Name: human dupuytren's ex vivo culture for the study of myofibroblasts and extracellular matrix interactions
Uploaded: 2015-04-18T14:00:00.000+00:00
Duration: 8 min 59 s
Description: Watch this Scientific Journal Video about Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions at JoVE.com

The authors are thankful to the nurses at the AMC that have facilitated the tissue collection. We would also like to acknowledge A.M.A. van der Laan for excellent technical support with the SHG and the two-photon imaging.

该协议遵循人类研究伦理委员会的LUMC和AMC的指导方针。 1.手术程序和组织收集注:虽然各种技术腱膜挛缩症的手术切除存在，目前的黄金标准是部分fasciectomy 15。大多数患者治疗当天手术的诊所。 <ol><li>根据患者的和麻醉师的偏好和患者的共病在全身或局部麻醉下进行手术。这些因素都超出了本文的范围。前切口，触及皮肤用尖锐物体来验证麻醉是令人满意的。使用止血带，以方便手术可视化在不流血的领域。 </li><li>无菌值预铺巾后，标记出预期的切口，用钢笔。无论是纵向切开Ø下放大镜放大手术刀皮肤ř采用锯齿切口，以防止更多的挛缩，也允许足够的曝光。 </li><li>用剪刀清扫，摆脱底层筋膜收缩掌的皮肤和皮下组织层。识别和保护手指的神经血管束，因为患病帘线可以取代它向中线。一旦纤维化组织（结节和/或电源线）概述，用手术刀尖锐和地区（小计）切除它。在该过程结束时进行下止血带控制细致止血，防止血肿形成。 </li><li>要关闭的皮肤，使用附加的Z-plasties，得到皮肤的局部延长。 </li><li>这些研究使用纤维化线，该疾病的最活跃的和细胞部分仅结节的部分。有外科医生进行宏观鉴定。分离的结节在手掌经常前体或帘线但未落切除，因为它们通常不引起症状。我们切除了那些人说是合同手指的线的一部分结节。由帘线的硬厚部分在handpalm（ 图1A）识别这些结节。 </li><li>一旦外科医生移除组织，使用无菌镊子至50ml管含有补充了10％FCS和1％（P / S）的DMEM培养基中，立即将其传送。保持组织最多2小时的在该培养基上的框湿冰上（4℃）的（ 例如，运输从操作室的细胞培养设备的组织的）。 </li><li>保持样品在湿冰上（4℃），同时准备用于下一步骤。材料和细胞培养容器的制备需要10-20分钟。 </li></ol> 2.准备仪器，培养基和培养插入内容注:所有的程序都在室温进行，除非特别声明。 <ol><li>制成500毫升之DMEM中补充有10％FCS和1％（P / S）和过滤sterilize。 Prewarm在37℃的介质。 </li><li>高压釜镊子，一对弯曲叶片梅奥剪刀（150-170毫米长）和一对梅岑鲍姆剪刀和存储在无菌容器中。 </li><li>在层流中，打开一个无菌的12孔培养板。在12孔板（ 图1B，下图）的每个孔中放置一个培养插入物（0.45微米孔径，直径12mm）。 </li><li>用600微升预热（37℃）的培养基通过移液在井，而不是直接对插入物的膜填充的12孔板。将板放入培养箱中（37℃，5％的CO 2）（ 图1B，上图）。 <ol><li>或者，使用24孔板，并添加培养基的300微升每孔。每孔培养基的体积是最佳时，液体和膜插入件的下部之间形成的弯月形层。 注意:在井底部的介质通过硝化纤维素membr扩散烷形成的弯液面层， 如图1B所示 （下图）的方案中描述。 </li></ol></li></ol> 3，组织准备和体外组织培养设置注:所有的程序都在室温进行，除非特别声明。 <ol><li>从50毫升管传输线的结节部上的100毫米的培养皿，并添加预热的（37℃）培养基为10ml。确保该组织是在整个过程中，与液体接触。 <ol><li>删除使用梅岑鲍姆剪刀perinodular脂肪层。丢弃脂肪组织。与使用镊子和弯曲刃的剪刀，切割横向组织切成小块（200微米的最大厚度）。 </li></ol></li><li>从培养箱转移的12孔板进层流罩。检查该插入物的膜已经变成透明的（湿），并因此与所述介质接触。 </li><li>使用镊子小心地提起一个组织部分通过触摸外层（不施加张力的组织）。将一个组织部分到细胞培养插管的膜，使得所述组织保持在空气介质相间。培养最多两个组织部分上的相同的插入/孔。 </li><li>确保组织被平放在该膜，在纵向接触的介质，使得所述组织既不浮离膜也没有被完全覆盖的介质。避免气泡。 注意:在此阶段，有可能添加的生长因子或感兴趣的媒体抑制剂;例如，测试化合物（A，B，C，D，E），在重复和/或在浓度曲线。包括在正常生长条件下的至少2对照（未处理）件的组织，以确保在实验已正确设置。孵育组织为3-7天，在组织培养箱。 </li><li>感染组织与任何慢病毒或腺病毒结构，如果过表达或击倒é的基因的感兴趣需要xperiments要执行（ 图2）。从12孔板转移的组织部分进入使用镊子35毫米培养皿中。 <ol><li>使用10微升高滴度的病毒的最大音量。 </li><li>与装载用50μl的PBS含10微升所选病毒的胰岛素注射器进行注射解剖显微镜下。放置针垂直于所述组织的中心。 </li><li>慢慢注入注射器的内容。不要直接收回该针头。