评估人体骨骼肌微组织骨骼肌健康的功能指标
Summary
本手稿描述了一种详细的方案，用于生成3D人骨骼肌微系统和微创下游功能原位测定阵列，包括收缩力和钙处理分析。
Abstract
骨骼肌的三维（3D）体外模型是生物医学研究的宝贵进步，因为它们提供了以可扩展格式研究骨骼肌改造和功能的机会，该格式适合实验操作。3D肌肉培养系统是可取的，因为它们使科学家能够在人类细胞的背景下离体研究骨骼肌。3D体外模型密切模仿成人骨骼肌天然组织结构的各个方面。然而，它们的普遍应用受到平台可用性的限制，这些平台易于制造，成本高，用户友好，并且产生相对大量的人体骨骼肌组织。此外，由于骨骼肌在许多疾病状态下起着重要的功能作用，随着时间的推移会受损，因此当微创钙瞬时和收缩力测量可以直接在平台本身内进行微创钙瞬时和收缩力测量时，微组织研究的实验平台是最实用的。在该协议中，描述了称为”MyoTACTIC”的96孔平台的制造，以及大规模生产3D人体骨骼肌微组织（hMMTs）。此外，还报告了微创电刺激应用的方法，该方法能够随着时间的推移重复测量每个微量组织的骨骼肌力和钙处理。
Introduction
骨骼肌是人体最丰富的组织之一，支持关键的身体功能，如运动，热稳态和新陈代谢1。从历史上看，动物模型和二维（2D）细胞培养系统已被用于研究生物过程和疾病发病机制，以及用于测试药物治疗骨骼肌疾病的药理化合物2，3。虽然动物模型大大提高了我们对健康和疾病骨骼肌的了解，但它们的转化影响受到高成本，伦理考虑和种间差异的阻碍2，4。在转向基于人类细胞的系统来研究骨骼肌时，2D细胞培养系统由于其简单性而具有优势。但是，存在限制。这种格式通常无法概括体内自然发生的细胞 – 细胞和细胞外基质相互作用5，6。在过去的几年中，三维（3D）骨骼肌模型已成为整个动物模型和传统2D培养系统的强大替代品，因为它允许对离体7，8的生理和病理学相关过程进行建模。事实上，大量研究报告了以生物人工3D培养格式1模拟人体骨骼肌的策略。许多这些研究的一个局限性是，在从培养平台上去除肌肉组织并附着在力传感器上之后，主动力是定量的，这是破坏性的，因此，仅限于作为终点测定9，10，11，12，13，14，15，16，17，18 ，19，20，21.其他人已经设计了允许非侵入性方法测量主动力的培养系统，但并非所有方法都适合高内涵分子测试应用7，8，9，10，14，18，22，23，24，25，26，27，28 ，29.
该协议描述了在骨骼肌（Myo）微Tissue Array deviCe中制造人类肌肉微组织（hMMTs）的详细方法，以研究forCe（MyoTACTIC）平台;支持批量生产3D骨骼肌微系统的96孔板设备30。MyoTACTIC板制造方法能够在单个铸造步骤中生成96孔聚二甲基硅氧烷（PDMS）培养板和所有相应的孔特征，因此每个孔需要相对较少的细胞来形成微组织。在MyoTACTIC中形成的微组织包含对齐，条纹和多核的肌管，这些肌管可以从设备的孔到孔重复，并且在成熟时可以对原位的化学和电刺激做出反应30。本文概述并讨论了从聚氨酯（PU）复制品制造PDMS肌定向培养板装置的技术，实现永生化人肌原祖细胞以制造hMMT的优化方法，以及工程hMMT力产生和钙处理特性的功能评估。
Protocol
1. PDMS肌定向板制造 注：PDMS肌定向板制造需要PU负模，其制造方式可如前所述30。用于 MyoTACTIC 板设计的计算机辅助设计 （CAD） SolidWorks 文件已在 GitHub （https://github.com/gilbertlabcode/MyoTACTIC-SolidWork-CAD-file） 上提供。 使用有机硅弹性体试剂盒中的组分，以1：15的比例在一次性塑料杯中以单体与固化剂的比例制备〜110g PDMS聚合物溶液。使用5 mL一次性血清移…
Representative Results
本文描述的方法是从PU模具中铸造基于96孔PDMS的MyoTACTIC培养平台，以制造hMMT复制组织阵列，并分析培养装置内hMMT功能的两个方面 – 力生成和钙处理。图1提供了在hMMT播种前制备MyoTACTIC培养孔的示意图。PDMS是一种广泛使用的有机硅基聚合物，可以很容易地模塑成复杂的器件32。基于PDMS的协议旨在从制造的负PU模具30中铸造无限数量的96孔培养…
Discussion
本手稿描述了制造和分析3D hMMT培养模型的方法，该模型可应用于基础肌肉生物学，疾病建模或候选分子测试的研究。MyoTACTIC平台成本友好，易于制造，并且需要相对较少的细胞来产生骨骼肌微组织。在MyoTACTIC培养平台内形成的hMMTs由对齐，多核和横纹肌管组成，并通过启动触发收缩的钙瞬变来响应电刺激（图2，图3，图4）。先前?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们要感谢Mohammad Afshar，Haben Abraha，Mohsen Afshar-Bakooshli和Sadegh Davoudi为MyoTACTIC培养平台的发明做出了贡献，并建立了本文所述的制造和分析方法。HL获得了自然科学与工程研究委员会（NSERC）器官芯片工程和创业奖学金培训计划和多伦多大学野猫研究生奖学金的资助。PMG是加拿大内源性修复研究主席，并获得了安大略省再生医学研究所，干细胞网络和加拿大第一研究卓越计划Medicine by Design对这项研究的支持。逻辑示意图是使用 BioRender.com 创建的。
Materials
| 0.9% Saline Solution, Sterile | House Brand | 1010 | 10 mL aliquots of the solution are made and stored at 4°C |
| 25G Needle | BD, Medstore, University of Toronto | 2548-CABD305127 | |
| 6-Aminocaproic Acid, ≥99% (titration), Powder | Sigma – Aldrich | A2504-100G | A 50 mg / mL stock solution is generated by dissolving 5 mg of 6-aminocaproic acid powder in 100 mL of autoclaved, distilled water. The solution is vaccum filtered and 10 mL aliquots are stored at 4°C |
| 6.35 mm ID Tubing | VWR | 60985-528 | |
| AB1167 Myoblast Cell Line | Institut de Myologie (Paris, France) | ||
| Arbitrary Waveform Generator | Rigol | DG1022Z | |
| Basement Membrane Extract (Geltrex) | Thermo Fisher Scientific | A14132-02 | Stored as aliquots of 50 µL or 100 µL at -80°C |
| Benchtop Vacuum Chamber | Sigma – Aldrich | D2672 | |
| BNC to Aligator Clip Cable | Ordered from Amazon | ||
