Name: a rapid, scalable method for the isolation, functional study, and analysis of cell-derived extracellular matrix
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我们非常感谢贝琳达·威拉德博士学，蛋白质组学和代谢组学实验室，勒纳研究所，克利夫兰诊所，为进行RCS ECM的蛋白质组学分析。我们承认theMedical研究理事会英国的财政支持，授权号K018043，以JCA。

1.去除细胞与氢氧化铵溶液<ol><li>通过在适当的密度电镀准备贴壁细胞。 注:细胞可以是用于分析产生足够的ECM任何贴壁细胞类型。在这里，我们描述了使用COS-7细胞，它包含SV-40病毒DNA序列的非洲绿猴肾成纤维细胞样细胞系的; RCS，大鼠软骨肉瘤细胞系;或正常人真皮成纤维（HDF）从幼年包皮菌株。 HDF用于从通道1到唯一通道8。 <ol><li>板上盖玻片活细胞和ECM，对固定的ECM的荧光显微镜研究的摄像单元，或用于制备细胞衍生的ECM的小规模功能测定。板上进行SDS-PAGE分析，免疫印迹，或蛋白质组学研究细胞培养皿的细胞。 注:细胞培养条件（细胞板和培养基的数目）将取决于细胞类型。细胞数量板需要根据经验为特定细胞系或细胞株建立。 </li><li>允许一个适当的时间，将细胞沉积的ECM，通常&gt; 16小时。注:时间段将取决于细胞类型，将需要根据经验确定。如果导电异位表达时，允许合适时间感兴趣的转染蛋白的表达和细胞外基质的沉积。 </li></ol></li><li>在一个排油烟机，准备的20mM氢氧化铵在合适的容器通过与去离子H 2 O稀释该储备溶液1/14 <ol><li>从孵化器中取出细胞，并轻轻取出培养基。通过对盘的壁轻轻倒出不含Ca 2+ / Mg 2+离子添加磷酸盐缓冲盐水（PBS）。岩盘两次并用塑料移液管除去液体。重复两次。 </li></ol></li><li>在排油烟机，倾斜每一道菜，然后从步骤1.2用塑料移液管取出PBS。添加3每100毫米培养皿氢氧化铵中的溶液，并培育它们在室温下5分钟。在5分钟温育期，轻轻搅动菜每分钟，以确保所有的细胞的裂解。步骤1.4-1.7还将在排油烟机进行。 注:替代细胞去除试剂包括2 M或8M尿素，这是与细胞10分钟孵育。 </li><li>丰富的金额去离子H 2的O添加到每一道菜，每100毫米的菜至少20毫升，用摇摆。氢氧化物溶解的材料，它由氢氧化铵，裂解的细胞，以及去离子H 2 O型通过用移液管抽吸处置铵。转让本废液转化为液体废物的容器。 </li><li>在丰富的去离子H 2Ø四次洗不溶性ECM层，以确保彻底清除所有的氢氧化铵，溶解的材料。 </li><li>对于通过免疫荧光ECM的检查，加入2％的固定ECM多聚甲醛（PFA;每100mm培养皿3mL）中在室温下10分钟。 注意:PFA是皮肤或眼睛接触和严重的过度曝光会产生肺部损伤，窒息，昏迷，甚至死亡的情况非常危险。它应该只在排油烟处理，和个人防护装备应戴。 </li><li>用PBS洗每个固定的样品两次，如在步骤1.2和PFA溶液到合适的液体废物容器的处理。如果需要的话，他们准备荧光显微镜之前在4℃下储存在PBS中的细胞外基质的样品。 </li></ol> 2.一个ECM蛋白沉积跟踪由活细胞的共聚焦显微镜成像<ol><li>计数血球下细胞。对于COS-7细胞，板250,000细胞上转染60毫米的细胞培养皿。 注:对于活细胞成像，细胞必须与编码感兴趣的ECM蛋白，在框内融合与遗传编码发出荧光的载体转染的NT标记。这是为了使所关注的细胞外基质蛋白的过程中活细胞成像的鉴定。 </li><li>后的适当时间用于蛋白质表达（ 例如，16小时），从用胰蛋白酶-EDTA溶液皿除去细胞，用血细胞计数器计数它们，并且板〜150,000个细胞到用于成像的35毫米网格，玻璃底皿。允许对电池2小时，以重新连接，并开始ECM沉积（的时间段将取决于细胞类型和必须凭经验建立）。 注意:使用不含有酚红，因为这可以得到红色色调影响图像质量的细胞培养基。 </li><li>在上述2小时内，设立了共聚焦显微镜用37℃培养箱室和CO 2的供应。允许在腔室和在盘的温度稳定至成像之前37℃;这可能需要1小时。 </li><li>孵育镀到网格碟30分钟的荧光细胞标记35毫米的细胞。然后，洗涤​​细胞在成像之前无酚红培养基中两次。 注:细胞质的荧光标记物可以用于可视化细胞卷或膜掺入标记物可以用于可视化小区边缘。 </li><li>使用20X物镜和相衬的共聚焦显微镜，标识包含了一些（&gt; 3）贴的，健康的细胞适当的方格。确认所选择的靶蛋白在使用63X或100X的目标和在荧光显微镜下的相应波长的选择的网格内的细胞中的表达。 </li><li>使用Z-栈软件设置荧光和相衬时间推移成像。将"开始"堆栈仅仅是个ECM层和"结束"堆栈下面是公正细胞体的上方。取决于细胞类型设置的z步长到0.3微米，在z体积为5和10微米之间的深度在总。这将每个时间点15-30 Z-部分之间给。 </li><li> Capturë荧光（红色荧光蛋白（RFP），激发= 555 nm，发射= 584纳米）和相位对比图像周期性超过设定的时间段。例如，使用时间推移软件设置在2分钟的时间间隔，持续时间为2小时。选择"开始"按钮在定义的时间段，开始z轴部的图像的捕获。 </li><li>在时间周期结束时，从盘除去细胞，如在步骤1.1-1.5中所述，以确认在ECM感兴趣的荧光标记蛋白的定位。 </li><li>使用相衬成像和20X客观搬迁相关的方格。 </li><li>切换到63X物镜和荧光显微镜下识别的ECM层。