JDM得到了INSERM /RégionAquitaine博士研究生的支持，现在得到博士后ARC研究金和西奈山医学院Tisch癌症研究所的支持。 EH得到了Ministèrede l&#39;EnseignementSupérieuret de la Recherche博士的支持。 ZE得到了国立国民大学（ANR）的博士后研究金的支持。 CM由西奈山医学院Tisch癌症研究所支持，JJBC得到NIH / NCI授权K22CA196750，TCI青年科学家癌症研究奖JJR基金和西奈山医学院Tisch癌症研究所的支持。这项工作得到ANR-13-JJC-JSV1-0005的资助。 FS由"Ligue nationale contre le cancer"支持。 VM和FS由"EquipeLabelliséeLigue Nationale contre le Cancer 2016"和Institut National du Cancer，INCA_8036和PLBio2012的资助提供支持。

注意:在固定之前，所有步骤都在无菌层流罩中完成。 二维矩阵注意:2D矩阵用于确定形成invadosomes的细胞的百分比和每个细胞的基质降解面积。 <img alt="图1" src="/files/ftp_upload/54911/54911fig1.jpg" /> 图1:协议。用于制备明胶和胶原蛋白I基质的方案总结。只能制成明胶基质或明胶和胶原蛋白I混合基质。 <a href="http://ecsource.jove.com/files/ftp_upload/54911/54911fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol><li> 明胶基质 注意:荧光明胶基质用于定量基质降解，非荧光明胶用于促进胶原I纤维的粘附。荧光明胶垫嵴也用于荚膜细胞和侵袭过程研究（ 见图1 ）。 <ol><li>在无菌层流罩下，将一块石蜡膜（20厘米x 15厘米）放在工作区域，并用70％乙醇消毒。 </li><li>将一批1毫克/毫升明胶的液滴分成20毫升，间隔约1厘米;参见图1的液滴组织。 </li><li>用高压灭菌的玻璃盖玻片（直径12毫米）覆盖每个20微米的明胶液滴。 </li><li>在室温下孵育20分钟。如果明胶是荧光的，保护它免受光照。 </li><li>将等分的40μL0.5％戊二醛液滴在1x磷酸盐缓冲盐水（PBS）中，每行间隔约1厘米，低于步骤1.1.3的盖玻片线。 注意:戊二醛是一种有毒的固定剂。使用手套，不要吸入。 </li><li>使用宝石镊子，将每个明胶包被的盖玻片转移到戊二醛液滴。 注意:正常情况下，小的明胶滴留在石蜡膜上。不要将其重用于另一个实验。 </li><li>在室温下孵育40分钟。对于荧光盖玻片，保护它们免受光线影响。 </li><li>取24孔细胞培养板，每孔加入500μL无菌1x PBS。将盖玻片，明胶一面朝上放入各孔中。 </li><li>在1x PBS中洗涤两次，每次5分钟。 注意:在此步骤中，盖玻片可用于实验或在4℃下储存在1x PBS中。非荧光盖玻片可以储存一周，荧光盖玻片可以储存48小时。 </li></ol></li><li> 纤维胶原I型基质 注意:I型原纤维胶原用于研究线性侵袭体形成和相关基质降解。使用荧光胶原I和非荧光明胶盖玻片的组合将invadosome标记物与胶原纤维共定位（ 例如， F-肌动蛋白i在红色通道，红色通道中的胶原蛋白I，绿色通道中的Tks5，以及用于染色的Hoechst染料）。另一方面，使用非荧光胶原I与非荧光明胶盖玻片的组合来最大化染色的侵染体标记物的数目（ 例如，远红色通道中的F-肌动蛋白，红色通道中的cortactin，Tks5在绿色通道和Hoechst染料用于染色）。最后，使用非荧光胶原I与荧光明胶盖玻片的组合来定量明胶相关的降解（ 例如，远红色通道中的F-肌动蛋白，红色通道中的Tks5，绿色通道中的Hoechst染料为核染色）（见图1）。 Hoechst染料用于染色细胞核，并且还检查支原体污染，以及定期在所用细胞系上进行的PCR检测。 <ol><li>荧光胶原蛋白I <ol><li>使用从0.02N乙酸丙酮中的大鼠尾肌提取的商业胶原蛋白I天。取出胶原蛋白I的量，以每个盖玻片制备500μL溶液，终浓度为0.4mg / mL胶原I. </li><li>加入最终浓度为10μg/ mL的5-羧基x罗丹明琥珀酰亚胺酯，计算出胶原蛋白I溶液（nx 500μL）的最终体积。 </li><li>通过上下移液混合溶液，并在室温下孵育5分钟，防止光照。 </li><li>用含有钙和镁的1X Dulbecco的磷酸盐缓冲盐水（DPBS）稀释至最终体积，并直接使用。 </li></ol></li><li>纤维胶原I盖玻片<ol><li>使用先前在步骤1.1中制备的非荧光或荧光明胶盖玻片。 </li><li>在含有钙和镁的1X DPBS中制备终浓度为0.4mg / mL的荧光或非荧光胶原I的溶液。 </li><li>加入500μL荧光或非荧光胶原蛋白I溶液盖玻片在24孔细胞培养板中。 </li><li>在37℃下孵育4小时，进行胶原蛋白I聚合。 </li><li>孵育后，通过倾斜板和移液孔（不直接盖在盖玻片上）来移除过量的胶原蛋白I，以避免损坏基质。 注意:此矩阵无法存储。去除多余的胶原蛋白I后立即种细胞。 </li></ol></li></ol></li></ol>细胞播种，培养时间和固定<ol><li>根据细胞类型，将细胞制备在合适的培养基中，并加入细胞，每孔最终体积为500μL。 </li><li>为了表征细胞系形成线性侵袭体并降解基质的能力，将细胞在胶原I基质上铺板4小时，12小时和24小时，以确定线性侵袭体形成的动力学和相关的降解活性。 注意: 表1包含细胞系的实例在实验室测试。 <table border="1"><tbody><tr><td>细胞系</td><td>每盖玻片的细胞数</td><td>与矩阵接触线性侵袭体定量的时间</td><td>与矩阵接触的时间用于定量线性侵袭体降解活性</td></tr><tr><td> MDA MB 231 </td><td> 40000 </td><td> 4小时</td><td> 12小时</td></tr><tr><td> HUH7 </td><td> 40000 </td><td> 12小时</td><td> 24小时</td></tr><tr><td> HEP 3B </td><td> 40000 </td><td> 12小时</td><td> 24小时</td></tr><tr><td> SNU 398 </td><td> 40000 </td><td> 12小时</td><td> 24小时</td></tr><tr><td> NIH 3T3 src </td><td> 30000 </td><td> 4小时</td><td> 12小时</td></tr><tr><td> A431 </td><td> 50000 </td><td> 6小时</td><td> 24小时</td></tr><tr><td> A549 </td><td> 40000 </td><td> 12小时</td><td> 24小时＃160; </td></tr><tr><td>生的</td><td> 40000 </td><td> 12小时</td><td> 12小时</td></tr><tr><td> PAE </td><td> 40000 </td><td> 12小时</td><td> 12小时</td></tr></tbody></table> 表1:细胞接种和孵育时间参考。该表列出了用于研究线性侵袭体的细胞系列表。它表示在基质上种子的细胞数，观察线性侵袭体必需的时间以及观察相关基质降解所需的时间。线性invadosomes的寿命短。为了能够在最大数量的细胞中观察线性侵袭体，细胞必须在短时间内与胶原I基质接触。另一方面，为了能够使所报告的明胶降解可视化，细胞必须与胶原蛋白I基质接触时间比上述那些长。 </li><li>除去细胞培养基后，在500μL的4％多聚甲醛在1X PBS中将盖玻片固定10分钟。 </li><li>用1x PBS洗两次。 </li><li>储存于4°C，避光保存。 </li></ol> 3.免疫荧光<ol><li>用新鲜制备的溶液将细胞渗透10分钟 的0.2％Triton X-100在1×PBS中。不要重复使用此解决方案。 </li><li>用1x PBS洗两次盖玻片。 </li><li>将一块石板玻璃（20厘米x 15厘米）放在长凳上。 </li><li>在1×PBS中的4％牛血清白蛋白（BSA）中稀释一抗，如材料表中所述。 </li><li>在一条线上分装50μL一抗一抗溶液液滴，每个间隔约1厘米。 </li><li>将每个盖玻片放在一滴上。 </li><li>在室温下孵育40分钟，防止光照。 </li><li>孵育期后，用1x PBS洗涤两次。 </li><li>在1％的4％BSA中稀释二抗，鬼笔环肽和Hoechst染料PBS。 </li><li>如前所述，将50μL混合物的液滴分别分隔在第一行盖玻片下方约1厘米处。 </li><li>将每个盖玻片放在一滴上。 </li><li>在室温下孵育30分钟，防止光照。 </li><li>孵育后，用1x PBS洗涤两次。 </li><li>用蒸馏水洗涤一次以除去盐。 </li><li>将玻璃盖安装在玻璃载玻片上，聚合安装介质。 </li><li>在显微镜下，盖玻片在室温下干燥过夜，防止光照。 </li></ol>量化<img alt="图2" src="/files/ftp_upload/54911/54911fig2.jpg" /> 图2:线性侵袭体定量。