当穿刺组织，不要让针穿过对方，但仍维持在组织本身内部的针（周围组织的中心）。 </li><li>转移组织放回12孔板，封闭培养板，并把它放入培养箱中（37℃，5％的CO 2）。 </li></ol></li></ol> 4.全安装免疫染色和三维重建注:所有亲cedures是在室温进行，除非特别声明。该协议为整装免疫染色和成像下面描述与用于其它组织16-19先前报道的方法的改编。缓冲液和材料在表1和材料清单中有描述。 <ol><li>转移的组织从培养板于含有PBS 2ml微量管中。洗2×5分钟，在旋转平台上的PBS溶液。修复组织在4％缓冲的PFA（全组织1.5毫升溶液在2ml微量离心管），O / N，在4℃的旋转平台上。 </li><li>除去PFA和在PBS中洗涤3次，每次5分钟。 </li><li>脱水组织浸在2小时在RT的旋转平台上的25％甲醇溶液。增加甲醇百分比逐渐（50％，75％，100％）与浸渍在各溶液2小时，在室温在旋转平台上的组织。 </li><li>转移的组织在DMSO /甲醇溶液（透工序），连续3周，在4℃，如前面描述的18。 注意:在此阶段，组织可以在-20℃储存长期在100％甲醇。 </li><li>进行染色过程16，17;逐渐水合组织（75％-50％-25％-0％甲醇系列）。在4℃下执行在RT或O / N 2小时每补液步骤，在恒定的搅拌。 </li><li>孵育在PBS中含有1％牛血清白蛋白（BSA）和20％DMSO中O / N在4℃下一级抗体样品。使用的抗体在使用商业股在PBS以下比率:抗α平滑肌肌动蛋白（1:500，小鼠），抗I型胶原（1:500，山羊），抗胶原III型（1:500，山羊）。 </li><li>为48小时，在4℃（变更PBS溶液3〜4倍）的PBS广泛清洗。 </li><li>在1稀适当的二级抗体（Alexa的555抗小鼠，Alexa的488抗山羊）:250稀释在PBS中含有1％（BSA）和20％的DMSO。孵育O / N在4℃。 </li><li>为48小时，在4在PBS广泛洗76℃和孵育核染液，TO-PRO-3 1:500稀释在PBS中，在最后的洗涤步骤。 注意:TO-PRO-3是一个远红光发射DNA染料（用氦氖633nm的激光线），并结合对同时成像对其它信道;在这种类型的案件I型胶原/ III型胶原（488纳米），α平滑肌肌动蛋白（555纳米），TO-PRO-3（647纳米）。 </li><li>转移组织的甲醇系列（25％-50％-75％），以缓慢脱水的组织;执行在恒定搅拌下进行2小时，在室温或O / N的每个脱水步骤在4℃下。 <ol><li>转移组织中的聚丙烯管中。在水杨酸甲酯或清除的组织（在化学罩手柄）18作为替代使用BABB液19。在RT下持续搅拌孵育直至组织变得透明。山间的幻灯片和硬组的安装介质（ 图1C，上图）密封盖玻片。 </li></ol></li><li>在一个倒置的共聚焦显微图像样本范围配备高NA 63X和/或100X油浸物镜（ 图1C）。 <ol><li>清洗与由显微镜的制造商提供的清洗液中的透镜。 </li><li>适用的油的一滴在物镜。放置与显微镜舞台上和聚焦在样品上的检体的幻灯片。 </li></ol></li><li>设置在显微镜的三通道荧光与激光线488纳米，514纳米和633纳米的同时成像的配置。 </li><li>获得单个的XY图像或多个焦平面图像（Z堆栈）（ 图3A）。使用ž堆栈增加的宽度进行三维重建。为了获得高分辨率的图像使用较低的扫描速度，平均扫描（≥3）的数目，并获得1024×1024像素分辨率的16位图像。 </li><li>分析Z轴堆栈采集的图像:第一重命名并保存图像文件（XYZ）。使用共焦显微镜的软件程序中，配置一个最大强度幻灯在Z堆叠图像转换成三维的ction重建图像。 <ol><li>选择XYZ文件，在"进程工具"下，单击"可视化"和"3D投影"。在凸起（或"平均"，如果荧光信号饱和的）的方法中进入"最大"。对于一个单一的投影不改变在X，Y和Z平面，并应用投影工具（ 图3B，R1）。 </li></ol></li><li>使用Z堆叠图像创建3D投影与动画（共聚焦软件）用于观看的目的。选择XYZ文件，在这个过程工具，单击下的"可视化"和"3D投影"。 <ol><li>点击"创建电影"。度输入开始旋转角度（ 如 0°为电影，Y轴的起点），然后点击"设置开始"。进入度结束旋转角度（Y轴）作为影片的结束点（ 如 180°或3606;一个完整的旋转），然后点击"设置结束"。 </li><li>选择"最大"作为凸起（或"平均"，如果荧光信号饱和的）的方法。进入"帧数"用于3D重建的旋转（ 例如，20转或者更高，以降低旋转速度）。点击"应用"。导出的电影视觉文件（ 例如，"AVI"）（补充电影1）或导出为"TIFF"文件，该文件会为每个旋转角度的图像文件（ 图3B;旋转R4，R10，R18）。 </li></ol></li></ol> 5.合并的第二次谐波产生（胶原蛋白），并在培养过程前体内组织双光子激发荧光（弹力）成像注:所有的程序都在室温进行，除非特别声明。 <ol><li>从孵化器中取出Transwell小板和放置一个单（不固定）TIssue部分中含有500微升培养基的35毫米细胞培养板。 </li><li>定位所述组织，以使长轴平盘上。继续组织湿时刻然而，不浮。 </li><li>放置多光子显微镜的浸水目标直接与组织（ 图1C）接触。 </li><li>获得的图像为800纳米的激发波长和371-425纳米（SHG，胶原蛋白）和474-532纳米（自体荧光，弹性蛋白）（图3C）之间收集发射光。在直立的共聚焦显微镜配备一个飞秒激光用20X / 1.0 NA水浸物镜进行双光子显微镜。过程共聚焦栈与制造商的软件。 注意:励磁与胶原分子的近红外线激光产生的天然胶原结构中的不同深度的高对比度成像。 </li></ol>

