The authors would like to acknowledge Drs. Jonathan Reichner and Angle Byrd for cell experiment insight. The National Science Foundation Graduate Research Fellowship Program (GRFP) supported this work.

1. 3D模具设计和零件<ol><li>模<ol><li>之前的工作，得到的有机硅弹性体的试剂盒，一个活细胞成像室，一个22毫米的玻璃盖玻片，和一个加工铝金属立方体，尺寸为10.07毫米×3.95毫米×5.99毫米。通过将盖玻片在底部支架和所指示的厂商组装腔室的其余部分制备活细胞成像室，用于成型。 </li><li>接着，使用镊子，将加工铝金属立方体中的活细胞成像室外壳的中间，并且在盖玻片的顶部，然后待用。 </li><li>根据制造商的协议，使5毫升弹性体的混合硅氧烷弹性体的解决方案。 </li><li>使用一次性实验室锅铲，倒入有机硅弹性体解决方案进入活细胞成像室的设置，并确保不移动放置加工铝金属立方体。将系统中的实验台上，在安全的位置O / N进行固化。 </li><li>第二天早上，解构活细胞成像室，使用镊子所推荐的制造商，拔出的模具。使用镊子，仔细提取模具加工铝金属立方体。转到水槽和用去离子水冲洗模具。将模具在纸巾上干燥。 </li><li>干燥后，用一个爱好美工刀，通过模具切缝，间隔2.34毫米相距纵向到底，硅胶模具内。保证了模具停留在一个安全干燥的地方，直到您准备构建系统的实验。 </li></ol></li><li>亲水性和疏水盖玻片准备<ol><li>以允许胶原基质粘附到表面上，创建亲水盖玻片: <ol><li>使用一次性吸液管，测量出150微升3-氨基丙基 - 三甲氧基硅烷和倒在50ml管溶液。加30毫升100％乙醇至50ml管中的与第二一次性吸，并盖上盖子。涡的解决方案，持续2分钟，以确保完全混合。 P我们在一玻璃培养皿溶液待用。 </li><li>倒入将15ml 100％乙醇在第二培养皿中，并预留（请务必标注菜肴）。 </li><li>使用新的一次性吸移管，量出30ml去离子水和倒溶液到50毫升管中。其次，采用了全新的一次性吸管量出1,875微升戊二醛，倒液进入同样的50毫升管，并盖上盖子。涡的解决方案，持续2分钟，以确保完全混合。将混合物倒入第三个培养皿中，并预留。 </li><li>使用镊子，取出22之一毫米的圆形玻璃盖玻片并使用一次性吸冲洗两面用100％的乙醇的混合物。 </li><li>放置漂洗玻璃盖玻片成3-氨基丙基 - 三甲氧基硅烷用30毫升100％乙醇溶液皿，并允许坐在溶液5分钟。 </li><li>用钳子，取出玻璃盖玻片，并与一次性吸管100％乙醇溶液再次冲洗。 </li><li>冲洗滴玻璃盖玻片到1,875微升戊二醛和30ml去离子水的混合物，并搁置30分钟。 </li><li> 30分钟后，取出玻璃盖玻片钳和用去离子水冲洗，并放置到干燥的组织O / N干在室温。 </li><li>重复浸盖玻片，并根据需要使用相同的解决方案产生的移动尽可能多的盖玻片。 </li></ol></li><li>在给定的协议使疏水盖玻片。 <ol><li>加入500μl十三氟-1,1,2,2-四氢辛基，100微升乙酸和在50毫升的管中，涡旋2分钟19.4毫升己烷。倒入玻璃培养皿的解决方案，并预留。 </li><li>用干净的镊子，取出一块玻璃盖玻片拖放到准备混合溶液2分钟。经过2分钟过去了，取出玻璃盖玻片钳和使用一次性吸管和地点盖玻片上干组织O / N干在室温用去离子水冲洗。商店盖玻片在塑料培养皿直至使用。 </li><li>重复盖玻片DIPP荷兰国际集团，并根据需要使用相同的解决方案产生的移动尽可能多的盖玻片。 </li></ol></li></ol></li></ol> 2.模具大会<ol><li>在使用的模具，采用一次性吸管冲洗模具90％的乙醇在化学废物容器。接下来，放置在培养皿中装有去离子水的模具和允许坐O / N。 </li><li>取模子里出来的使用镊子和地方的毛巾晾干解决方案开始在模具组装之前。 </li><li>而模具是空气干燥，切正方形亲水盖玻片成两个矩形比3.95毫米×5.99毫米使用高精度金刚石刻划工具略大。将每个切矩形盖玻片到切入硅胶模具的插槽。 </li><li>翻转模上下颠倒并应用真空润滑脂沿着使用一次性吸硅氧烷模具的底部。接下来，翻转模具靠背右侧朝上，然后按模具的底部到圆形亲水玻璃罩唇形成密封。 </li><li>与一次性手套，拿起组装模具，并放置到一次性培养皿中，然后进入生物罩。打开紫外线生物罩灯1小时的实验发生之前进行消毒模具。 </li></ol> 3.胶原蛋白混合物和3D矩阵<ol><li>之前制备胶原混合物，染色剂和制备细胞用于根据标准方法28成像。 </li><li>使用标准实验室技术和协议29,30，在一个生物罩，混合细胞培养基和趋化在15ml管所需的化学引诱物的浓度。