该宏需要F-actin和Tks5染色的共焦图像（格式:1,024×1,024）。 （ A ）首先打开F肌动蛋白图片并制作面膜。 （ B ）然后，打开Tks5图片和阈值。应用宏。 （ C ）分析结果将出现在两个表格中。每个细胞的线性侵袭体的数量和其他信息，如线性invadosomes在细胞中使用的百分比面积将在Summary表中。每个线性invadosome的大小将在"结果"表中。 <a href="http://ecsource.jove.com/files/ftp_upload/54911/54911fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol><li> 形成线性侵袭体的细胞百分比 <ol><li>计数形成线性侵袭体的细胞，每盖玻片约100个细胞（使用技术一式三份）。每n个量化至少300个细胞，n = 3。 </li><li>通过取三次独立实验的平均值计算形成线性侵袭体的细胞的百分比，得到900个定量细胞。 </li></ol></li><li> 线性侵袭体大小和细胞数 <li>使用带有补充宏的ImageJ。 </li></ol></li><li> 每个细胞降解 <ol><li>每个条件使用三个盖玻片。 </li><li>拍摄30张荧光明胶和Hoechst染色核的盖玻片。 </li><li>使用ImageJ软件，如Martin KH 等人所述。 9 。 </li></ol></li></ol> 5. 3D胶原蛋白入侵检测<img alt="图3" src="/files/ftp_upload/54911/54911fig3.jpg" /> 图3:入侵测定方案。入侵试验方案总结。胶原蛋白I与琥珀酰亚胺酯染料交联并在37℃下聚合1小时。将细胞加入胶原蛋白I塞顶部的无血清培养基中。将插入物放置在介质wi中10％FBS。在实验条件下，将细胞接种1h后胶原Ⅰ栓塞固定在细胞接种后3天，测定细胞侵袭能力。 <a href="http://ecsource.jove.com/files/ftp_upload/54911/54911fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <ol><li> 胶原I插头准备 <ol><li>进行第一次实验的4小时，12小时，24小时，48小时和72小时的时间过程，以确定根据细胞类型侵入凝胶的动力学。对于每个实验，添加1小时的时间点作为参考时间点，当没有入侵发生时。 </li><li>在无菌层流罩下在冰上制备胶原蛋白I溶液。为3个Boyden室插入物准备500μL溶液。 </li><li>吸出1mg市售的大鼠尾胶原I制备终浓度为2mg / mL胶原的溶液I. </li><li>加入5-羧基 - 罗丹明琥珀酰亚胺酯最终浓度为1μg/ mL，计算出胶原I溶液的最终体积（在这种情况下为500μL）。 </li><li>混合均匀，在无菌层流罩下冰上孵育5分钟，防止光照。 </li><li>加入50μL冰冷的，过滤的10X PBS和6.25μL冰冷的1M氢氧化钠（NaOH）。 </li><li>加入无菌水按照公式443.75 - xμL胶原Ⅰ。 </li><li>每Boyden腔插入物加入100μL溶液。 </li><li>在37℃孵育1小时以允许聚合。 </li><li>将富含胎牛血清（FBS）的细胞培养基加入新孔中;将插入物插入这些孔。 </li><li>在胶原蛋白I上方的种子30,000个细胞插入无FBS的培养基中。 </li><li>在37℃下培养细胞后，将胶原I栓塞在1×PBS中的4％多聚甲醛中固定30分钟。 </li><li>完全免疫荧光，如步骤3，除了以下步骤:渗透用0.2％Triton X-100在1x PBS中30分钟，而不是10分钟;分别用一次和二次抗体溶液孵育1.5小时，而不是40分钟和30分钟。染色F-肌动蛋白或细胞核定位细胞。 </li><li>免疫荧光后，使用手术刀切割插入物的膜，小心地将胶原蛋白I塞子转移到玻璃底盘。确保胶原蛋白I插头的顶部与玻璃接触。保持插入膜附着在胶原蛋白I插头的底部，以保持插头的完整性。 注意:胶原蛋白I塞在玻璃底盘中保持完整。玻璃底部和围绕玻璃底部的塑料之间的深度等于插头的厚度。 </li><li>在胶原蛋白I上插入盖玻片并用聚合安装介质安装，以防止插头变干。 </li><li>使用倒置的共聚焦显微镜来成像胶原蛋白I塞;的顶部插头与盘的玻璃底部接触。 <ol><li>从胶原蛋白I插头的顶部到底部获取一个z-stack来显现细胞侵袭。通过检查细胞侵入成像领域的深度来确定z-stack的厚度。使用Z轴步长为0.25μm的512 x 512格式进行采集。 注:作为参考，播种3天后MDA-MB-231细胞的平均z-叠层为30μm。 </li></ol></li></ol></li></ol>