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Optical second-harmonic microscopy, crossed-beam summation, and small-angle scattering in rat-tail tendon.</a> Biophysical Journal. 50 (4), 693-712 (1986).</li><li>Mohler, W., Millard, A. C., Campagnola, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second+harmonic+generation+imaging+of+endogenous+structural+proteins.">Second harmonic generation imaging of endogenous structural proteins.</a> Methods. 29 (1), 97-109 (2003).</li><li>Boulesteix, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micrometer+scale+ex+vivo+multiphoton+imaging+of+unstained+arterial+wall+structure.">Micrometer scale ex vivo multiphoton imaging of unstained arterial wall structure.</a> Cytometry Part A. 69A (1), 20-26 (2006).</li><li>Tomasek, J. J., Gabbiani, G., Hinz, B., Chaponnier, C., Brown, R. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+culture+force+monitor+for+measurement+of+contraction+forces+generated+in+human+dermal+fibroblast+cultures:+evidence+for+cell-matrix+mechanical+signalling.">A culture force monitor for measurement of contraction forces generated in human dermal fibroblast cultures: evidence for cell-matrix mechanical signalling.</a> Biochimica et Biophysica Acta (BBA) - General Subjects. 1201 (2), 186-192 (1994).</li><li>Verjee, L. S., Midwood, K., Davidson, D., Eastwood, M., Nanchahal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-transcriptional+regulation+of+&#945;-smooth+muscle+actin+determines+the+contractile+phenotype+of+Dupuytren's+nodular+cells.">Post-transcriptional regulation of &#945;-smooth muscle actin determines the contractile phenotype of Dupuytren's nodular cells.</a> Journal of Cellular Physiology. 224 (3), 681-690 (2010).</li></ol>

