencies in ECM composition and cell-ECM interactions are important contributors to a variety of different diseases. One example is LAMA2-congenital muscular dystrophy (LAMA2-CMD), where the absence or reduction of functional laminin 211 and 221 can lead to severe hypotony, detectable at or soon after birth. Previous work using a mouse model of the disease suggests that its onset occurs during fetal myogenesis. The present study aimed to develop a 3D in vitro model permitting the study of the interactions between muscle cells and the fetal muscle ECM, mimicking the native microenvironment. This protocol uses deep back muscles dissected from E18.5 mouse fetuses, treated with a hypotonic buffer, an anionic detergent, and DNase. The resultant decellularized matrices (dECMs) retained all ECM proteins tested (laminin α2, total laminins, fibronectin, collagen I, and collagen IV) compared to the native tissue. When C2C12 myoblasts were seeded on top of these dECMs, they penetrated and colonized the dECMs, which supported their proliferation and differentiation. Furthermore, the C2C12 cells produced ECM proteins, contributing to the remodeling of their niche within the dECMs. The establishment of this in vitro platform provides a new promising approach to unravel the processes involved in the onset of LAMA2-CMD, and has the potential to be adapted to other skeletal muscle diseases where deficiencies in communication between the ECM and skeletal muscle cells contribute to disease progression.
细胞外基质（ECM）在为细胞提供结构支持以及传递对各种细胞过程至关重要的信号方面发挥着关键作用。二维（2D）细胞培养模型过度简化了细胞与ECM之间的复杂相互作用，因为缺乏完整的三维（3D）支持会改变细胞行为，使其不足以用于理解体内过程。ECM组成和细胞 - ECM相互作用的缺陷是多种不同疾病的重要促成因素。一个例子是LAMA2 - 先天性肌营养不良症（LAMA2 - CMD），其中功能性层粘连蛋白211和221的缺失或减少可导致严重的肌张力减退，在出生时或出生后不久即可检测到。先前使用该疾病小鼠模型的研究表明，其发病发生在胎儿肌生成期间。本研究旨在建立一种三维体外模型，用于研究肌肉细胞与胎儿肌肉ECM之间的相互作用，模拟天然微环境。该方案使用从E18.5小鼠胎儿解剖的深部背肌，用低渗缓冲液、阴离子洗涤剂和DNA酶处理。与天然组织相比，所得的脱细胞基质（dECM）保留了所有测试的ECM蛋白（层粘连蛋白α2、总层粘连蛋白、纤连蛋白、胶原蛋白I和胶原蛋白IV）。当将C2C12成肌细胞接种在这些dECM上时，它们穿透并定殖于dECM，dECM支持它们的增殖和分化。此外，C2C12细胞产生ECM蛋白，有助于其在dECM内生态位的重塑。这种体外平台的建立为揭示LAMA2 - CMD发病过程中涉及的机制提供了一种新的有前景的方法，并且有可能适用于其他骨骼肌疾病，其中ECM与骨骼肌细胞之间通讯缺陷导致疾病进展。