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B., Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amoeboid+shape+change+and+contact+guidance:+T-lymphocyte+crawling+through+fibrillar+collagen+is+independent+of+matrix+remodeling+by+MMPs+and+other+proteases.">Amoeboid shape change and contact guidance: T-lymphocyte crawling through fibrillar collagen is independent of matrix remodeling by MMPs and other proteases.</a> Blood. 102, 3262-3269 (2003).</li><li>Geraldo, S., 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=Do+cancer+cells+have+distinct+adhesions+in+3D+collagen+matrices+and+in+vivo?.">Do cancer cells have distinct adhesions in 3D collagen matrices and in vivo?.</a> Eur. J. Cell Biol. 91, 930-937 (2012).</li><li>Monteiro, P., 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=Endosomal+WASH+and+exocyst+complexes+control+exocytosis+of+MT1-MMP+at+invadopodia.">Endosomal WASH and exocyst complexes control exocytosis of MT1-MMP at invadopodia.</a> J. Cell Biol. 203, 1063-1079 (2013).</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> Semin. Cell Dev. Biol. 20, 931-941 (2009).</li></ol>

作者没有什么可以披露的。

经典地， 在体外研究侵袭体，而不考虑微环境和细胞被电镀的基质。目前使用几种类型的基质，包括明胶，纤连蛋白，玻连蛋白或高密度纤维胶原（HDFC） 7,11 ;然而，这些通常不代表细胞存在并且不具有生理学相关性的微环境。在这里，使用由明胶和纤维I型胶原之间的关联组成的新型基质。使用I型胶原原纤维可以使我们突出出一种新颖的侵袭体，被称为线形侵袭?...