作者们提交了一份专利申请掌腱膜挛缩症:3D器官培养。 ＃GB1307200。商业授权和服务合同是可用的。

培养离体人结缔组织的最关键的步骤是即时使用的手术切除，以确保生存力后的组织的;该组织应保留在任何时候都培养基或盐溶液;保持无菌培养;组织的横截面应该有最大200微米的厚度;建立体外培养体系是最优的时组织是在与介质接触，但尚未完全浸没。介质应当只在Transwell小的外部腔室中的小容积（ 例如，500-600微升每12孔板表面积）加入。 从掌腱膜挛缩的衍生结缔组织纠缠在一个艰难的结构与细胞外基质蛋白，如胶原蛋白，蛋白多糖和弹性蛋白含量高;由于这些性能，另外，由于肌成纤维细胞挛缩它可以是具有挑战性的穿过组织来切割。以使用适当的手术工具是很重要的;弯曲BL交锋梅奥剪刀用于切割较大的部件在相同的厚度和更小的外科剪刀用于切割较小的组织部位（ 例如 ，如果更多的组织部位，需要使用24孔板）。 方法DD的研究是切除标本纤维化病理组织学分析，或主要的成纤维细胞的推导。来自患者的材料做细胞推导是两种方式;在其中的一种方法，组织部分在塑料培养皿数周的，直到有纤维细胞的生长培养。第二种方法是酶处理的组织与胶原酶和胰蛋白酶。第一种方法是耗时的，成纤维细胞性质改变因培养条件而有相同的细胞系的传代中通过依赖变性。酶促方法是更快，它可以用于细胞分选测定法，但是， 在体外维持这些细胞的出先天的ECM的不反映在体内 </EM&gt;属性。这里提出的模型是适用于细胞和细胞外基质的形态研究，几种技术已成功地使用，例如免疫荧光，流式细胞术，核糖核酸分析，整装3D成像和胶原沉积测量（分泌在培养基中）。然而，从组织部位，细胞信号传导实验和蛋白免疫印迹的总蛋白质分离需要优化，并且不作为单层成纤维细胞培养物作为稳健。该方法的另一个缺点是缺乏机械张力工程化到培养体系。根据一个恒定的或基底的机械力存在最结缔组织，即使在静止的条件下，它不仅提供了机械支持，而且还影响细胞的行为和信号25（即mechanoregulation和机械传导）。损失紧张导致αSMA纤维多快好省拆卸的时间短26期。 αSMA在体外丰</em&gt;培养组织表示MFB收缩。然而，我们的模型可以受益于一个等距力传感器27，28。关于技术和生物变异的掺入，我们的方法是可重复的（可行性，ECM网络），不容易改变的组织，由于文化的时间很短。例如，成纤维细胞生长的方法需要好几个星期可能引起的生化变化（ 如，遗传突变，复制性衰老的积累）。但是，患者特异性遗传特性和生物变异性是本身在DD研究领域的挑战，并且不能与任何现有的方法解决。 如该协议中，手术切除后纤维化的DD标本直接培养在体外的3D系统和/或快速冷冻。根据科学问题，组织的变形例是可能的，通过加入生长因子，化学化合物，V病毒属介导的基因递送，反义寡核苷酸和微RNA。化合物可递送介质或与在3D培养室中的组织的局部显微注射。 3之后和最多7天培养的组织既可以均化细胞的分离和FACS分析或总RNA和蛋白质表达谱分析的隔离，以及处理用于组织学分析。纤维蛋白（αSMA，I型胶原和II，纤连蛋白），该线和纤维网络的一致性的结节部的组织结构中的表达状态之前和处理之后进行评估。为了在培养过程中，以监视对ECM重排我们结合在SHG（胶原）和双光子激发荧光（弹性蛋白）成像。 胞重排可以被量化或使用图像量化软件建模。这些参数给出的纤维化或molec的药物作用的指示在培养过程中已被诱导ular改变。此外ECM阵列也可以在单片进行产生的效果更好概述。整体而言，我们已经建立了一个强大的系统，使纤维化体外的研究。