吸移管加入5ml特定细胞媒体趋化浓度溶液到15毫升管和移动溶液到加热的水浴中。 </li><li>使用移液管，提取5毫升只是细胞培养基到第二15ml试管并置于加热的水浴升温显微镜实验溶液。 </li><li>在生物罩，吸管30微升10×磷酸盐缓冲编溶液（PBS）中成微离心管中。加入6微升1N氢氧化钠（氢氧化钠）到相同的微离心管中，涡旋15秒。 </li><li>得到胶原I鼠尾溶液从冰箱，并使用标准实验室技术和协议29移动到生物罩。确保胶原蛋白是依然寒冷。吸取168.3微升胶原的成同一微离心管中。 </li><li>接着，用移液管以相同的微离心管中添加18微升的黄绿色荧光羧化物修饰的微球。涡流的微离心管中进行30秒的所有解决方案。 </li><li>添加77.7微升所需的细胞培养基细胞混合物（约2×10 6细胞/ ml的浓度）的入微量离心管中并混合使用的吸头20秒。必要时，由适于加入荧光珠和细胞活力表（ 表1）的自定义胶原混合物以下改变基质密度。 </li><li>吸管300＃181;升胶原细胞混合物的进入中心孔（孔＃2）制得的模具组件的。放置在模具上的一次性培养皿并将其移到一个标准培养箱20分钟，在37℃，5％的CO 2。 </li><li>从孵化器中删除系统，并将放回生物罩。使用镊子，通过夹紧到每个盖玻片的顶部和拉起和远离模具中取出疏水盖玻片。 </li><li>将整个系统所需的显微镜和图像，以确保胶原模具两侧仍然直，以允许一个平面扩散梯度。 </li><li>与系统仍然在显微镜下，使用100微升单通道移液器，加入100微升细胞培养基到孔＃1。接着，使用100微升单通道移液器加入100微升的细胞培养基具有所需的化学引诱物的浓度成孔＃3。 </li><li>马上加入到解决方案两口井后，开始成像。 </li></ol> 4.成像和弥散造型<ol><li>如果一个人想找到的扩散系数的具体胶原蛋白的密度来计算特定浓度，按照以下协议: <ol><li>遵循给定的协议上述标题"胶原混合和3D矩阵"，而不是产生细胞介质具有所需的化学引诱物的浓度，使在一个15毫升管5微米罗丹明使用标准计算和协议30。 </li><li>图像的3D胶原基质共聚焦系统，安装在使用氩激光（488纳米）来捕捉荧光倒置显微镜。图像，每2或3秒长达7小时。 </li></ol></li><li>为扩散建模:使用以下所示的数学模型，写处理和分析的计算软件代码包含有这些一般步骤。 <img alt="式（1）" src="/files/ftp_upload/52948/52948eq1.jpg" /><ol><li>导入影像到了重新调整和规范计算软件由最大INTEN减到。图像转换为灰色颜色映射图像的选择的边缘。挑一轴线沿所述图像的中心，该中心的右侧，到中心的左边。 </li><li>使用的计算软件代码，绘制归一化的强度，随着真实强度和这两者之间的区别。 </li><li>接着，输入必要的用于装配（浓度，时间和长度）的参数。 </li><li>绘制的归一化浓度对时间和整个x轴的浓度分布与多项式拟合到数据。 </li><li>计算扩散使用的计算软件代码的系数。 </li></ol></li></ol> 5.实验测量<ol><li>返回参照的扩散方程，重写方程找到特定浓度在系统内的任何位置，如图所示。 <img alt="公式（2）" src="/files/ftp_upload/52948/52948eq2.jpg" /> 其中: 次的L =长度Ë胶原基质 在调查X =位置 D =系数扩散 T =经过时间</li><li>在实验中趋化，确保记录的那一刻趋化因子添加到系统的具体时间。一旦细胞迁移运动被发现，确保记录的共焦时间推移记录，并注意从边缘成像的地方发生的具体时间和地点。 </li><li>在数据分析期间，插注意到经过时间，地点和扩散系数为特定的小区入方程找到在胶原为扩散和细胞实验中的任何点的归一特定的浓度。 </li></ol> 6.跟踪细胞迁移利用TFM <ol><li>要使用TFM / DVC技术遵循以下协议跟踪细胞迁移: <ol><li>在加入所需的化学引诱物的浓度成孔＃3之后，开始成像的3D胶原基质与安装在一个倒MICR共焦系统oscope需要找一个小区进行调查。 </li><li>一旦移动单元找到，将细胞在视场的中间，并利用一压电马达（到图像中的z轴），图像每1或2分钟。 </li><li>为了计算单元力产生变形，产生一个计算程序，找到下面的TFM / DVC协议31的荧光珠的位移。 </li></ol></li></ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+and+chemical+modifications+of+collagen+gels:+impact+on+diffusion.">Physical and chemical modifications of collagen gels: impact on diffusion.</a> Biopolymers. 