Supplementary Material for: Creating Interactions between Tissue-Engineered Skeletal Muscle and the Peripheral Nervous System

Effective models of mammalian tissues must allow and encourage physiologically (mimetic) correct interactions between co-cultured cell types in order to produce culture microenvironments as similar as possible to those that would normally occur in vivo. In the case of skeletal muscle, the development of such a culture model, integrating multiple relevant cell types within a biomimetic scaffold, would be of significant benefit for investigations into the development, functional performance, and pathophysiology of skeletal muscle tissue. Although some work has been published regarding the behaviour of in vitro muscle models co-cultured with organotypic slices of CNS tissue or with stem cell-derived neurospheres, little investigation has so far been made regarding the potential to maintain isolated motor neurons within a 3D biomimetic skeletal muscle culture platform. Here, we review the current state of the art for engineering neuromuscular contacts in vitro and provide original data detailing the development of a 3D collagen-based model for the co-culture of primary muscle cells and motor neurons. The devised culture system promotes increased myoblast differentiation, forming arrays of parallel, aligned myotubes on which areas of nerve-muscle contact can be detected by immunostaining for pre- and post-synaptic proteins. Quantitative RT-PCR results indicate that motor neuron presence has a positive effect on myotube maturation, suggesting neural incorporation influences muscle development and maturation in vitro. The importance of this work is discussed in relation to other published neuromuscular co-culture platforms along with possible future directions for the field.构建功能完备的哺乳动物组织模型，需能够支持并促进共培养细胞类型间发生符合生理（仿生）标准的相互作用，从而使培养微环境尽可能贴近体内天然状态。以骨骼肌为例，开发此类整合了多种相关细胞与仿生支架（biomimetic scaffold）的培养模型，将对骨骼肌组织的发育、功能表现及病理生理学研究具有重要价值。尽管已有研究报道了与中枢神经系统（CNS）组织器官型切片或干细胞源性神经球共培养的体外肌肉模型的相关特性，但目前针对在三维仿生骨骼肌培养平台中维持分离运动神经元的潜力的相关探索仍较为匮乏。本文综述了体外构建神经肌肉连接的当前技术前沿，并展示了用于原代肌细胞与运动神经元共培养的三维胶原基模型的构建原始数据。该自主研发的培养系统可促进成肌细胞分化，形成平行排列的肌管阵列；通过对突触前与突触后蛋白进行免疫染色，可在该阵列上检测到神经-肌肉接触区域。定量逆转录聚合酶链反应（quantitative RT-PCR）结果显示，运动神经元的存在对肌管成熟具有积极调控作用，表明神经整合可在体外影响肌肉的发育与成熟过程。本文还讨论了本研究相较于其他已发表的神经肌肉共培养平台的重要意义，并展望了该领域未来的发展方向。