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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle&#8217;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>0.45um pore size, 12mm diameter</td>
		</tr>
		<tr>
			<td>anti-collagen type I</td>
			<td>Southern Biotech</td>
			<td>1310-08</td>
			<td></td>
		</tr>
		<tr>
			<td>anti- &#945; 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>24x60mm</td>
		</tr>
		<tr>
			<td>Microscope SuperFrost slides</td>
			<td>Menzel-Glaser</td>
			<td>AA00000102E</td>
			<td>76x26mm</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&#039;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

10.3K 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

Ex Vivo Culture of Patient Tissue &amp; Examination of Gene Delivery

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery. Fresh patient tissue obtained at the time of surgery is sliced and maintained in culture. The ex vivo model system allows for the physical delivery of genes into intact patient tissue and gene expression is analysed by bioluminescence imaging using the IVIS detection system. The bioluminescent detection system demonstrates rapid and accurate quantification of gene expression within individual slices without the need for tissue sacrifice. This slice tissue culture system may be used in a variety of tissue types including normal and malignant tissue and allows us to study the effects of the heterogeneous nature of intact tissue and the high degree of variability between individual patients. This model system could be used in certain situations as an alternative to animal models and as a complementary preclinical mode prior to entering clinical trial.

Ex Vivo Culture of Patient Tissue &amp; Examination of Gene Delivery

This article describes the culture of patient tissue slices for gene delivery studies and subsequent analysis of gene expression using IVIS bioluminescence imaging.

患者组织及基因传递考试前的体外培养

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery.  Fresh patient tissue obtained at the time of surgery ...

科研

医学

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人类神经内分泌肿瘤细胞株为研究人类神经内分泌肿瘤治疗的三维模型

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets.

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods.

一个人力<em&gt;前体内</em&gt;动脉粥样硬化斑块模型研究病变的生物学

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic ...

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal hyperplasia (IH) that eventually leads to vessel occlusion and graft failure. Mechanical forces, particularly low shear stress and high wall tension, are thought to initiate and to sustain these cellular and molecular changes, but their exact contribution remains to be unraveled. To selectively evaluate the role of pressure and shear stress on the biology of IH, an ex&#160;vivo perfusion system (EVPS) was created to perfuse segments of human saphenous veins under arterial regimen (high shear stress and high pressure). Further technical innovations allowed the simultaneous perfusion of two segments from the same vein, one reinforced with an external mesh. Veins were harvested using a no-touch technique and immediately transferred to the laboratory for assembly in the EVPS. One segment of the freshly isolated vein was not perfused (control, day 0). The two others segments were perfused for up to 7 days, one being completely sheltered with a 4 mm (diameter) external mesh. The pressure, flow velocity, and pulse rate were continuously monitored and adjusted to mimic the hemodynamic conditions prevailing in the femoral artery. Upon completion of the perfusion, veins were dismounted and used for histological and molecular analysis. Under ex&#160;vivo conditions, high pressure perfusion (arterial, mean = 100 mm Hg) is sufficient to generate IH and remodeling of human veins. These alterations are reduced in the presence of an external polyester mesh.

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mechanisms leading to the development of intimal hyperplasia (IH) and vein graft failure are still poorly understood. This study describes an ex&#160;vivo system to perfuse human veins under controlled flow and pressure. Furthermore the efficiency of external mesh reinforcement to limit the development of IH was evaluated.

步骤人类隐静脉<em&gt;前体内</em&gt;灌注和外部强化

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

一个<em&gt;体外</em&gt;模型在乳腺癌研究激素的作用

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

在体内和体外途径研究银河系结构现货卵巢癌转移定植腹膜脂肪

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Microtumors使用3D人体Biogel培养体系和病人源性胶质母细胞瘤的Kinomic剖析和药物反应测试的生成

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

战略内皮细胞形成实验：比较外基质和生长因子，细胞外基质

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

磷31磁共振波谱分析：一种测量工具<em&gt;在体内</em&gt;线粒体氧化磷酸化能力人骨骼肌

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

小鼠视网膜微动脉的反应性测定的制备步骤前体

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...