89 (2), 135-143 (2008).</li><li>Moghe, P. V., Nelson, R. D., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine-stimulated+chemotaxis+of+human+neutrophils+in+a+3-D+conjoined+fibrin+gel+assay.">Cytokine-stimulated chemotaxis of human neutrophils in a 3-D conjoined fibrin gel assay.</a> J. Immunol. Methods. 180 (2), 193-211 (1995).</li><li>Sundd, 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=Live+cell+imaging+of+paxillin+in+rolling+neutrophils+by+dual-color+quantitative+dynamic+footprinting.">Live cell imaging of paxillin in rolling neutrophils by dual-color quantitative dynamic footprinting.</a> Microcirculation. 18 (5), 361-372 (2011).</li><li>Sanders, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aseptic+Laboratory+Techniques:+Volume+Transfers+with+Serological+Pipettes+and+Micropipettors.">Aseptic Laboratory Techniques: Volume Transfers with Serological Pipettes and Micropipettors.</a> J. Vis. Exp. (63), 2754 (2012).</li><li>Stephenson, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Calculations for Molecular Biology and Biotechnology: A Guide to Mathematics in the Laboratory 2e. , (2010).</li><li>Bar-Kochba, E., Toyjanova, J., Andrews, E., Kim, K., Franck, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Fast+Iterative+Digital+Volume+Correlation+Algorithm+for+Large+Deformations.">A Fast Iterative Digital Volume Correlation Algorithm for Large Deformations.</a> Exp. Mech. In press, (2014).</li><li>Shenoy, V., Rosenblatt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+of+Macromolecules+in+Collagen+and+Hyaluronic+Acid,+Rigid-Rod-Flexible+Polymer+Composite+Matrixes.">Diffusion of Macromolecules in Collagen and Hyaluronic Acid, Rigid-Rod-Flexible Polymer Composite Matrixes.</a> Macromolecules. 28 (26), 8751-8758 (1995).</li><li>Leddy, H., Guilak, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-Specific+Molecular+Diffusion+in+Articular+Cartilage+Measured+using+Fluorescence+Recovery+after+Photobleaching.">Site-Specific Molecular Diffusion in Articular Cartilage Measured using Fluorescence Recovery after Photobleaching.</a> Ann. Biomed. Eng. 31 (7), 753-760 (2003).</li></ol>