细胞外基质（ECM）重塑发生在生理和病理过程中，如血管生成和肿瘤细胞侵袭。在生理条件下，许多细胞类型，主要来自造血系，能够降解ECM元件。例如，巨噬细胞能够跨越解剖障碍以达到组织，并且破骨细胞降解骨基质以确保钙稳态。在全球范围内，身体中的所有基质更新以维持其物理和化学性质。在癌组织中，肿瘤微环境组成被改变。例如，在乳腺癌和肺癌中，I型胶原蛋白被过度表达并在肿瘤周围积聚。此外，这种积累与发生转移的风险增加相关1,2 。癌转移依赖于癌细胞降解ECM并侵入邻近组织的能力。 该细胞的侵袭活性归因于被称为invadosome的专门的肌动蛋白丰富的结构。该术语包括分别存在于正常和癌细胞（ 例如，巨噬细胞，内皮细胞和癌细胞如MDA-MB-231乳腺癌细胞系）中的囊体和隐球菌。 体外 ，invadosomes可以组织成不同的形状:点，聚集体或花环3 。经典地，侵袭体由含有若干蛋白质的F-肌动蛋白核心组成，如骨架蛋白Tks5和皮质激素，被粘附斑块分子包围，如整联蛋白和连锁蛋白4 。最近，显示Tks5和RhoGTPase Cdc42可以用作功能性侵袭体5的最小分子标记。这些结构能够通过募集和激活特异性金属蛋白酶蛋白（如MT1-MMP和MMP-2 6）来降解ECM。在之前的研究中，我们报道了I型胶原原纤维与I型胶原原纤维的特异性受体的盘状蛋白结构域受体1（DDR1）之间的相互作用导致形成一类称为线性侵袭体的新型invadosome。沿着I型胶原纤维7形成线性侵袭体。这些结构能够通过募集和激活MT1-MMP / MMP2来降解I型胶原原纤维。此外，它们的形成取决于Tks5和Cdc42 5 。 在体外 ，我们证明线性侵袭体的存在和DDR1参与其形成和活动8使用由各种基质元素的组合组成的不同策略，包括:i）荧光明胶包被的盖玻片，ii）荧光I型胶原原纤维涂层盖玻片，和iii）3D I型胶原蛋白塞。由于使用不同的矩阵，我们能够学习和品格ize线性侵袭体形成及其降解活性7,8 。 最后，为了更好地了解入侵细胞的生物学，使用2D和3D培养系统中的ECM组分的组合，模拟肿瘤微环境组成的复杂性。以下是在2D和3D培养系统中用明胶/ I型胶原涂覆盖玻片的方案。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Gelatin solution</td>
			<td>Sigma</td>
			<td>G1393</td>
			<td>Stock 20 mg/ml used at 1 mg/ml</td>
		</tr>
		<tr>
			<td>Gelatin from pig skin Oregon green 488 conjugate</td>
			<td>Molecular probe life technologies</td>
			<td>G13186</td>
			<td>5mg dilute at 1 mg/ml in sterile water</td>
		</tr>
		<tr>
			<td>Microscope cover glasses</td>
			<td>Marlenfeld GmbH &#38; Co</td>
			<td>111520</td>
			<td>&#216;12mm No.1</td>
		</tr>
		<tr>
			<td>2.5% Glutaraldehyd in 0.1M sodium cacodylate buffer pH 7.4</td>
			<td>Electron microscopy science</td>
			<td>15960</td>
			<td>Dilute at 0.5 % in sterile water</td>
		</tr>
		<tr>
			<td>1X PBS pH 7.4</td>
			<td>Gibco by life technologies</td>
			<td>10010-015</td>
			<td>Use this PBS in all steps before fixation</td>
		</tr>
		<tr>
			<td>Collagen I Rat tail</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>5 carboxy X rhodamin siccinimidyl ester</td>
			<td>Life technologies</td>
			<td>C-6125</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS 1X + calcium + magnesium</td>
			<td>Gibco by life technologies</td>
			<td>14040-091</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16% solution</td>
			<td>Electron microscopy science</td>
			<td>15710</td>
			<td>Dilute at 4% in 1X PBS</td>
		</tr>
		<tr>
			<td>Triton X 100</td>
			<td>Sigma</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>10X PBS buffer pH 7.4</td>
			<td>Ambion</td>
			<td>AM9625</td>
			<td>Dilute at 1X in water Use in steps after fixation</td>
		</tr>
		<tr>
			<td>Tks5 antibody</td>
			<td>Santa Cruz</td>
			<td>sc-30122</td>
			<td>Invadosome markers Dilution : 1/100</td>
		</tr>
		<tr>
			<td>Cortactin 4F11 antibody</td>
			<td>Millipore</td>
			<td>5180</td>
			<td>Invadosome markers Dilution :1/100</td>
		</tr>
		<tr>
			<td rowspan="2">DDR1 antibody</td>
			<td rowspan="2">Cell signaling</td>
			<td rowspan="2">5583</td>
			<td>Linear invadosome receptor</td>
		</tr>
		<tr>
			<td>Dilution :1/100</td>
		</tr>
		<tr>
			<td>Phalloidin FluoProbes</td>
			<td>Interchim</td>
			<td>FT-AZ0330</td>
			<td>Fibrillary actin marker Dilution :1/200</td>
		</tr>
		<tr>
			<td>Hoechst</td>
			<td>Sigma</td>
			<td>33258</td>
			<td>Nucleus marker Dilution :1/200</td>
		</tr>
		<tr>
			<td>Secondary antibodies FluoProbes</td>
			<td>Interchim</td>
			<td>FP-488 FP-547H or FP-647H</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin from bovine serum</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td>Dilute at 4% in 1X PBS</td>
		</tr>
		<tr>
			<td>Fluoromount G mounting medium</td>
			<td>Interchim</td>
			<td>FP 483331</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>Public domain</td>
			<td></td>
			<td>http://www.macbiophotonics.ca/imagej/</td>
		</tr>
		<tr>
			<td>Cell culture insert</td>
			<td>Corning</td>
			<td>353097</td>
			<td>8.0&#181;m pore size / 24 wells</td>
		</tr>
		<tr>
			<td>Sodium hydroxide (NaOH)</td>
			<td>Sigma</td>
			<td>221465</td>
			<td></td>
		</tr>
	</tbody>
</table>