这一形象比较提取前映像</li></ol> 3.细胞来源的ECM的无菌制剂的细胞功能测定<ol><li>盖玻片上，另一珀普生长细胞的一个群体（"黑客帝国生产者"细胞）特征研在至少24小时100毫米细胞培养皿（"测试"细胞）在标准培养。 注意:这些可以是任何两个不同的贴壁细胞类型，实验设计可以包括一种或两种细胞群的转染以表达荧光标记的ECM蛋白。 </li><li>在层流罩，作为用于细胞培养，制备PBS中的无菌溶液不含Ca 2+ / Mg 2+离子 ，20mM的氢氧化铵通过使每通过无菌0.2（参见步骤1.3），和去离子H 2ö微米的过滤器到一个合适的无菌容器中。 </li><li>保持无菌技术，轻轻三次用无菌PBS（见步骤1.2）洗盖玻片上的"矩阵制片人"的细胞。 </li><li>通过抽吸用无菌移液管除去PBS中并在合适的液体废物容器处理它。添加20毫摩尔氢氧无菌铵（3毫升/ 100mm皿）中，用在间隔摇动孵育他们5分钟。 </li><li>添加丰富的金额无菌去离子H型2 O的"矩阵生产者"菜，每100mm培养皿至少20毫升，用摇摆，并倒掉细胞裂解物到合适的废物容器中。 </li><li>重复步骤3.5四次，以确保完全除去氢氧化铵溶解的材料制成。 注:盖玻片存放于"矩阵生产者"细胞ECM然后提供与任何感兴趣的测试细胞功能检测无菌ECM。 </li><li>孵育从细胞库存培养皿试验细胞用1.5毫升的每100mm培养皿胰蛋白酶-EDTA溶液（0.05％w / v的胰蛋白酶和0.02％w / v的EDTA中Hank氏平衡盐溶液），直到测试细胞从培养皿分离的。添加细胞培养基，在400 XG 5分钟离心试验细胞，除去上层液体。 </li><li>重悬测试细胞在新鲜，无菌的培养基并用血细胞计数器计数。板，这些测试单元的适当数量到上coversli无菌的，孤立的ECMPS。培养他们在适当的细胞培养基中进行2-3小时，或用于为实验设计所需的时间周期。 </li><li>为了检查细胞骨架的组织，固定在测试细胞中的2％的PFA和异硫氰酸荧光素染色它们（FITC）-phalloidin（激发= 490 nm，发射= 525纳米）来可视化F-肌动蛋白并用4&#39;，6 -diamidino基-2-苯基吲哚（DAPI;激发= 358 nm，发射= 461纳米）以可视化的核11。 注:比较在铺于异源细胞来源的ECM与镀上自己的ECM测试细胞的试验细胞的形态和肌动蛋白的组织。 </li></ol> 4.分离的ECM的用于SDS-PAGE，免疫印迹分析，或蛋白质组学<ol><li>成长100 MM细胞培养皿（5至10道菜蛋白质组学或1-2菜肴免疫）利息贴壁细胞24-96小时，为适合的细胞类型，允许分泌和ECM沉积。 </li><li>除去细胞作为DESCRIBED步骤1.1-1.5。 </li><li>倾斜每块板以一定的角度2 O排出残留轰到培养皿底部，并小心地取出任何剩余的H 2 O运用P200的吸管，直到板完全干燥。 </li><li>平行地，含热的SDS-PAGE样品缓冲液的100mM二硫苏糖醇（DTT）至95℃使用热块2分钟，然后将其添加到板。每100mm板样品缓冲液200微升适合于通过免疫印迹进行检测的样本。 <ol><li>分析通过质谱法的ECM，由从盘收集的SDS-PAGE样品缓冲液（如在步骤4.5和4.6，如下所述）实现更浓缩的样品，将其重新加热至95℃，并在使用相同的等分试样额外的2-3片。 </li></ol></li><li>使用细胞刮彻底刮除ECM关闭的菜，确保菜的所有区域都被擦。 </li><li>用细胞刮刀，收集样品到培养皿的一侧，并用200移除μL枪头，成筒。 </li><li>传送从细胞刮刀尖端的任何残余的SDS-PAGE样品缓冲液提取到管以减少的ECM材料的损失。对于浓缩的样品，再热同的SDS-PAGE样品缓冲液等分试样到95℃，并在整个额外2-3板重新使用它。 </li><li>解决通过SDS-PAGE 12，即使用7％或4-7％的梯度聚丙烯酰胺凝胶中的ECM蛋白。 注意:为方便起见，使用预先染色的蛋白标记。 </li><li>为了增加高分子量ECM蛋白的分辨率，延长凝胶运行时间，使得37 kDa的分子量标记运行接近凝胶和分子量标记物&lt;37 kDa的底部已跑出凝胶。 </li><li>用于ECM蛋白的蛋白质组分析，染色用蛋白质染色的凝胶和捕获图像。切感兴趣频带从凝胶中，用胰蛋白酶消化它们，并通过基质辅助激光解吸分析它们/电离（MALDI）-mass光谱&lt;SUP&gt; 13。 </li><li>可替代地，用于对ECM提取物的更全面的分析，并分析整个样本，切片凝胶泳道段，消化它们与胰蛋白酶，以及执行液相色谱-质谱（LC-MS）14。 </li><li>可替代地，对于免疫印迹15，在15 V转移从SDS-PAGE凝胶的蛋白质到聚偏二氟乙烯（PVDF）膜至少1小时10分钟（半干法），以确保高分子量ECM蛋白的转移。可视转印到膜上用丽春红硫染色的总蛋白。 </li><li>以下步骤4.11中，使用抗体来建立的存在和/或使个别ECM蛋白15的半定量分析。 <ol><li>该膜用2％在TBS-吐温20（封闭缓冲液）（重量/体积）奶粉在4℃下过夜阻塞。稀释至ECM蛋白的特异性抗体在封闭缓冲液中并在室温下与膜孵育1.5小时（抗体对fibronectin稀释1/600和抗体血小板-1稀释1/150; 补充表1）。 </li><li>通过重新探测相同的印迹与抗体丰富细胞内蛋白质，如肌动蛋白或微管蛋白（抗肌动蛋白稀释1 / 10,000和抗微管蛋白稀释1/5000）测试的ECM隔离的质量。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