迪皮特朗病（DD），良性纤维增生疾病导致手指的永久弯曲，由于结节和帘线中的手掌的形成。虽然疾病传播是北欧的白种人的特别高，该疾病的潜在的遗传病因仍然是未知的1。 DD的主要特征是过量生产细胞外基质（ECM）蛋白（ 例如，胶原），其形成坚韧的纤维组织占据的肌腱和手和手指的手掌的皮肤之间的空间中的，永久地破坏精细动作手2，3。该疾病的复发表明潜在的遗传改变如纤维化1,4的一个原因。一种有效的治疗方法可能是直接针对在细胞和分子水平上的不可控的纤维化的机制。 我们最近对肝纤维化的工作使我们的发展新颖的三维培养系统，它允许人类纤维化组织的短期培养以药物测试的潜力。该系统有助于克服2D成纤维细胞培养物的限制的方式，为在介导纤维化5定义角色为偏向下调节，通过外显 ​​子跳跃实现，TGFβ途径激活。 我们已经开发出一种方法，以从培养的DD患者体外人体切除标本，研究和肌成纤维细胞之间的相互作用的周围的ECM 5,6。的DD结缔组织纤维化的研究以及其他纤维化疾病依赖于外科切除的组织病理学分析标本，从组织和建立原代培养物或细胞分选程序中的成纤维细胞的分离。这些方法是相当静态的，因为它们不允许外感操纵疾病性质或治疗干预由实验者的。此外，主CELL文化倾向于适应培养条件和它们的基因表达的特性的每个通道在基本上从体内情况不同，即使是在早期传代（通道3之间和6）7，8，我们已成功地保持了废物外科材料在体外培养条件下的时间周期，允许的患者特异性特征的研究和抗纤维化或消炎药的化合物的筛选。 该系统是基于硝化纤维素膜，其允许组织的与介质但不与塑料接触，因此，防止在附接所述组织的改变，如培养的DD成纤维细胞以及其它细胞类型9时以前观察。无胶原凝胶或其他ECM蛋白基质是必需的，因为在DD组织自身产生大量的这些蛋白质。这有利于本土ECM微环境和成交量罪维修CE矩阵基板是组织结构和功能10,11的重要调节器。举例来说，细胞外基质蛋白，如纤连蛋白，层粘连蛋白和胶原蛋白，可能会影响在上皮细胞12同样示于心尖-基底极性的成纤维细胞的前-后极性，13 。极化细胞具有细胞外的分子的非对称分布，它确定细胞迁移和基因表达， 例如，在膜α1β1整联无障碍影响细胞粘附到I型胶原14。由于此3D模型的一个主要目标是保持天然的微环境，无人工的ECM矩阵基板。 简言之:切除标本也同样切在无菌环境中，并放置在nitrocellular膜。如果治疗通过注射给药是必需的组织被注射它们已被放置在该膜后。如果治疗不要求通过注射进行给药挠度则该化合物被添加到培养基（Dulbecco氏改良的Eagle氏培养基（DMEM）中，用1％胎牛血清（FCS），1％青霉素 - 链霉素（P / S））。将培养物维持最多十天，之后将组织固定在4％多聚甲醛（PFA），通过30％蔗糖溶液进行处理，包埋在OCT化合物，并储存在-80℃下，如前所述5。

<table><tbody><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium</td><td>Invitrogen</td><td>11965-084</td><td></td></tr><tr><td>fetal calf serum (FCS)</td><td>Gibco</td><td></td><td>high glucose, heat inactivated</td></tr><tr><td>penicillin-streptomycin</td><td>Invitrogen</td><td>15070-063</td><td></td></tr><tr><td>Cell culture inserts</td><td>Millicell</td><td>PIHA01250</td><td>0.45 &amp;#956;m pore size, 12&amp;nbsp;mm diameter</td></tr><tr><td>anti-collagen type I</td><td>Southern Biotech</td><td>1310-08</td><td></td></tr><tr><td>anti-&amp;alpha; smooth muscle actin</td><td>Sigma</td><td>A2547</td><td></td></tr><tr><td>anti-collagen type III</td><td>Southern Biotech</td><td>1330-01</td><td></td></tr><tr><td>Alexa Fluor555 Donkey Anti-Mouse IgG (H+L)</td><td>Invitrogen</td><td>A-31570</td><td></td></tr><tr><td>Alexa Fluor 488 Donkey Anti-Goat IgG (H+L) Antibody</td><td>Invitrogen</td><td>A-11055</td><td></td></tr><tr><td>TOPRO-3</td><td>Invitrogen</td><td>T3605</td><td></td></tr><tr><td>methylsalicylate</td><td>Sigma</td><td>M6752</td><td></td></tr><tr><td>paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Tissue Tek OCT</td><td>Sakura</td><td>25608-930</td><td></td></tr><tr><td>Microsccope glass coverslips</td><td>Menzel-Glaser</td><td>BB024060A1</td><td>24 x 60 mm</td></tr><tr><td>Microscope SuperFrost slides</td><td>Menzel-Glaser</td><td>AA00000102E</td><td>76 x 26 mm</td></tr><tr><td>VECTASHIELD HardSet Mounting Medium</td><td>Vector laboratories</td><td>H-1400</td><td></td></tr><tr><td>Leica TCS SP5 II confocal microscope</td><td>Leica Microsystems</td><td></td><td>Argon-488, 514 nm and HeNe-633 nm laser lines</td></tr><tr><td>Zeiss 710 NLO upright confocal microscope</td><td>Jena, Germany</td><td></td><td>Equipped with femtosecond Spectra - Physics Deep See MP laser (Santa Clara, United States) using a Plan-Apochromat 20X/1.0 NA water-immersion objective.</td></tr></tbody></table>

human dupuytren's ex vivo culture for the study of myofibroblasts and extracellular matrix interactions

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.
To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. 
As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first &#8220;organ&#8221; culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.