2d and 3d matrices to study linear invadosome formation and activity

细胞粘附，迁移和侵袭参与许多生理和病理过程。例如，在转移形成期间，肿瘤细胞必须跨越解剖障碍以侵入和迁移穿过周围组织以便达到血液或淋巴管。这需要细胞和细胞外基质（ECM）之间的相互作用。在细胞水平上，包括大多数癌细胞在内的许多细胞能够形成invadosomes，这是能够降解ECM的基于F-肌动蛋白的结构。侵袭体是募集和激活基质金属蛋白酶（MMP）的突出肌动蛋白结构。 ECM的分子组成，密度，组织和刚度在调节invadosome形成和激活中是至关重要的。 在体外 ，明胶测定是用于观察和定量侵袭体降解活性的标准测定法。然而，变性胶原I的明胶不是生理基质e字元素。开发了使用I型胶原原纤维的新型测定法，并用于证明该生理基质是侵入体的有效诱导剂。由于它们在纤维上的线性组织，沿着胶原纤维形成的侵袭体被称为线性侵袭体。此外，线性invadosomes的分子分析表明，盘状结构域受体1（DDR1）是参与其形成的受体。这些数据清楚地表明了使用生理相关基质以了解细胞与ECM之间的复杂相互作用的重要性。

使用两种类型的基质，明胶和I型胶原的组合（ 图1和图4 ），我们突出了一种称为线性侵袭体的新型invadosome。这些基质的标记允许观察沿胶原I纤维的线性侵袭体形成及其降解能力（ 图5 ）。然后可以通过前面描述的宏来量化侵入性结构的数量（步骤4.2），还可以使用ImageJ软件来确定降解活性，如Martin 等人所述。和Diaz <em...