该议定书中的关键步骤该方法必须遵循以确保ECM的成功分离和分析一些关键的步骤。例如，氢氧化铵是用来除去细胞，并用于分析分离ECM。虽然氢氧化铵是在黑暗的水溶液稳定，可以在室温下保存，瓶必须严格重新密封在每次使用之间。因为气体可能从溶液中挥发，从而降低浓度，我们强烈建议在开始一个新的瓶每6-8周。此外，该工作液应新鲜作出每个实验。它同样重要的是将细胞完全在去离子水荧光棒洗涤除去。如果没有充分执行这些步骤，蜂窝材料可保留在盘中，污染下游分析。如果细胞已生长了10天或更长的时间，则可能需要额外的水洗涤。它是为n重要OTE所分离的ECM层通常相比于电池11非常薄，所以在成像期间标识ECM时需要小心。为隔离的ECM成的SDS-PAGE样品缓冲液中有效提取，至关重要的是，在样品缓冲液中含有还原剂。对于最有效地除去ECM的，必须使用沸腾热样品缓冲液。对于实验，其中的ECM样本将通过SDS-PAGE电泳进行蛋白质染色或蛋白质组学解析，这是至关重要通过刮擦几个板价值的ECM的到样品缓冲液的相同等分部分以获得浓缩的样品。 修改和故障排除 ECM蛋白的生成和分泌能细胞类型之间显著变化，所以对于外基质分泌及沉积时间周期应当确定从头每种细胞类型。同样重要的是要知道，ECM组合物将在关系式T动态地改变培养18 O细胞密度和时间。这个因素应该设计实验时，应考虑到。显然，这种方法的细胞类型的选择是重要的，对于通过质谱法，这可能需要总的ECM蛋白的显着量的分析中提取ECM时尤为如此。 该技术的局限性分泌ECM蛋白的水平非常低的细胞将是使用此方法使用更具挑战性。非贴壁细胞，三维细胞培养物，或细胞侵袭测定法是不适合在通过该方法本为ECM的隔离。该方法是最适合用于标准的"二维"的细胞培养物的使用。因为氢氧化铵萃取去除完全，ECM与细胞的上侧相关联，并且分泌的细胞，但非交联的，ECM蛋白也被去除，从下面和周围的基地上留下盘组装的ECM的薄层细胞11,17的</SUP&gt;。它是复杂的量化多少ECM由氢氧化铵萃取释放，因为所释放的材料还包括细胞外基质蛋白，要么之前分泌或从细胞表面摄取后是细胞内。 关于到现有/替代方法的技术意义进行了广泛的孤立的ECM实验的能力是在细胞生物学和组织生理学的上下文中重要的是，它也具有用于组织再生和组织工程领域的影响。该方法比在纯化的胶原蛋白或其他纯ECM蛋白细胞组装复杂的ECM结构在家乡的大小，与本地交联机制，并与目前在适当起源细胞类型比单独的细胞外基质蛋白形成的凝胶的优势。例如，RCS ECM的蛋白质组研究表明丰富matrilins和COMP / TSP5，如预期一个软骨的ECM 19,20（ 图4）。在一般情况下，细胞衍生ECM的分析是通过其性质阻碍作为广泛的多蛋白网络，其导致共价交联和许多ECM蛋白的不溶性，而且还由ECM的潜在污染与胞内蛋白提取物。旨在隔离从组织蛋白质组分析ECM的级分的多步骤过程中的总集合确定了21蛋白质的产生ECM蛋白的8％。然而，从ECM蛋白肽是确定的总肽的73％。这对于细胞源ECM的分离和分析的方法迅速且可靠地除去细胞物质，同时保持ECM蛋白。这种方法可用于各种细胞类型和下游应用的并且便于ECM生物学和细胞-ECM相互作用的在细胞培养物的分析。我们如何该方法可以在不同的尺度上使用的协议细节，以满足广泛的EXP关于ECM组织，动力学，或组合物erimental的问题，并以分离的ECM对细胞的作用效果。 掌握这一技术后，未来的应用方向或展望未来，该方法可以适用于三维细胞培养使用;这将扩大其生理意义。脱细胞的天然组织有很大的潜力，作为丢失或受损组织的再生，作为替代移植，如心脏衰竭22后的支架。它克服了供体的可用性和免疫排斥的问题的可能性。本报告中所描述的方法的未来的应用可以调查其与三维细胞培养物或组织脱细胞，这将进一步增加了该方法的范围使用。