The authors declare that they have no competing financial interests.

对于成功的扩​​散实验或不含有细胞的最重要的步骤是:正确设置的模具组件;开发必要的手巧，以防止提取疏水盖玻片的过程中损坏;确保找到一个很好的线性起跑线正确计算扩散系数;既纠正胶原蛋白和趋化实验计算;正确使用活细胞成像系统，以确保基质不干燥的;并保持无菌，健康培养。 当进行扩散实验，就必须确保扩散曲线匹配的线性扩散模型的传统形状（ 图4）?...

细胞的优选运动朝向的浓度梯度，被称为趋化作用，起着在体内的病理和生理过程中起重要作用。这样的例子是皮肤和粘膜伤口愈合1，形态2，炎症3，和肿瘤生长4,5。还已经表明，癌细胞可通过个人和集体细胞迁移策略6迁移。此外，扩散不稳定机制可以诱导单或聚簇的细胞从肿瘤体/对象分离，然后可以移民朝营养素源，从而侵入更广泛的领域和组织7。 此外，已经表明，不同的迁移机制可以是主动的二维和三维中，由于粘附分子8的不同的作用。因此，此举是为了生理有关体外实验来研究细胞运动的measureable和简单的方法就是意义的理解细胞的运动现象9。不幸的是，在分析细胞迁移的困难，全面量化的趋化性试验通常需要很长的费力的方法，建立在公正的细胞运动和迁移现象模型测量。 过去的实验方法来研究细胞趋化包括在Boyden小室10和下琼脂糖测定11。然而，这些早期试验中，细胞迁移实验没有监测方面的运动时间。更重要的是，用于该实验的浓度梯度没有明确定义或完全地理解，而只维持信令为不超过几个小时以上。此外，早期的趋化室的尝试限制细胞迁移到两个维度，并没有允许一个监视迁移12的动力学。纵观Boyden小室，端点检测不会允许研究者以目视观察的迁移，无法直接区分化学激活（随机运动）的趋化（定向运动）。此外，几个变量-差异的孔径和厚度的膜制造的腔室非常难以轻易再现和隐蔽细胞对趋化因子13,14的流动反应。 随着微流体的新的认识，新的商会和微型设备进行了调查作为一种工具来调查间质性流动条件下的细胞运动和趋化性15,16。根据这些新的设备，新的小区的度量进行了介绍和研究，象剪切应力对细胞17,18的效果。不幸的是，过去和现在的微流控趋庭，因为许多生物过程，包括肿瘤细胞的侵袭和转移，IM限制细胞迁移的研究，以2D基板，一个重要挫折宗细胞迁移，涉及到3D的迁移。 直接观察室-其中趋化溶液与含有细胞三维凝胶接触也被也报道19,20。这些室具有两个隔室，一个包含一个趋化和一种含有细胞，旁彼此水平21或作为同心环22接合。这些系统指出了正确的方向，但不保持一个趋系统的长时间。 此外，研究人员还透析细胞，以及示踪分子通过胶原样品进行静水压力23-25 ​​扩散检验扩散通过胶原膜。在胶原凝胶一些扩散实验依靠凝胶的使用磁场和化学掺入26物理和化学修饰。在建模科亚扩散性流行的方法genous组织依赖于连续漂白点的荧光成像。这种方法已经揭示各向异性大分子在面向胶原组织中的扩散系数。然而，光漂白已被用于在关节软骨和不胶原基质。虽然类似的，必要的模拟实验，必须通过具体理解胶原凝胶的扩散系数来进行。更重要的是，系统不使用用于测量细胞力产生的方法。 不幸的是，大多数系统似乎缺少了一个理想的系统中的一个或两个关键要素:细胞跟踪，通过矩阵，一个相对简单的设置了一个轻松的再现性，最小化的趋化因子的扩散梯度的理解使细胞-细胞相互作用，并且以测量的尺寸单位为量化（ 即 ，速度，力，具体浓度）的能力。 Moghe 等。 </e米&gt; 27提出了一个系统，该系统满足大多数的这些要求，其中细胞在整个凝胶最初分散，而不是集中在过滤表面上，但是难以测量的力，所述细胞产生。 为此目的，我们提出了一个平面梯度扩散系统中的3D胶原基质，它允许一个克服现有测定法，它是基于时间推移显微镜，加上图像分析技术来测量细胞的现代扩散腔的限制，调查趋力在3D环境中。这个协议提供了创建一个简单的3D扩散室，其可用于研究3D趋于不同小区的一个简单但创新的方式。

