Name: assessing functional metrics of skeletal muscle health in human skeletal muscle microtissues
Uploaded: 2021-02-18T15:00:00
Description: Watch this Scientific Journal Video about Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues at JoVE.com

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。

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。
<ol><li>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...

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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?...

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。

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	<tbody>
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			<td>0.9% Saline Solution, Sterile</td>
			<td>House Brand</td>
			<td>1010</td>
			<td>10 mL aliquots of the solution are made and stored at 4&#176;C</td>
		</tr>
		<tr>
			<td>25G Needle</td>
			<td>BD, Medstore, University of Toronto</td>
			<td>2548-CABD305127</td>
			<td></td>
		</tr>
		<tr>
			<td>6-Aminocaproic Acid, &#8805;99% (titration), Powder</td>
			<td>Sigma - Aldrich</td>
			<td>A2504-100G</td>
			<td>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&#176;C</td>
		</tr>
		<tr>
			<td>6.35 mm ID Tubing</td>
			<td>VWR</td>
			<td>60985-528</td>
			<td></td>
		</tr>
		<tr>
			<td>AB1167 Myoblast Cell Line</td>
			<td>Institut de Myologie (Paris, France)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Arbitrary Waveform Generator</td>
			<td>Rigol</td>
			<td>DG1022Z</td>
			<td></td>
		</tr>
		<tr>
			<td>Basement Membrane Extract (Geltrex)</td>
			<td>Thermo Fisher Scientific</td>
			<td>A14132-02</td>
			<td>Stored as aliquots of 50 &#181;L or 100 &#181;L at -80&#176;C</td>
		</tr>
		<tr>
			<td>Benchtop Vacuum Chamber</td>
			<td>Sigma - Aldrich</td>
			<td>D2672</td>
			<td></td>
		</tr>
		<tr>
			<td>BNC to Aligator Clip Cable</td>
			<td></td>
			<td></td>
			<td>Ordered from Amazon</td>
		</tr>
		<tr>
			<td>Culture Plastics</td>
			<td>Sarstedt</td>
			<td></td>
			<td>Includes culture plates, serological pipettes, etc</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma - Aldrich</td>
			<td>D8418-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, Powder, No Calcium, No Magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>21600069</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM) (1X)</td>
			<td>Gibco</td>
			<td>11995-065</td>
			<td>This is a high glucose DMEM with L-glutamine and sodium pyruvate</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Fisher Scientific</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen from Bovine Plasma</td>
			<td>Sigma - Aldrich</td>
			<td>F8630-5G</td>
			<td>Aliquots ranging from 7 - 10 mg of fibrinogen powder are made and stored at -20&#176;C</td>
		</tr>
		<tr>
			<td>Filtropur Syringe Filter, 0.22um Pore Size</td>
			<td>Sarstedt</td>
			<td>83.1826.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Gibco</td>
			<td>16050-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Recombinant Insulin</td>
			<td>Sigma - Aldrich</td>
			<td>91077C</td>
			<td>Stock solution is 100X and made by dissolving 1 mg of human recombinant insulin in 1 mL of DMEM and 1 &#181;L of NaOH 10N. Solution is filtered and stored as 1 mL aliquots at 4&#176;C</td>
		</tr>
		<tr>
			<td>Image Acquisition Software</td>
			<td>Olympus</td>
			<td>cellSens Dimension</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Processing Software</td>
			<td>National Institutes of Health</td>
			<td>ImageJ</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotemp Oven</td>
			<td>Thermo Fisher Scientific</td>
			<td>201</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>IX83</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope - Camera Mount</td>
			<td>Labcam</td>
			<td>Labcam for iPhone</td>
			<td>Ordered from Amazon</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Disposable Syringes, 1cc</td>
			<td>BD</td>
			<td>2606-309659</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Disposable Syringes, 50cc</td>
			<td>BD</td>
			<td>2612-309653</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127, Powder, BioReagent</td>
			<td>Sigma - Aldrich</td>
			<td>P2443-250G</td>
			<td>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&#176;C</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (Sylgard 184 Silicone Elastomer Kit)</td>
			<td>Dow</td>
			<td>4019862</td>
			<td>Kits are also available at Thermo Fisher Scientific, Sigma - Aldrich, etc.</td>
		</tr>
		<tr>
			<td>Polyurethane Negative Mold</td>
			<td>In House</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Release Agent</td>
			<td>Mann Release Technologies</td>
			<td>200</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary Vane Vacuum Pump</td>
			<td>Edwards</td>
			<td>A65401906</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Almedic, Medstore, University of Toronto</td>
			<td>2586-M36-0100</td>
			<td></td>
		</tr>
		<tr>
			<td>Single Edge Razor Blade</td>
			<td>VWR</td>
			<td>55411-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Skeletal Muscle Cell Basal Medium</td>
			<td>Promocell</td>
			<td>C-23260</td>
			<td>30 mL aliquotes are generated and at stored at 4&#176;C.</td>
		</tr>
		<tr>
			<td>Skeletal Muscle Cell Growth Medium (Ready-to-use)</td>
			<td>Promocell</td>
			<td>C-23060</td>
			<td>42 mL aliquots are generated and stored at 4&#176;C.</td>
		</tr>
		<tr>
			<td>Smartphone (iPhone)</td>
			<td>Apple</td>
			<td>SE</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Duty Dry Vacuum Pump</td>
			<td>Welch</td>
			<td>2546B-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilization Bag</td>
			<td>Alliance</td>
			<td>211-SCM2</td>
			<td></td>
		</tr>
		<tr>
			<td>Thimble</td>
			<td>Igege</td>
			<td></td>
			<td>Ordered from Amazon</td>
		</tr>
		<tr>
			<td>Thrombin from human plasma</td>
			<td>Sigma - Aldrich</td>
			<td>T6884-250UN</td>
			<td>100 units of thrombin is dissolved in 1 mL of a 0.1% BSA solution. 10 &#181;L aliquots are prepared and stored at - 20&#176;C.</td>
		</tr>
		<tr>
			<td>Tin coated copper wire</td>
			<td>Arco</td>
			<td>B8871K48</td>
			<td>Ordered from Amazon</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>Thermo Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, 0.25%</td>
			<td>Thermo FIsher Scientific</td>
			<td>25200072</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Chamber 2</td>
			<td>SP Bel-Art</td>
			<td>F42027-0000</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing functional metrics of skeletal muscle health in human skeletal muscle microtissues