Watch this Scientific Journal Video about 2D and 3D Matrices to Study Linear Invadosome Formation and Activity at JoVE.com

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

该协议描述了如何制备由明胶和胶原蛋白I组成的2D混合基质，以及3D胶原I插塞来研究线性侵袭体。这些方案允许研究线性侵袭体形成，基质降解活性和原代细胞和癌细胞系的侵袭能力。

2D和3D矩阵研究线性侵袭体形成和活性

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

2d-and-3d-matrices-to-study-linear-invadosome-formation-and-activity

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

9.9K 次观看.  INSERM U1053. 该协议描述了如何制备由明胶和胶原蛋白I组成的2D混合基质，以及3D胶原I插塞来研究线性侵袭体。这些方案允许研究线性侵袭体形成，基质降解活性和原代细胞和癌细胞系的侵袭能力。 

视频: 2D and 3D Matrices to Study Linear Invadosome Formation and Activity

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

科研

生物工程

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

电场控制的二维和三维环境中的神经祖细胞定向迁移

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

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 ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Mammalian Cell Division in 3D Matrices via Quantitative Confocal Reflection Microscopy

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic significance. Possible reasons for the lack of exploration are the experimental limitations and technical challenges that render the study of cell division in 3D culture inefficient. Here, we describe an imaging-based method to efficiently study mammalian cell division and cell-matrix interactions in 3D collagen matrices. Cells labeled with fluorescent H2B are synchronized using the combination of thymidine blocking and nocodazole treatment, followed by a mechanical shake-off technique. Synchronized cells are then embedded into a 3D collagen matrix. Cell division is monitored using live-cell microscopy. The deformation of collagen fibers during and after cell division, which is an indicator of cell-matrix interaction, can be monitored and quantified using quantitative confocal reflection microscopy. The method provides an efficient and general approach to study mammalian cell division and cell-matrix interactions in a physiologically relevant 3D environment. This approach not only provides novel insights into the molecular basis of the development of normal tissue and diseases, but also allows for the design of novel diagnostic and therapeutic approaches.

This protocol efficiently studies mammalian cell division in 3D collagen matrices by integrating synchronization of cell division, monitoring of division events in 3D matrices using live-cell imaging technique, time-resolved confocal reflection microscopy and quantitative imaging analysis.

3D 基质中哺乳动物细胞分裂的定量共焦反射显微镜观察

The study of how mammalian cell division is regulated in a 3D environment remains largely unexplored despite its physiological relevance and therapeutic ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

Bioprintable 藻酸盐/明胶水凝胶 3D体外模型诱导细胞球形形成

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Novel Process for 3D Printing Decellularized Matrices

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1,2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 &#176;C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 &#176;C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

This protocol describes the production of polycaprolactone (PCL) filament with embedded polylactic acid (PLA) microspheres which contain decellularized matrices (DM) for 3D printing of structural tissue engineering constructs.

一种新的三维打印解光矩阵处理方法

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

三维水凝胶中全球细胞基质金属蛋白酶和代谢活性的测定

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current in vitro screening models that have sufficient throughput only have limited physiological relevance, which hinders the translation of findings from in vitro to in vivo. On the other hand, microfluidic cell culture platforms have shown unparalleled physiological relevancy in vitro, but often lack the required throughput, scalability and standardization. We demonstrate a robust platform to study angiogenesis of endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) in a physiological relevant cellular microenvironment, including perfusion and gradients. The iPSC-ECs are cultured as 40 perfused 3D microvessels against a patterned collagen-1 scaffold. Upon the application of a gradient of angiogenic factors, important hallmarks of angiogenesis can be studied, including the differentiation into tip- and stalk cell and the formation of perfusable lumen. Perfusion with fluorescent tracer dyes enables the study of permeability during and after anastomosis of the angiogenic sprouts. In conclusion, this method shows the feasibility of iPSC-derived ECs in a standardized and scalable 3D angiogenic assay that combines physiological relevant culture conditions in a platform that has the required robustness and scalability to be integrated within the drug screening infrastructure.

This method describes the culture of iPSC-derived endothelial cells as 40 perfused 3D microvessels in a standardized microfluidic platform. This platform enables the study of gradient-driven angiogenic sprouting in 3D, including anastomosis and stabilization of the angiogenic sprouts in a scalable and high-throughput manner.

标准化和可扩展的测定，以研究iPSC衍生内皮细胞在Vitro的渗透3D血管生成发芽

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current ...

2D和3D矩阵研究线性侵袭体形成和活性

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