Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada.
Donnelly Centre for Cellular and Biomolecular Research, Toronto, Canada.
Sci Rep. 2020 Apr 24;10(1):6918. doi: 10.1038/s41598-020-62837-8.
Three-dimensional (3D) in vitro models of human skeletal muscle mimic aspects of native tissue structure and function, thereby providing a promising system for disease modeling, drug discovery or pre-clinical validation, and toxicity testing. Widespread adoption of this research approach is hindered by the lack of easy-to-use platforms that are simple to fabricate and that yield arrays of human skeletal muscle micro-tissues (hMMTs) in culture with reproducible physiological responses that can be assayed non-invasively. Here, we describe a design and methods to generate a reusable mold to fabricate a 96-well platform, referred to as MyoTACTIC, that enables bulk production of 3D hMMTs. All 96-wells and all well features are cast in a single step from the reusable mold. Non-invasive calcium transient and contractile force measurements are performed on hMMTs directly in MyoTACTIC, and unbiased force analysis occurs by a custom automated algorithm, allowing for longitudinal studies of function. Characterizations of MyoTACTIC and resulting hMMTs confirms the capability of the device to support formation of hMMTs that recapitulate biological responses. We show that hMMT contractile force mirrors expected responses to compounds shown by others to decrease (dexamethasone, cerivastatin) or increase (IGF-1) skeletal muscle strength. Since MyoTACTIC supports hMMT long-term culture, we evaluated direct influences of pancreatic cancer chemotherapeutics agents on contraction competent human skeletal muscle myotubes. A single application of a clinically relevant dose of Irinotecan decreased hMMT contractile force generation, while clear effects on myotube atrophy were observed histologically only at a higher dose. This suggests an off-target effect that may contribute to cancer associated muscle wasting, and highlights the value of the MyoTACTIC platform to non-invasively predict modulators of human skeletal muscle function.
三维（3D）体外人类骨骼肌模型模拟了天然组织的结构和功能，从而为疾病建模、药物发现或临床前验证和毒性测试提供了一个很有前途的系统。这种研究方法的广泛采用受到缺乏易于使用的平台的阻碍，这些平台制造简单，能够在培养物中产生阵列式的人类骨骼肌微组织（hMMTs），具有可重复的生理反应，可以进行非侵入性检测。在这里，我们描述了一种设计和方法，用于制造可重复使用的模具来制造 96 孔平台，称为 MyoTACTIC，它能够批量生产 3D hMMTs。所有 96 个孔和所有孔特征都是在可重复使用的模具中一步铸造而成。hMMTs 直接在 MyoTACTIC 中进行非侵入性钙瞬变和收缩力测量，通过自定义自动算法进行无偏力分析，从而实现功能的纵向研究。MyoTACTIC 和产生的 hMMTs 的特性验证了该设备支持形成能够重现生物学反应的 hMMTs 的能力。我们表明，hMMT 收缩力反映了对其他化合物的预期反应，这些化合物被证明可以降低（地塞米松、西立伐他汀）或增加（IGF-1）骨骼肌强度。由于 MyoTACTIC 支持 hMMT 的长期培养，我们评估了胰腺癌化疗药物对收缩能力的人类骨骼肌肌管的直接影响。单次应用临床相关剂量的伊立替康降低了 hMMT 的收缩力产生，而仅在更高剂量下才观察到组织学上对肌管萎缩的明显影响。这表明存在一种非靶标效应，可能导致与癌症相关的肌肉消耗，这突显了 MyoTACTIC 平台在非侵入性预测人类骨骼肌功能调节剂方面的价值。