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。

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...

Watch this Scientific Journal Video about Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues at JoVE.com

Assessing Functional Metrics of Skeletal Muscle Health in Human Skeletal Muscle Microtissues

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。

评估人体骨骼肌微组织骨骼肌健康的功能指标

Three-dimensional (3D) in vitro models of skeletal muscle are a valuable advancement in biomedical research as they afford the opportunity to study ...

我们的实验方案描述了制造和分析3D人骨骼肌微组织培养物的详细方法。肌肉微组织可以应用于基础肌肉生物学，疾病建模或候选分子测试的研究。该技术的主要优点是它允许对收缩力和钙瞬变进行原位评估。因此，您可以对肌肉组织功能进行纵向研究。演示该程序的将是Brennen Musgrave和Heta Lad，他们来自我的实验室。在细胞接种前两到三小时，将六孔温和的策略板部分放入10厘米的细胞培养皿中。通过加入100微升5%多聚体F-127溶液来制备每个单独的肌细胞培养物。使用盖子盖住孔，并涂上石蜡膜以密封盘子。在装有板式旋转适配器的离心机中以1， 550 g离心一分钟。将培养皿储存在四摄氏度，直到细胞准备好接种。然后在培养罩中的冰上缓慢解冻150微升等分试样的基底膜提取物和110微升凝血酶等分试样。在细胞培养罩中工作，将700微升0.9%盐水溶液加入含有7毫克纤维蛋白原粉末的1.5毫升微量离心管中。将试管置于37摄氏度的细胞培养箱中三到五分钟，而不会涡旋。取出并轻轻拂管，然后在微量离心机中脉冲旋转溶解的溶液，然后将其返回培养罩。使用配备0.22微米注射器过滤器的一毫升注射器过滤纤维蛋白原溶液。然后将溶解的纤维蛋白原溶液转移到基底膜提取物和凝血酶等分试样的冰中。准备并在37摄氏度下预热组织生长培养基，在接种组织后将其引入培养井。从培养箱中取出细胞培养板并吸出培养基。然后通过在每个培养板中加入五毫升DPBS来洗涤细胞一次。吸出DPBS并通过在每个培养皿中加入一毫升0.25%胰蛋白酶EDTA来分离细胞。将板置于细胞培养箱中三分钟，通过向培养皿中加入三毫升洗涤培养基来保持胰蛋白酶，然后将细胞转移到适当大小的锥形管中。通过以400倍G离心10分钟来沉淀细胞。小心地吸出培养基而不损坏细胞沉淀，然后将细胞重新悬浮在一毫升洗涤培养基中。在明场显微镜下使用血细胞计数器和台盼蓝染料计数细胞。为了播种六个组织，为八个组织准备足够的细胞和细胞外基质，从而考虑损失和气泡形成。将120万个细胞转移到新的锥形管中，然后用洗涤介质将体积增加到10毫米。通过以400倍G离心10分钟来沉淀细胞。在1.5毫升的微型离心管中制备150微升ECM混合物，并将混合物储存在冰上直至使用。从含有细胞的锥形管中吸出培养基，注意避免细胞沉淀。用戴着手套的手指用力轻拂管的末端，直到沉淀显示为细胞浆液。将120微升的ECM溶液转移到含有细胞浆液的管中，并小心地上下移液以完全重新悬浮ECM内的细胞以产生单个细胞悬浮液。避免产生气泡，然后将细胞ECM悬浮液放在冰上直到使用。将含有肌细胞培养罩内的冰袋顶部的10厘米冷藏皿放在细胞培养罩内的冰袋上，并从每个孔中吸出多聚体F-127溶液。让残留的多质F-127溶液从多孔PDMS中释放出来，并通过让孔在冰上静置五分钟，然后再次吸出而沉降到井底。小心地移取细胞ECM悬浮液以重新悬浮细胞，然后通过抓住靠近顶部的1.5毫升将105微升悬浮液转移到新鲜的，预冷的1.5毫升管中。向105微升细胞ECM悬浮液中加入0.84微升每毫升凝血酶储备溶液100单位。快速彻底地移液混合，避免引入气泡。向每个孔中加入15微升细胞ECM混合物，而无需将移液器压入孔的底部。通过两个轻动作，将细胞悬浮液散布在孔中每个柱子后面。将盖子放在10厘米培养板上，并将其转移到37摄氏度的组织培养箱中约5分钟。在细胞ECM混合物聚合后，向每个肌钾孔中加入200微升预热的组织生长培养基。盖上10厘米培养皿的盖子，并将其保存在培养箱中，直到分化。