离体的方法中，结缔组织的三维培养是一个简单而稳健的建立系统了解的ECM纤维化和构成DD组织的其它细胞组分和潜在的其他类型之间的关系也是如此。此外，这种系统允许一种可靠的方法，以测试在不同的细胞类型和它们对纤维化负载20作用的化合物的效果。 从采集自迪皮特朗的纤维化而得外科废料的步骤中，培养腔室和分析工具的例子的组装示于图1。如图1所示 ，该方法对于大屏幕药物屏幕提供了实验工具， 例如抗纤维化药物化合物以及ECM建模在实时和可再现的方式的研究。纤维化索结节部位都切片，放在Trans well板;每个组织部分（100-200微米）是在培养插入物（0.45微米孔径，直径12mm）的膜​​的顶部进行培养。培养基中加入在底部室中，允许与通过膜的组织接触。平均30-40组织部分可以衍​​生自单一切除标本，其允许药物大规模筛选;化合物，小分子或生长因子。实验寻址的浓度和/或药物的时间依赖性效应是可行的。 如图2中 ，基因的表达可以被操纵（过表达/击倒）通过使用腺/慢病毒直接注射到组织中。荧光标记的病毒转导在新鲜厚的组织标本通过注射进行的。组织保持在培养24-48小时后喷射并随后固定，包埋和切片在0.7微米冷冻切片。另外，以分析不同的化合物或培养条件的组织部分的作用s的固定，并进行整个装载免疫如图3英寸厚的组织的三维成像是通过共聚焦显微镜（图3A-B）中完成的。另一种方法是在保持在培养组织中的实时三维成像;内源性胶原重构了研究实时在新鲜解剖，非固定的组织份二次谐波产生（SHG）（图3C）。也跟着在不同时间点相同组织部位潜在的抗纤维化药物的效果。厚组织的胶原蛋白结构直立多光子显微镜利用SHG成像。在成像期间，组织标本被放置在介质和成像用与水浸泡的目的。成像后，将组织可以被转移回培养系统。效果可以在细胞和胞外环境中提供优于在体外基于细胞的测定水平进行研究。有对ANA多种可能性裂解的生物效应，如组织学的固定的组织切片，整装免疫荧光和三维重建成像通过共聚焦显微镜的SHG和内源性胶原蛋白和弹性蛋白的双光子激发荧光成像。一些非SHG成像的优点是使用新鲜的，非固定的组织，即无抗体染色制剂是必需的，而且在同一组织可以（在培养条件时程）被成像多次。 SHG和双光子激发荧光成像先前已描述用于肌肉和结缔组织21-23和未染色的动脉壁结构24，但是，在这里我们报告迪皮特朗的纤维化的研究的替代应用程序。 <img alt="图1" src="/files/ftp_upload/52534/52534fig1.jpg" /> 图体外三维组织培养体系的方案1。 </stron克&gt;（A）中的DD永久弯曲在患者的手前手术切除挛缩。这个数字已经被修改以前的研究5。 体外培养建立的（B）为例，实验方法，可用于ECM沉积分析（C）的例子。 <a href="/files/ftp_upload/52534/52534fig1large.jpg" target="_blank">请点击此处查看图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52534/52534fig2.jpg" /> 证明型原理图2. 体外病毒介导的基因表达调控的整装组织。（A）细胞核可视化与TO-PRO-3染料（蓝色）。酒吧75微米。（B）荧光的直接可视化染料红色荧光蛋白（红色），注射后与腺病毒表达，红色荧光蛋白。 条为75μm。（C）的合并的图像（蓝色核染色，DsRed的红色）。红色荧光蛋白的细胞质定位，表示24小时内送货腺病毒在细胞内。条为75μm。（DF）的慢病毒转导。（D）的细胞核可视化用的TO-PRO-3染料（蓝色）。条为25μm。（E）的绿色荧光蛋白在组织切片的直接可视化（绿），后喷射用的Lenti-GFP颗粒。酒吧25微米。（F）D的合并图像和E表明伦蒂-GFP的细胞内定位。杆25微米（GH）实施例针对感兴趣（指定为"A"）基因的慢病毒介导的敲低的;（G）的慢病毒携带加扰的shRNA序列注射体外进入组织。免疫荧光染色的感兴趣蛋白质（指定为"A"）显示为红色。酒吧75微米。（H）的慢病毒携带基因A-shRNA的顺序注射体外进入组织。免疫荧光蛋白"A"。细胞核用TO-PRO-3染料。酒吧75微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52534/52534fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52534/52534fig3.jpg" /> 图3.三维重建成像，二次谐波和双光子激发荧光成像。（A）三维成像激光共聚焦显微镜。单（XY）的图像中沿不同的组织深度（Z = 50-200微米）的焦平面。整个安装IMMUnofluorescence染色;肌成纤维细胞标记物α平滑肌肌动蛋白（αSMA，红色），核染色（TO-PRO-3，蓝色）。酒吧:500微米（B）3D重建图像的原始图像（Z堆栈389微米），在A组的表示为"R1"，"R4"，"R10"，"R18"不同的3D旋转显示。酒吧500微米。（C）由新鲜，厚组织部分二次谐波（SHG）成像（左图，绿色）内源性胶原蛋白含量建模。 ECM的弹性结构的双光子激发荧光（TPF）捕获由多光子显微镜（中间面板，红色）。胶原蛋白（绿色）和弹性蛋白（红色）（右图）的合并后的图像。酒吧:100微米<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52534/52534fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 补充电影1 Represen对准焦点3D掌腱膜挛缩的整装组织重建后的电影七天的体外培养和处理整装免疫荧光染色进行DD组织的共聚焦成像。肌成纤维细胞标记物α平滑肌肌动蛋白（αSMA，红色），核染色（TO-PRO-3，蓝色）。比例尺= 500微米。帧= 20，Z = 389微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52534/Movie 1.avi" target="_blank">请点击此处观看该视频。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> PBS（磷酸缓冲盐水） </td><td> 8.00克氯化钠（0.137 M） 0.20克氯化钾（2.7毫米） 0.20克KH 2 PO 4（1.1毫摩尔） 0.10克氯化镁 ·6H 2 O（0.5毫米） 2.16克的Na 2 HPO 4·7H 2 O（8.1毫米） 0.10克无水氯化钙 （0.9毫米） H 2 O来1升</td></tr><tr><td&gt; 4％多聚甲醛/ PBS中 </td><td>溶解4克多聚甲醛在含有100ml磷酸盐缓冲盐水（PBS）中，在通风橱中的烧瓶，覆盖烧瓶。将烧瓶加热/搅拌块上，轻轻搅拌溶液。监测温度，以便为达到最大值65℃。避免过度加热。一旦多聚甲醛溶解和解决方案出现明确的，关闭的热量，但离开搅拌。不要为安全起见处理。允许冷却。当冷却时，等分试样的溶液，并存储长期在-20℃的冷冻器中或在4℃冰箱中的最大一个星期。 </td></tr><tr><td> BSA（牛血清白蛋白），10％（重量/体积） </td><td>溶解10克BSA的100毫升的 H 2 O.过滤消毒使用低蛋白结合0.22微米的过滤器。保存在4℃。 </td></tr><tr><td> DMSO /甲醇（20％）透化溶液 </td><td>混合二甲基亚砜与甲醇（1:4的类比，总体积100ml）中。 </td></tr></tbody></table> 表1缓冲溶液。