在过去的几十年中，细胞外基质（ECM）已成为公认的是参与多种细胞生理和病理过程多样化，动态和复杂的环境。在组织水平，则ECM影响细胞信号传导，运动，分化，血管生成，干细胞生物学，肿瘤，纤维化等 1,2-。 ECM组织和ECM依赖过程的研究因而对细胞生物学和生理学组织广泛的影响。到达细胞外基质组合物，组织和功能性质的一种机械的理解，需要用于ECM的准确分离方法。而从组织3在隔离依靠细胞外基质蛋白的鉴定最早，ECM从培养细胞的准备现已成为更为普遍。 细胞来源的ECM的分离和分析是复杂主要有两个原因。首先，细胞的存在和IR丰富细胞内蛋白质可能使得难以给ECM分离为离散的细胞外结构。事实上，一些ECM蛋白具有在细胞内以及在ECM 4的作用;因此，从细胞来源的ECM的有效去除胞内蛋白质是至关重要的，如果蛋白质的ECM内的研究是不与在细胞内的角色混淆。其次，细胞来源的ECM是由许多大，寡聚蛋白质，这往往是共价交联后的ECM组件，因此在标准的洗涤剂不溶性的。这些属性可以变得复杂提取和ECM的进一步分析。为了解决这些问题，需要一种方法，可实现从细胞成分ECM蛋白的有效的分离。 几种方法已在文献中的ECM无论从细胞培养物或组织提取物的分离进行了描述。许多这些方法都瞄准了丰富的ECM PR的提取otein，胶原蛋白，从组织和包括使用中性盐3，在酸性条件5,6，或胃蛋白酶7。从组织提取总细胞外基质的隔离通常涉及的组织的脱细胞的ECM的隔离之前。例如，一个连续提取方法已经描述了分离自人心脏组织8的ECM。首先，松散结合的ECM蛋白用0.5M NaCl的前十二烷基硫酸钠（SDS）萃取用来除去细胞。最后，使用4M的胍8提取剩余的ECM蛋白质。 ECM还可以从使用8M脲9脱细胞的样品溶解。其它技术用洗涤剂，如脱氧胆酸10，通过离心从可溶细胞裂解物中分离不溶性ECM蛋白前提取两个小区和ECM。 分离和本报告中所述的细胞来源的ECM的分析的方法提供了摄制除去细胞材料，留下可以通过原位免疫荧光进行分析或用于进一步的生化分析中提取细胞源ECM诱导型方法。这种方法可以适用于任何贴壁细胞类型，并且可以按比例增加下游过程，例如免疫印迹或质谱法，或用于功能研究的分离的细胞外基质的利用率。该方法也可以在用活细胞的共聚焦显微镜一起使用，以跟踪的实时感兴趣的标签的蛋白的ECM的沉积。这是通过使用一网格，玻璃底皿而实现的。总体而言，该方法提供了细胞来源的ECM和也以确定并监控该沉积和单个ECM蛋白的动力学范围的精确隔离。