<table><tbody><tr><td>Silicone elastomer kit</td><td>Dow Corning Corp</td><td>182 SIL ELAST KIT .5KG</td><td>a two-part misture with a 10:1 mix</td></tr><tr><td>Live cell imaging chamber</td><td>Live Cell Instrument</td><td>CM-B18-1</td><td>CMB for 18&amp;nbsp;mm round coverslips</td></tr><tr><td>22&amp;nbsp;mm Glass Coverslip</td><td>Fisher Scientific</td><td>NC0180281</td><td>Neuvitro Corp. cover slip 22&amp;nbsp;mm 1.5</td></tr><tr><td>Machined aluminum metal cube</td><td></td><td></td><td></td></tr><tr><td>Hobby utility knife</td><td>X-Acto</td><td>X3201</td><td></td></tr><tr><td>3-(aminopropyl) trimethoxysilane</td><td>Sigma-Aldrich</td><td>281778-5ML</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Polysciences, Inc</td><td>00216A-10</td><td>Glutaraldehyde, EM Grade, 8%</td></tr><tr><td>50 ml tube</td><td>Fisher Scientific</td><td>14-432-22</td><td>Standard floor model and tabletop centrifuges</td></tr><tr><td>Glass Petri dish</td><td>Fisher Scientific</td><td>08-747A</td><td>Reusable Petri Dishes: Complete (60 x 15&amp;nbsp;mm)</td></tr><tr><td>Forceps</td><td>Fisher Scientific</td><td>22-327-379</td><td>Fine Point Forceps</td></tr><tr><td>Cover glasses</td><td>Fisher Scientific</td><td>12-518-105A</td><td>Rectangle; 30 x 22&amp;nbsp;mm; Thickness No. 1</td></tr><tr><td>Tridecafluoro-1,1,2,2-tetrahydrooctyl</td><td>Gelest</td><td>SIT8174.0</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>320099</td><td>Acetic acid ACS reagent, &amp;ge;99.7%</td></tr><tr><td>Hexane</td><td>Sigma-Aldrich</td><td>296090</td><td>anhydrous, 95%</td></tr><tr><td>Ethyl alcohol</td><td>Sigma-Aldrich</td><td>E7023</td><td>200 proof, for molecular biology</td></tr><tr><td>High-precision diamond scribing tool</td><td>Lunzer</td><td>PV-081-3</td><td>Straight extended tip scribe, .020&quot; (.50&amp;nbsp;mm) diameter by .200&quot; (5.0&amp;nbsp;mm) tip length</td></tr><tr><td>Vacuum grease</td><td>Dow Corning</td><td>14-635-5C</td><td>High-Vacuum Grease</td></tr><tr><td>15 ml tube</td><td>Fisher Scientific</td><td>14-959-49D</td><td>15 ml conical centrifuge tubes with hydrophobic, biologically inert surface</td></tr><tr><td>10x phosphate buffered solution</td><td>Fisher Scientific</td><td>BP399-500</td><td>1.37 M Sodium Chloride, 0.027 M Potassium Chloride, and 0.119 M Phosphate Buffer</td></tr><tr><td>1&amp;nbsp;N sodium hydroxide</td><td>Sigma-Aldrich</td><td>38215</td><td>Sodium hydroxide concentrate</td></tr><tr><td>Collagen I, rat tail</td><td>BD Biosciences</td><td>354236</td><td>Rat tail&amp;nbsp;</td></tr><tr><td>Micro centrifuge tube</td><td>Fisher Scientific</td><td>02-681-332</td><td>Volume: 2.0 ml; &lt;br /&gt;O.D. x L: 13&amp;nbsp;x&amp;nbsp;40&amp;nbsp;mm; &lt;br /&gt;sterile; single-wrapped</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Carboxylate-modified microspheres</td><td>Invitrogen</td><td>&amp;nbsp;F-8813</td><td>Carboxylate-modified microspheres, 0.5 &amp;micro;m, yellow-green fluorescent (505/515), 2% solids</td></tr><tr><td>Rhodamine</td><td>Sigma-Aldrich</td><td>83689</td><td>Rhodamine B for fluorescence</td></tr></tbody></table>

