Institute of Biomedical Engineering, University of Toronto; Donnelly Centre for Cellular and Biomolecular Research, University of Toronto.
Institute of Biomedical Engineering, University of Toronto; Donnelly Centre for Cellular and Biomolecular Research, University of Toronto; Department of Cell and Systems Biology, University of Toronto;
J Vis Exp. 2021 Feb 18(168). doi: 10.3791/62307.
Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study skeletal muscle reformation and function in a scalable format that is amenable to experimental manipulations. 3D muscle culture systems are desirable as they enable scientists to study skeletal muscle ex vivo in the context of human cells. 3D in vitro models closely mimic aspects of the native tissue structure of adult skeletal muscle. However, their universal application is limited by the availability of platforms that are simple to fabricate, cost and user-friendly, and yield relatively high quantities of human skeletal muscle tissues. Additionally, since skeletal muscle plays an important functional role that is impaired over time in many disease states, an experimental platform for microtissue studies is most practical when minimally invasive calcium transient and contractile force measurements can be conducted directly within the platform itself. In this protocol, the fabrication of a 96-well platform known as 'MyoTACTIC', and en masse production of 3D human skeletal muscle microtissues (hMMTs) is described. In addition, the methods for a minimally invasive application of electrical stimulation that enables repeated measurements of skeletal muscle force and calcium handling of each microtissue over time are reported.
三维（3D）体外骨骼肌模型是生物医学研究的一项重要进展，因为它们提供了在可扩展的格式中研究骨骼肌重构和功能的机会，并且易于进行实验操作。3D 肌肉培养系统是理想的，因为它们使科学家能够在体外研究人类细胞中的骨骼肌。3D 体外模型非常接近成年骨骼肌的天然组织结构的某些方面。然而，它们的普遍应用受到限制，因为缺乏简单制造、成本低廉、用户友好且能够产生相对大量人体骨骼肌组织的平台。此外，由于骨骼肌在许多疾病状态下随着时间的推移会发挥重要的功能作用，因此当可以直接在平台本身内部进行微创钙瞬变和收缩力测量时，用于微组织研究的实验平台最实用。在本方案中，描述了一种称为“MyoTACTIC”的 96 孔平台的制造方法，以及批量生产 3D 人体骨骼肌微组织（hMMTs）的方法。此外，还报告了一种微创电刺激应用方法，该方法可以重复测量每个微组织的骨骼肌力和钙处理能力。