<table><tbody><tr><td>Antibody to COL1A1</td><td>Novus</td><td>NB600-408</td><td>Rabbit polyclonal. Reacts with other fibrillar collagens as well as collagen I. IF: 1/200</td></tr><tr><td>Antibody to fibronectin</td><td>Sigma</td><td>F3648</td><td>Rabbit polyclonal. WB: 1/600 for 1.5 h. IF: 1/200</td></tr><tr><td>Antibody to thrombospondin 1</td><td>ThermoFisher&amp;nbsp;</td><td>MA5-13398</td><td>Mouse monoclonal clone A6.1. WB: 1/150 for 1.5 h</td></tr><tr><td>Antibody to RFP</td><td>Abcam</td><td>ab62341</td><td>Rabbit polyclonal. WB: 1/2,000 for 2 h</td></tr><tr><td>Antibody to &amp;beta;-actin</td><td>Sigma</td><td>A1978</td><td>Mouse monoclonal clone AC-15. WB: 1/10,000 for 1.5 h</td></tr><tr><td>Antibody to &amp;alpha;-tubulin</td><td>Sigma</td><td>T9026</td><td>Mouse monoclonal clone DM1A. WB: 1/5,000 for 1.5 h</td></tr><tr><td>Cell Tracker Green</td><td>ThermoFisher&amp;nbsp;</td><td>C2925</td><td>Fluorescent cell marker. Use at 1 &amp;micro;M for 30 min</td></tr><tr><td>Fluorescein isothiocyanate (FITC)-Phalloidin</td><td>Sigma</td><td>P-5282</td><td>Stock = 50 mg/mL in DMSO. 1/50 for 45 min</td></tr><tr><td>FITC-conjugated goat anti-mouse IgG</td><td>Sigma</td><td>F8771</td><td>IF: 1/50 for 1 h</td></tr><tr><td>FITC-conjugated sheep anti-rabbit IgG</td><td>SIgma</td><td>F7512</td><td>IF: 1/50 for 1 h</td></tr><tr><td>Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG</td><td>LI-COR</td><td>926-80010</td><td>WB: 1/50,000 for 1 h</td></tr><tr><td>HRP-conjugated goat anti-rabbit IgG</td><td>LI-COR</td><td>926-80011</td><td>WB: 1/100,000 for 1 h</td></tr><tr><td>Vectorshield mounting medium with DAPI</td><td>Vector</td><td>H-1200</td><td>Store at 4 &amp;deg;C in the dark</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium</td><td>Sigma</td><td>D6429</td><td>Warm before use</td></tr><tr><td>Fibroblast growth medium</td><td>PromoCell</td><td>C-23010</td><td>Warm before use, supplement with 50 &amp;micro;g/mL ascorbic acid</td></tr><tr><td>Phenol red-free DMEM</td><td>ThermoFisher</td><td>21063-029</td><td>Warm before use</td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma</td><td>T3924</td><td>Warm before use</td></tr><tr><td>Gridded glass-bottomed dish</td><td>MatTek</td><td>P35G-2-14-C-GRID</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;OH, 28-30% solution</td><td>Sigma</td><td>221228</td><td>Dilute to 20 mM in de-ionised water. Once opened store in the dark, tightly capped.</td></tr><tr><td>Paraformaldehyde, 16% solution</td><td>Alfa Aesar</td><td>43368</td><td>Dilute to 2% (v/v) in PBS</td></tr><tr><td>Deoxycholic acid</td><td>Sigma</td><td>D2510</td><td>Use at 2% (v/v) final concentration</td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td>Dilute to 0.5% (v/v) final concentration</td></tr><tr><td>GelCode Blue stain reagent</td><td>ThermoFisher&amp;nbsp;</td><td>24590</td><td>Protein stain for SDS-PAGE gels</td></tr><tr><td>Precision Plus protein standards</td><td>Biorad</td><td>161-0374</td><td>Protein standards for SDS-PAGE gels</td></tr><tr><td>Trans-Blot SD Semi Dry transfer cell</td><td>Biorad</td><td>1703940</td><td>Efficient transfer of high molecular weight proteins</td></tr><tr><td>PVDF transfer membrane</td><td>Millipore</td><td>ISEQ00010</td><td>Immobilon-PSQ&amp;nbsp;</td></tr><tr><td>Ponceau S stain</td><td>Sigma</td><td>P7170</td><td>Reversible protein stain for PVDF membrane</td></tr><tr><td>WesternSure Enhanced chemiluminescence (ECL) substrate</td><td>LI-COR</td><td>926-80200</td><td></td></tr><tr><td>High performance chemiluminescence film</td><td>GE Healthcare</td><td>28906837</td><td></td></tr><tr><td>Sterile filtration unit, MILLEX-GV</td><td>Millipore</td><td>ML481051</td><td></td></tr><tr><td>SP8 AOBS confocal laser scanning microscope</td><td>Leica</td><td></td><td>Environmental chamber needed for temperature and CO&lt;sub&gt;2&lt;/sub&gt; control (Life Imaging Services)</td></tr><tr><td colspan="4">Key: IF, Immunofluorescence; WB, Western Blot</td></tr></tbody></table>