planar gradient diffusion system to investigate chemotaxis in a 3d collagen matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

该测定的准确评估细胞迁移的能力依赖于该系统的一个良好的设置。因此，它是为要确保设计的扩散系统模具准确和非常谨慎地放置两个疏水和亲水盖玻片， 如图1关键的。如果系统设计正确，并且在扩散建模台确保找到一个很良好的线性起跑线，一个是能够实现很好的荧光图像，描绘在图2中 ，用于分析所述系统的扩散。 在成功的关键细胞趋化分析...

Watch this Scientific Journal Video about Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix at JoVE.com

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

平面梯度扩散系统的调查趋化三维胶原基质

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

planar-gradient-diffusion-system-to-investigate-chemotaxis-3d

Research

JoVE Journal

Bioengineering

8.5K 次观看.  California State University, Long Beach. Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

视频: Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

创建的胶粘剂和可溶性梯度荧光显微镜成像细胞迁移

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

科研

生物工程

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

在一个三维胶原蛋白矩阵分析细胞迁移

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

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 ...

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 ...

An All-on-chip Method for Rapid Neutrophil Chemotaxis Analysis Directly from a Drop of Blood

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil migration and chemotaxis owing to their advantages in real-time visualization, precise control of chemical concentration gradient generation, and reduced reagent and sample consumption. Recently, a growing effort has been made by the microfluidic researchers toward developing integrated and easily operated microfluidic chemotaxis analysis systems, directly from whole blood. In this direction, the first all-on-chip method was developed for integrating the magnetic negative purification of neutrophils and the chemotaxis assay from small blood volume samples. This new method permits a rapid sample-to-result neutrophil chemotaxis test in 25 min. In this paper, we provide detailed construction, operation and data analysis method for this all-on-chip chemotaxis assay with a discussion on troubleshooting strategies, limitations and future directions. Representative results of the neutrophil chemotaxis assay testing a defined chemoattractant, N-Formyl-Met-Leu-Phe (fMLP), and sputum from a chronic obstructive pulmonary disease (COPD) patient, using this all-on-chip method are shown. This method is applicable to many cell migration-related investigations and clinical applications.