在14天的培养期间，成肌细胞通过在温和的策略井中自组织来显示天然组织的各个方面，以形成具有多核横纹肌管的3D微组织。肌管具有肌节条纹，通过肉瘤α肌动蛋白的免疫染色来可视化。为了实现井面管的排列，应避免技术错误，例如移液时形成气泡，或在抽吸过程中损坏浮滤膜。这里显示了肌细胞孔板柱响应于低频和高频电刺激的代表性位移，表明肌管对不同的电刺激做出反应并相应地收缩。置换后定量允许将HMMT转换为绝对收缩力.还分析了由GCaMP6转导的成肌细胞制成的HMMT上的钙瞬变，以响应低频和高频刺激。荧光强度的定量可用作HMMT钙处理特性的测量。大多数第一次进行这种组织接种的人，在将细胞细胞外基质添加到孔中以及形成气泡时都会遇到困难。我们建议在第一次尝试细胞之前，在一些备用的微组织培养井上用非常简单的粘性溶液练习该技术。

Research

JoVE Journal

Bioengineering

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

工程骨骼肌组织中的小鼠成肌细胞的内皮祖细胞与应用电刺激

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

科研

生物工程

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

在肌肉再生的老鼠模型中移植诱导多能干细胞衍生的中生代细胞样肌原体

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

最大等长收缩力的测量通过透骨骼肌纤维生成

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

Engineered Vascularized Muscle Flap

工程化血管肌瓣

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen ...

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

计算机断层扫描和成骨血管生成耦合光学成像评估头盖骨自体移植和移植物的整合

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

中的应用<em&gt;在体内</em&gt;评定组织的鼠胫骨前的功能测试工程化骨骼肌修复

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Cardiac Muscle-cell Based Actuator and Self-stabilizing Biorobot - PART 1

心肌肌细胞基致动器和自稳定Biorobot  - 第1部分

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living ...

Cardiac Muscle Cell-based Actuator and Self-stabilizing Biorobot - Part 2

心肌肌细胞基致动器和自稳定Biorobot  - 第2部分

In recent years, hybrid devices that consist of a living cell or tissue component integrated with a synthetic mechanical backbone have been developed. ...

Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model

半自动纵 Microcomputed Tomography-based 裸鼠骨质疏松症相关椎体骨折模型的定量结构分析

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

Melt Electrospinning Writing of Three-dimensional PolyÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Âµ-caprolactone Scaffolds with Controllable Morphologies for Tissue Engineering Applications

用于组织工程应用的三维聚 (ε-内) 支架的熔体静电纺丝

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...