a rapid, scalable method for the isolation, functional study, and analysis of cell-derived extracellular matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

中所描述的协议步骤1.1-1.5行为以去除细胞和离开细胞源ECM的后面。该协议是在COS-7细胞生长在玻璃盖玻片96小时进行，所述细胞源ECM通过荧光显微镜进行分析。的氢氧化铵处理除去该细胞有效，由细胞核和肌动蛋白细胞骨架的损失所确立，根据DAPI和鬼笔环肽染色，分别为（ 图1）。细胞用2M尿素提取不是氢氧化铵作为有效;治疗后，检测DAPI和鬼笔环肽染色的痕迹。 8M尿素不得不氢氧化铵类似的效果（ 图1）。接着，细胞来源的ECM被前后的氢氧化铵处理可视化（步骤2.1-2.11）。 COS-7细胞用单体红色荧光蛋白（MRFP）转染-tagged血小板反应蛋白1 C-末端三聚体，（mRFPovTSP1C）11，生长在35毫米的菜用网格玻璃基板。 两个小时后电镀，合适的方格包含表达mRFPovTSP1C多个健康细胞的共聚焦显微镜下观察。参考相位对比图像拍摄该区域的，然后荧光延时成像进行每隔1分钟2小时（ 图2A）。然后将细胞用氢氧化铵除去，并在盘的同一网格正方形混合物在相衬搬迁，然后通过荧光显微镜重新分析。将细胞不再存在于网格正方形，但mRFPovTSP1C保持在ECM内，通过荧光显微镜作为特征泪点（ 图2A）进行检测。沉积之前细胞去除对ECM任何mRFPovTSP1C鉴定通过比较荧光图案前和去除后的细胞。在两幅图像中确定的荧光泪点（Figu再如图2A所示，例如箭头）被推定为与ECM相关联。 然后与氢氧化铵处理被用于天然ECM从培养的贴壁细胞中分离。人真皮成纤维细胞（HDF）中生长在玻璃盖玻片96小时。细胞用氢氧化铵除去，则ECM被用抗胶原蛋白I抗体，其表现出的特性纤维状和小梁染色图案16（ 图2B）进行探测。纤连蛋白图案形成围绕固定的，非透化的大鼠软骨肉瘤细胞（RCS）和ECM内检测了除去RCS细胞（ 图2C）的后。 ECM的氢氧化铵隔离还无菌条件下进行，以便"测试"电池可以重新铺板于ECM的表型或功能性研究。例如，"测试"的COS-7细胞2小时镀上由RCS的细胞产生的无菌的ECM，和叔母鸡他们被固定，透，以及用于通过FITC标记的鬼笔环肽染色F-肌动蛋白组织分析，相比于COS-7细胞产生自己的ECM（步骤3.1-3.9）（ 图3）。 在更大的规模，使用该方法，以用于生化程序隔离的ECM。例如，RCS细胞生长于两个100 MM细胞培养皿为7天，并在步骤4.1-4.7所述的方法来进行。在细胞来源的ECM蛋白被刮他们进入热SDS-PAGE样品缓冲液收集并在还原条件下通过SDS-PAGE上分离7％聚丙烯酰胺凝胶。将凝胶染色蛋白质，和四个主要条带分别分离并通过质谱法（ 图4A）进行分析。一些ECM蛋白，包括纤维连接蛋白，血小板反应蛋白1（TSP1），血小板反应蛋白5 /软骨寡聚基质蛋白（TSP5 / COMP），以及胞外基质蛋白1被鉴定（ 图4B）。另外，ECM的小规模的制剂可以通过免疫印迹进行评估。例如，从异位表达mRFPovTSP1C通过SDS-PAGE分离，转移至PVDF膜，并探查RFP与抗RFP抗体（ 图4C）COS-7细胞的细胞源ECM。这种技术是足够敏感以检测TSP1（mRFPovTSP1C）的天然三聚体和工程化的五聚体的TSP1 C-末端区域（MRFP-TSP-5-1C）17（ 图4C）之间在沉积的差异。调查HDF的内源ECM的，在细胞裂解物中或分离的细胞外基质的特定蛋白质的存在通过免疫印迹检查。在ECM中检测到的特征的ECM蛋白纤连蛋白和血小板反应蛋白1，而丰富的胞内蛋白质α微管蛋白和β肌动蛋白分别从分离ECM（ 图4D）不存在。 图1:细胞去除方法的比较。将COS-7细胞以高密度生长在盖玻片上96小时，固定在2％的PFA，而在0.5％的Triton X100透化，或作为细胞去除指示进行处理。盖玻片然后用FITC-鬼笔环肽染色来可视化F-肌动蛋白和DAPI形象化细胞核。比例尺= 25微米。这个数字是从日报的原始出版物细胞科学11修改。 <a href="http://ecsource.jove.com/files/ftp_upload/55051/55051fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55051/55051fig2.jpg" /> 图 2: 隔离ECM ECM蛋白的鉴定。 （A）的相衬和荧光图像COS-7细胞表达mRFPovTSP1C，生长在35毫米菜2小时玻璃底，网格基地。图像之前和氢氧化铵除去细胞之后被示出。网格信的边缘用虚线相位对比图像中指示。白色箭头指示在场前后细胞去除后，荧光泪点的例子。我在间接免疫荧光的HDF的ECM胶原的（B）的检测。 HDF中生长96小时，并用氢氧化铵除去，则ECM被染色的人类纤维状胶原一，ECM也被单独染色用FITC缀合的抗兔次级抗体。 （ 三 ）各地RCS细胞或分离的RCS ECM内的纤维连接蛋白的定位，用间接免疫荧光所检测。 RCS细胞生长48小时，固定在2％的PFA和染色纤连蛋白和用DAPI。在平行的菜肴，将细胞用20mM氢氧化铵除去和ECM被染色FIBRonectin和DAPI。细胞也沾上单独的FITC缀合的抗小鼠IgG抗体。 <a href="http://ecsource.jove.com/files/ftp_upload/55051/55051fig2large.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/55051/55051fig3.jpg" /> 图 3: 功能研究使用无菌ECM的。 RCS"矩阵生产者"细胞生长在玻璃盖玻片48小时，然后用20mM氢氧化铵在无菌条件下处理以除去细胞。铺板的COS-7"测试"细胞在盖玻片上具有或不具有隔离的RCS的ECM 2小时，固定在2％的PFA，透在0.5％的Triton X100和用FITC-鬼笔环肽染色来可视化F-肌动蛋白和DAPI来可视化的细胞核。 COS-7细胞接种在RCS ECM传播更加广泛，形成较大的微丝束（例如箭头）和边缘RUFFLES。 <a href="http://ecsource.jove.com/files/ftp_upload/55051/55051fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55051/55051fig4.jpg" /> 图4: 通过SDS-PAGE，质谱，或免疫印迹从隔离的ECM蛋白的鉴定。 （A）中的RCS细胞中生长7天，用20mM氢氧化铵除去将ECM被刮成含有100mM DTT的热SDS-PAGE样品缓冲液中。 ECM的制剂在还原条件下分离的SDS-PAGE上的7％聚丙烯酰胺凝胶。四显著带（箭头1-4）是由蛋白质染色鉴定。从RCS ECM频带1-4（B）的蛋白质通过MALDI质谱分析鉴定。 （C）的COS-7细胞表达任一mRFPovTSP1C（三聚体）或MRFP-TSP-5-1C（五聚体）中培养48小时，用20mM氢氧化铵除去，并且是ECM中分离成含有100mM DTT的热SDS-PAGE样品缓冲液中。 ECM的制剂还原条件下，转移至PVDF膜下分离通过SDS-PAGE上的7％聚丙烯酰胺凝胶，并用抗RFP抗体探测。该面板是从生物科学报告17的原始发布修改。 （D）的HDF中生长96小时，然后要么用在PBS中的2％脱氧胆酸（细胞裂解物）或用20mM氢氧化铵处理以除去细胞。将ECM被刮入热SDS-PAGE样品缓冲液中。两个级分通过SDS-PAGE在10％聚丙烯酰胺凝胶在还原条件，转移至PVDF膜下分离，并用抗体探测，所指示的。在每个面板的分子量标记的位置以kDa表示。 <a href="http://ecsource.jove.com/files/ftp_upload/55051/55051fig4large.jpg" target="_blank">请点击此处查看该网络的放大版本</a>古尔。

Watch this Scientific Journal Video about A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix at JoVE.com

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

一种快速，为隔离，功能可扩展的研究方法，以及细胞衍生的细胞外基质的分析

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

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Research

JoVE Journal

Biology

16.4K 次观看.  University of Bristol. The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies. 