This article provides the detailed method of performing a rapid neutrophil chemotaxis assay by integrating the on-chip neutrophil isolation from whole blood and the chemotaxis test on a single microfluidic chip.

一种直接从一滴血液中快速中性粒细胞趋化分析的片上方法

Neutrophil migration and chemotaxis are critical for our body's immune system. Microfluidic devices are increasingly used for investigating neutrophil ...

免疫与感染

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and chemical complexity of living tissues and mimic in vivo tissue functions. Recent advances in microfabrication technologies have facilitated the development of 3D in vitro environments in which cells can be integrated into a well-defined extracellular matrix (ECM) and a defined set of soluble or matrix associated biomolecules. However, technological barriers have limited their widespread use in research laboratories. Here, we describe a method to construct simple devices for 3D culture and experimentation with cells and multicellular organoids in 3D microenvironments with a defined chemoattractant gradient. We illustrate the use of this platform for analysis of the response of epithelial cells and organoids to gradients of growth factors, such as epidermal growth factor (EGF). EGF gradients were stable in the devices for several days leading to directed branch formation in breast organoids. This analysis allowed us to conclude that collective gradient sensing by groups of cells is more sensitive vs. single cells. We also describe the fabrication method, which does not require photolithography facilities nor advanced soft lithography techniques. This method will be helpful to study 3D cellular behaviors in the context of the analysis of development and pathological states, including cancer.

We describe a method to construct devices for 3D culture and experimentation with cells and multicellular organoids. This device allows analysis of cellular responses to soluble signals in 3D microenvironments with defined chemoattractant gradients. Organoids are better than single cells at detection of weak noisy inputs.

趋化梯度多细胞响应的三维分析

Various limitations of 2D cell culture systems have sparked interest in 3D cell culture and analysis platforms, which would better mimic the spatial and ...

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

牵引显微镜与微流体集成，用于化学集体迁移

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

Time-lapse Imaging of Mouse Macrophage Chemotaxis

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a chemical. Chemokinesis and chemotaxis are fundamental for the mobilization and deployment of immune cells. For example, chemokines (chemotactic cytokines) can rapidly recruit circulating neutrophils and monocytes to extravascular sites of inflammation. Chemoattractant receptors belong to the large family of G protein-coupled receptors. How chemoattractant (i.e., ligand) gradients direct cell migration via G protein-coupled receptor signaling is not yet fully understood. In the field of immunology, neutrophils are popular model cells for studying chemotaxis in vitro. Here we describe a real-time two-dimensional (2D) chemotaxis assay tailored for mouse resident macrophages, which have traditionally been more difficult to study. Macrophages move at a slow pace of ~1 &#181;m/min on a 2D surface and are less well suited for point-source migration assays (e.g., migration towards the tip of a micropipette filled with chemoattractant) than neutrophils or Dictyostelium discoideum, which move an order of magnitude faster. Widely used Transwell assays are useful for studying the chemotactic activity of different substances, but do not provide information on cell morphology, velocity, or chemotactic navigation. Here we describe a time-lapse microscopy-based macrophage chemotaxis assay that allows quantification of cell velocity and chemotactic efficiency and provides a platform to delineate the transducers, signal pathways, and effectors of chemotaxis.

Here we describe methods using time-lapse, phase-contrast microscopy to image mouse resident peritoneal macrophages in a chemotactic complement C5a gradient. The protocols can be extended to other immune cells.

小鼠巨噬体化学术的延时成像

Chemotaxis is receptor-mediated guidance of cells along a chemical gradient, whereas chemokinesis is the stimulation of random cell motility by a ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

生成基于3-D胶原蛋白的水凝胶,分析神经系统发育过程中的Axonal生长和行为

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

神经科学

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells.

成像G蛋白偶联受体介导的趋化和信令活动在中性粒细胞样细胞HL60

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...