| Culture Plastics | Sarstedt | Includes culture plates, serological pipettes, etc | |
| Dimethyl Sulfoxide | Sigma – Aldrich | D8418-250ML | |
| DPBS, Powder, No Calcium, No Magnesium | Thermo Fisher Scientific | 21600069 | |
| Dulbecco's Modified Eagle Medium (DMEM) (1X) | Gibco | 11995-065 | This is a high glucose DMEM with L-glutamine and sodium pyruvate |
| Fetal Bovine Serum | Fisher Scientific | 10437028 | |
| Fibrinogen from Bovine Plasma | Sigma – Aldrich | F8630-5G | Aliquots ranging from 7 – 10 mg of fibrinogen powder are made and stored at -20°C |
| Filtropur Syringe Filter, 0.22um Pore Size | Sarstedt | 83.1826.001 | |
| Horse Serum | Gibco | 16050-122 | |
| Human Recombinant Insulin | Sigma – Aldrich | 91077C | Stock solution is 100X and made by dissolving 1 mg of human recombinant insulin in 1 mL of DMEM and 1 µL of NaOH 10N. Solution is filtered and stored as 1 mL aliquots at 4°C |
| Image Acquisition Software | Olympus | cellSens Dimension | |
| Image Processing Software | National Institutes of Health | ImageJ | |
| Isotemp Oven | Thermo Fisher Scientific | 201 | |
| Microscope | Olympus | IX83 | |
| Microscope – Camera Mount | Labcam | Labcam for iPhone | Ordered from Amazon |
| Penicillin-Streptomycin (10,000 U/mL) | Gibco | 15140-122 | |
| Plastic Disposable Syringes, 1cc | BD | 2606-309659 | |
| Plastic Disposable Syringes, 50cc | BD | 2612-309653 | |
| Pluronic F-127, Powder, BioReagent | Sigma – Aldrich | P2443-250G | A 5% stock solution of pluronic acid is made by dissolving 5 g of pluronic acid powder in 100 mL of chilled, autoclaved, distilled water. The solution is vaccum filtered and 10 mL aliquots are stored at 4°C |
| Polydimethylsiloxane (Sylgard 184 Silicone Elastomer Kit) | Dow | 4019862 | Kits are also available at Thermo Fisher Scientific, Sigma – Aldrich, etc. |
| Polyurethane Negative Mold | In House | ||
| Release Agent | Mann Release Technologies | 200 | |
| Rotary Vane Vacuum Pump | Edwards | A65401906 | |
| Scalpel | Almedic, Medstore, University of Toronto | 2586-M36-0100 | |
| Single Edge Razor Blade | VWR | 55411-050 | |
| Skeletal Muscle Cell Basal Medium | Promocell | C-23260 | 30 mL aliquotes are generated and at stored at 4°C. |
| Skeletal Muscle Cell Growth Medium (Ready-to-use) | Promocell | C-23060 | 42 mL aliquots are generated and stored at 4°C. |
| Smartphone (iPhone) | Apple | SE | |
| Standard Duty Dry Vacuum Pump | Welch | 2546B-01 | |
| Sterilization Bag | Alliance | 211-SCM2 | |
| Thimble | Igege | Ordered from Amazon | |
| Thrombin from human plasma | Sigma – Aldrich | T6884-250UN | 100 units of thrombin is dissolved in 1 mL of a 0.1% BSA solution. 10 µL aliquots are prepared and stored at – 20°C. |
| Tin coated copper wire | Arco | B8871K48 | Ordered from Amazon |
| Trypan Blue Solution, 0.4% | Thermo Scientific | 15250061 | |
| Trypsin-EDTA, 0.25% | Thermo FIsher Scientific | 25200072 | |
| Vacuum Chamber 2 | SP Bel-Art | F42027-0000 |
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Citazione di questo articolo
Lad, H., Musgrave, B., Ebrahimi, M., Gilbert, P. M. Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues. J. Vis. Exp. (168), e62307, doi:10.3791/62307 (2021).