视频: A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell&#8217;s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

同心凝胶系统来研究基质微环境的三维细胞迁移的生物物理作用

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

科研

生物工程

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

提高2D和3D皮肤<em&gt;体外</em&gt;模型使用大分子拥挤

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

细胞分化和牵引力的相关分析高通量细胞芯片平台

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

细胞外基质衍生的泡沫和微载体如组织特异性细胞培养和交付平台的制造

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides.
IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3.72 &plusmn; 0.74 x 10-6 M as determined by binding experiments using biolayer interferometry experiments. Endothelial cells (EC) proliferation and apoptosis were unchanged using increasing concentrations of IRF5D (0 to 100 &#181;g/mL, 24 h). Tsk/+ mice were treated with IRF5D (1 mg/kg/d subcutaneously, 21 d). IRF5 and ICAM expressions were decreased after IRF5D treatment. Endothelial function was improved as assessed by vasodilation of facialis arteries from Tsk/+ mice treated with IRF5D compared to Tsk/+ mice without IRF5D treatment. As a transcription factor, IRF5 traffics from the cytosol to the nucleus. Translocation was assessed by immunohistochemistry on cardiac myocytes cultured on the different cardiac extracellular matrices. IRF5D treatment of the Tsk/+ mouse resulted in a reduced number of IRF5 positive nuclei in comparison to the animals without IRF5D treatment (50 &#181;g/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.

We describe the isolation of cardiac extracellular matrix from C57Bl/6J control mice, tight-skin mice, and tight-skin mice treated with the IRF5 inhibitory peptide. We also describe the vasodilation studies on the isolated vessels from C57Bl/6J, tight-skin mice and tight-skin mice treated with the IRF5 inhibitory peptide.

隔离容器的血管扩张和细胞外基质的紧皮肤小鼠隔离

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ...

免疫与感染

Production of Extracellular Matrix Fibers via Sacrificial Hollow Fiber Membrane Cell Culture

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure and healing. Extraction of extracellular matrix from fibrogenic cell cultures in vitro has potential for generation of ECM from human- and potentially patient-specific cell lines, minimizing the presence of xenogeneic epitopes which has hindered the clinical success of some existing ECM products. A significant challenge in in vitro production of ECM suitable for implantation is that ECM production by cell culture is typically of relatively low yield. In this work, protocols are described for the production of ECM by cells cultured within sacrificial hollow fiber membrane scaffolds. Hollow fiber membranes are cultured with fibroblast cell lines in a conventional cell medium and dissolved after cell culture to yield continuous threads of ECM. The resulting ECM fibers produced by this method can be decellularized and lyophilized, rendering it suitable for storage and implantation.

The goal of this protocol is production of whole extracellular matrix fibers targeted for wound repair which are suitable for preclinical evaluation as part of a regenerative scaffold implant. These fibers are produced by the culture of fibroblasts on hollow fiber membranes and extracted by dissolution of the membranes.

牺牲纤维膜细胞培养生产细胞外基质纤维

Engineered scaffolds derived from extracellular matrix (ECM) have driven significant interest in medicine for their potential in expediting wound closure ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

从三维混合细胞球体共培养的乳腺上皮细胞的隔离

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

生物学

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

准备可调细胞外基质微环境，以评估施万细胞表型规范

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

从富集组织及消化细胞外基质蛋白的成肽质谱分析

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Invasion Assay Using 3D Matrices

The extracellular matrix (ECM) is a network of molecules that provide a structural framework for cells and tissues and helps facilitate intercellular communication. Three-dimensional cell culture techniques have been developed to more accurately model this extracellular environment for in vitro study. While many cell processes during migration through 3D matrices are similar to those required for movement across rigid 2D surfaces, including adherence, migration through ECM also requires cells to modulate and invade this polymeric-mesh of ECM. In this video, we will present the structure and function of ECM and the basic mechanisms of how cells migrate through it. Then, we will examine the protocol of an assay for tube formation by endothelial cells, whose steps can be generalized to other experiments based on 3D matrices. We will finish by exploring several other biological questions that can be addressed using ECM invasion assays.

Cell Invasion Assay Using 3D Matrices

使用 3D 矩阵的入侵检测

The extracellular matrix (ECM) is a network of molecules that provide a structural framework for cells and tissues and helps facilitate intercellular ...