Watch this Scientific Journal Video about Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions at JoVE.com

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

迪皮特朗病（DD）是手的手掌的纤维增生性疾病。在这里，我们提出了一个协议，从DD文化切除标本的三维（3D）培养体系。这种短期培养系统允许保存的三维结构和纤维化组织的分子性质。

人掌腱膜挛缩的<em&gt;离体</em&gt;文化为肌成纤维细胞的研究和细胞外基质相互作用

Organ fibrosis or &#8220;scarring&#8221; is known to account for a high death toll due to the extensive amount of disorders and organs affected (from ...

human-dupuytren-s-ex-vivo-culture-for-study-myofibroblasts

Nanyang Technological University

Research

JoVE Journal

Medicine

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

10.4K 次观看.  Leiden University Medical Center. 迪皮特朗病（DD）是手的手掌的纤维增生性疾病。在这里，我们提出了一个协议，从DD文化切除标本的三维（3D）培养体系。这种短期培养系统允许保存的三维结构和纤维化组织的分子性质。 

视频: Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

推导人类纤维原细胞的皮肤穿刺活检植文化

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

科研

医学

Fibroblast-Derived 3D Matrix System Applicable to Endothelial Tube Formation Assay

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ECM also participates in cell migration, proliferation, and differentiation. Fibroblasts are the main type of cells modifying ECM fiber arrangement and production. In cancer, CAFs (cancer associated fibroblasts) are in permanent activation status, participating in ECM remodeling, facilitating tumor cell migration, and stimulating tumor-associated angiogenesis, among other pro-tumorigenic roles. The objective of this method is to create a three-dimensional matrix with a fiber composition that is similar to in vivo matrices, using immortalized fibroblasts or human primary CAFs. Fibroblasts are cultured in pre-treated cell culture plates and grown under ascorbic acid stimulation. Then, fibroblasts are removed and matrices are blocked for further cell seeding. In this ECM model, fibroblasts can be activated or modified to generate different kinds of matrix, whose effects can be studied in cell culture. 3D matrices are also shaped by cell signals, like degradation or cross-linking enzymes that might modify fiber distribution. In this context, angiogenesis can be studied, along with other cell types such as epithelial tumor cells.

The aim of this method is to obtain fibroblast-derived 3D matrices as a natural scaffold for subsequent cellular assays. Fibroblasts are seeded in a pre-treated culture plate and stimulated with ascorbic acid for matrix generation. Matrices are decellularized and blocked to culture relevant cells (e.g., endothelial cells).

