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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G., 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=Dynamic+3D+Cell+Rearrangements+Guided+by+a+Fibronectin+Matrix+Underlie+Somitogenesis.">Dynamic 3D Cell Rearrangements Guided by a Fibronectin Matrix Underlie Somitogenesis.</a> PLoS ONE. 4 (10), e7429 (2009).</li><li>Yokomizo, 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=Whole-mount+three-dimensional+imaging+of+internally+localized+immunostained+cells+within+mouse+embryos.">Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos.</a> Nat. Protocols. 7 (3), 421-431 (2012).</li><li>Karkampouna, S., de Julio, M. K. r. u. i. t. h. o. f. -. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrosis:+a+novel+approach+for+an+old+problem.">Fibrosis: a novel approach for an old problem.</a> Receptors &amp; Clinical Investigation. 1 (5), (2014).</li><li>Wolf, K., 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=Collagen-based+cell+migration+models+in+vitro+and+in+vivo.">Collagen-based cell migration models in vitro and in vivo.</a> Seminars in Cell &amp; Developmental Biology. 20 (8), 931-941 (2009).</li><li>Freund, I., Deutsch, M., Sprecher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connective+tissue+polarity.+Optical+second-harmonic+microscopy,+crossed-beam+summation,+and+small-angle+scattering+in+rat-tail+tendon.">Connective tissue polarity. 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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofibroblasts+and+mechano-regulation+of+connective+tissue+remodelling.">Myofibroblasts and mechano-regulation of connective tissue remodelling.</a> Nat Rev Mol Cell Biol. 3 (5), 349-363 (2002).</li><li>Hinz, B., Mastrangelo, D., Iselin, C. E., Chaponnier, C., Gabbiani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+tension+controls+granulation+tissue+contractile+activity+and+myofibroblast+differentiation.">Mechanical tension controls granulation tissue contractile activity and myofibroblast differentiation.</a> The American Journal of Pathology. 159 (3), 1009-1020 (2001).</li><li>Eastwood, M., McGrouther, D. A., Brown, R. 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...

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?...

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

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

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 ...

科研

遗传学

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

从肌肉活检患者来源的细胞系疾病建模隔离和永生化

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

医学

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

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&#160;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 &#39;MyoTACTIC&#39;, 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.

This manuscript describes a detailed protocol to produce arrays of 3D human skeletal muscle microtissues and minimally invasive downstream in situ assays of function, including contractile force and calcium handling analyses.

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 ...

生物工程

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 ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

从小鼠和人类结肠组织初级肌成纤维细胞的分离

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

生物学

制造小鼠肌腱组合体以研究疾病中的细胞相互作用

Engineering Tendon Assembloids to Probe Cellular Crosstalk in Disease and Repair

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell populations. This load-bearing core is encompassed, nourished, and repaired by a synovial-like tissue layer comprising the extrinsic tendon compartment. Despite this sophisticated design, tendon injuries are common, and clinical treatment still relies on physiotherapy and surgery. The limitations of available experimental model systems have slowed the development of novel disease-modifying treatments and relapse-preventing clinical regimes. 
In vivo human studies are limited to comparing healthy tendons to end-stage diseased or ruptured tissues sampled during repair surgery and do not allow the longitudinal study of the underlying tendon disease. In vivo animal models also present important limits regarding opaque physiological complexity, the ethical burden on the animals, and large economic costs associated with their use. Further, in vivo animal models are poorly suited to systematic probing of drugs and multicellular, multi-tissue interaction pathways. Simpler in vitro model systems have also fallen short. One major reason is a failure to adequately replicate the three-dimensional mechanical loading necessary to meaningfully study tendon cells and their function. 
The new 3D model system presented here alleviates some of these issues by exploiting murine tail tendon core explants. Importantly, these explants are easily accessible in large numbers from a single mouse, retain 3D in situ loading patterns at the cellular level, and feature an in vivo-like extracellular matrix. In this protocol, step-by-step instructions are given on how to augment tendon core explants with collagen hydrogels laden with muscle-derived endothelial cells, tendon-derived fibroblasts, and bone marrow-derived macrophages to substitute disease- and injury-activated cell populations within the extrinsic tendon compartment. It is demonstrated how the resulting tendon assembloids can be challenged mechanically or through defined microenvironmental stimuli to investigate emerging multicellular crosstalk during disease and injury.

作者聚焦：通过开发小鼠组合来了解细胞机制来推进肌腱研究

Here, we present an assembloid model system to mimic tendon cellular crosstalk between the load-bearing tendon core tissue and an extrinsic compartment containing cell populations activated by disease and injury. As an important use case, we demonstrate how the system can be deployed to probe disease-relevant activation of extrinsic endothelial cells.

工程肌腱组件以探测疾病和修复中的细胞串扰

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell ...

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 ...

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

单肌纤维隔离与文化从埃默里-德雷福斯肌肉萎缩的早期后纳塔尔发展穆林模型

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

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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

从肌肉活检患者来源的细胞系疾病建模隔离和永生化

评估人体骨骼肌微组织骨骼肌健康的功能指标

成纤维细胞衍生的人类工程结缔组织用于筛查应用

从小鼠和人类结肠组织初级肌成纤维细胞的分离

工程肌腱组件以探测疾病和修复中的细胞串扰

工程肌腱组件以探测疾病和修复中的细胞串扰

工程肌腱组件以探测疾病和修复中的细胞串扰

超声波增强原成人成纤维细胞隔离

单肌纤维隔离与文化从埃默里-德雷福斯肌肉萎缩的早期后纳塔尔发展穆林模型