纤维细胞衍生的三维矩阵系统适用于内皮管形成测定

The extracellular matrix (ECM) is a three-dimensional scaffold that acts as the main support for cells in tissues. Besides its structural function, the ...

癌症研究

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.

This protocol describes the conversion of skin fibroblasts into myoblasts and their differentiation into myotubes. The cell lines are derived from patients with neuromuscular disorders and can be used to investigate pathological mechanisms and to test therapeutic strategies.

将人类成纤维细胞直接重新编程到肌细胞中，以研究神经肌肉疾病的治疗方法

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of ...

遗传学

Preparation of 3D Decellularized Matrices from Fetal Mouse Skeletal Muscle for Cell Culture

The extracellular matrix (ECM) plays a crucial role in providing structural support for cells and conveying signals that are important for various cellular processes. Two-dimensional (2D) cell culture models oversimplify the complex interactions between cells and the ECM, as the lack of a complete three-dimensional (3D) support can alter cell behavior, making them inadequate for understanding in vivo processes. Deficiencies in ECM composition and cell-ECM interactions are important contributors to a variety of different diseases. 
One example is LAMA2-congenital muscular dystrophy (LAMA2-CMD), where the absence or reduction of functional laminin 211 and 221 can lead to severe hypotony, detectable at or soon after birth. Previous work using a mouse model of the disease suggests that its onset occurs during fetal myogenesis. The present study aimed to develop a 3D in vitro model permitting the study of the interactions between muscle cells and the fetal muscle ECM, mimicking the native microenvironment. This protocol uses deep back muscles dissected from E18.5 mouse fetuses, treated with a hypotonic buffer, an anionic detergent, and DNase. The resultant decellularized matrices (dECMs) retained all ECM proteins tested (laminin &#945;2, total laminins, fibronectin, collagen I, and collagen IV) compared to the native tissue. 
When C2C12 myoblasts were seeded on top of these dECMs, they penetrated and colonized the dECMs, which supported their proliferation and differentiation. Furthermore, the C2C12 cells produced ECM proteins, contributing to the remodeling of their niche within the dECMs. The establishment of this in vitro platform provides a new promising approach to unravel the processes involved in the onset of LAMA2-CMD, and has the potential to be adapted to other skeletal muscle diseases where deficiencies in communication between the ECM and skeletal muscle cells contribute to disease progression.

In this work, a decellularization protocol was optimized to obtain decellularized matrices of fetal mouse skeletal muscle. C2C12 myoblasts can colonize these matrices, proliferate, and differentiate. This in vitro model can be used to study cell behavior in the context of skeletal muscle diseases such as muscular dystrophies.

胎儿小鼠骨骼肌3D脱细胞基质的制备用于细胞培养

The extracellular matrix (ECM) plays a crucial role in providing structural support for cells and conveying signals that are important for various ...

发育生物学

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

成纤维细胞衍生的人类工程结缔组织用于筛查应用

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

生物工程

Ultrasonic-augmented Primary Adult Fibroblast Isolation

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method&#8217;s degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

超声波增强原成人成纤维细胞隔离

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary ...

Isolation of Myofibroblasts from Mouse and Human Esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

从小鼠和人类食管隔离肌纤维母细胞

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

生物学

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

成人心脏纤维细胞和肌纤维细胞的分离与表征

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

单一肌纤维培养测定法用于评估体外成人肌肉干细胞功能

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches involve either differentiating myogenic progenitor cells into immature myotubes or the short-term ex vivo culture of isolated individual muscle fibers. A key benefit of ex vivo culture over in vitro is the retention of the complex cellular architecture and contractile characteristics. Here, we detail an experimental protocol for the isolation of intact flexor digitorum brevis muscle fibers from mice and their subsequent ex vivo culture. In this protocol, muscle fibers are embedded in a fibrin-based and basement membrane matrix hydrogel to immobilize the fibers and maintain their contractile function. We then describe methods to assess the muscle fiber contractile function using an optics-based, high-throughput contractility system. The embedded muscle fibers are electrically stimulated to induce contractions, after which their functional properties, such as sarcomere shortening and contractile velocity, are assessed using optics-based quantification. Coupling muscle fiber culture with this&#160;system allows for high-throughput testing of the effects of pharmacological agents on contractile function and ex vivo studies of genetic muscle disorders. Finally, this protocol can also be adapted to study dynamic cellular processes in muscle fibers using live-cell microscopy.

Skeletal muscle function can be assessed by quantifying the contractility of isolated muscle fibers, traditionally using laborious, low-throughput approaches. Here, we describe an optics-based, high-throughput method to quantify the contractility of hydrogel-embedded muscle fibers. This approach has applications for drug screening and therapeutic development.

使用基于光学的系统对水凝胶嵌入的完整小鼠肌纤维进行高通量收缩测量

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches ...

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