Name: concentric gel system to study the biophysical role of matrix microenvironment on 3d cell migration
Uploaded: 2015-04-03T15:00:00.000+00:00
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作者感谢W. Sun和K·詹森的批判性的讨论，并承认支持纳米生物力学实验室，在新加坡国立大学。 NAK承认支持由居里夫人IIF奖学金。

1.细胞收获<ol><li>得到的MD-MBA-231细胞从37℃，5％CO 2的培养箱中培养。分离，以0.5％胰蛋白酶-EDTA溶液从组织培养板上的细胞。使用1个毫升胰蛋白酶-EDTA溶液用于在T25培养瓶中的细胞进行培养。 </li><li>沉淀细胞在15ml锥形管中，离心，在200×g下4分钟，吸出上清液，并悬浮细胞在5ml培养基。 </li><li>计数细胞密度，ρ，使用血球。 注意:为了制备细胞接种内凝胶，将细胞悬液稍后会稀释10×达到最终细胞接种密度。因此，10×浓缩的细胞悬浮液是必需的。 </li><li>计算达到10×细胞浓度所需介质的体积: <img alt="式（1）" fo:content-width="2in" src="/files/ftp_upload/52735/52735eq1.jpg" width="175" /> 注:最后一个细胞接种密度，C 最终 ，大约215; 10 6个细胞/ ml的被推荐用于MD-MBA-231细胞，并用于在此协议。其他播种密度也可探索其他类型的细胞。 </li><li>沉淀细胞再一次在一个15毫升的锥形试管离心，在200×g下4分钟，吸出上清液。 </li><li>重悬的无血清细胞培养基中的细胞所需要的量（V 培养基在步骤1.3计算的）彻底以最小化细胞聚集。 注意:酚红是自体荧光，并且可以与荧光/反射成像干涉。使用不含酚红的培养基中可以考虑实现最佳的图像质量。 </li></ol> 2.准备胶原蛋白解决方案<ol><li>获取股票胶原溶液，10×PBS缓冲液，的Milli-Q H 2 O，0.1M NaOH和几个离心管。保留所有冰上以防止过早胶原蛋白的聚合，并保持无菌状态。 </li><li>平衡无菌玻璃 -底菜在37℃培养箱预热吧。 注:在本协议中的所有卷进行了优化的12 mm孔玻璃底菜。如果其他菜类型时，相应地调整卷。 </li><li>计算需要准备50微升2.4毫克/毫升的胶原溶液为基于所述胶原原料浓度的内凝胶（溶液Ⅰ）所需的体积。 注意:也可以使用其它的胶原蛋白的浓度为所述内凝胶。 </li><li>在无菌的环境（通常是生物安全罩），慢慢加入5微升10×PBS缓冲液至所需的胶原原液量（在步骤2.3计算的）伴以轻轻旋转。注意避免气泡的形成。 </li><li>调节混合物的7.4使用0.1M NaOH中使用校准的pH计的pH。作为一个粗略的指导，使用约5微升以使pH接近7.4（该量取决于该原料浓度和pH下）。 注意:请注意，参与这一卷步骤太小使用标准pH计。请使用以下招数之一: <ol><li>制备用于多个样品的胶原蛋白溶液。使用标准pH计调整在散装的pH值和整个样本分配胶原溶液。 </li><li>可替代地，调整的胶原溶液的pH值在大的体积（ 即 ，体积，允许使用标准的pH计）。注意，以使pH至最终pH所需的NaOH的量。缩小的容积，并使用氢氧化钠适量实验。确认使用石蕊试纸的pH值。 </li><li>否则，使用微pH电极能够更准确地调节小批量的pH。 </li></ol></li><li>使溶液于45体积微升使用的H 2 O.执行上冰的所有步骤，以防止过早胶原蛋白的聚合。 </li></ol> 3.形成同心凝胶文化<ol><li>就拿预热的玻璃底菜（见步骤2.2）从日Ë孵化器。 </li><li>添加5微升10×浓细胞悬液（步骤1.5准备）来解决一悬浮彻底。注意避免气泡的形成。该混合物现在有50微升的体积，并含有终胶原浓度（2.4毫克/毫升）和细胞密度（C 最终 = 2×10 6个细胞/ ml）。 </li><li>慢慢加入20微升所述含细胞溶液Ⅰ至井的中心，以便它形成一圆顶状的液滴（ 图1A）。注意避免泡沫形成在这一步。如果泡沫形成，但仔细赶快试试要么破裂，或用枪头吸出来。轻轻地放在培养皿放回培养箱让内部凝胶聚合45分钟。 </li><li>准备解决O（用于外，无细胞胶原凝胶）的孵育步骤结束前约15分钟。 注意:外凝胶条件可以在胶原蛋白浓度和聚合pH值而言，以改变获得网络的不同的微观结构10。在这个协议中，着眼于一个1.5 - 聚合在pH 7.4 4.0毫克/毫升的胶原凝胶。 <ol><li>基于该胶原原料浓度，计算需要准备200微升胶原溶液0在最终浓度所需的体积。 </li></ol></li><li>加入20微升10×PBS中缓慢缓冲到所需的胶原原液（在步骤3.4计算的）伴以轻轻旋转量。调节混合物的pH至用0.1M的NaOH与使用校准pH计的最终pH值。见注有关调节pH值至2.5步。 </li><li>使溶液至200的最终体积使用微升的H 2 O.执行上冰的所有步骤，以防止过早胶原蛋白的聚合。 </li><li>采取从孵化器的菜45分钟聚合内部凝胶后（见步骤3.3）。轻轻的内凝胶上添加180微升溶液O，以使溶液完全覆盖内凝胶和填充井（ 图1B）。 <ol><li>小心执行此步骤不搅拌的溶液中，这可能会导致在外部凝胶不均匀纤维取向。注意避免接触内部凝胶用枪头，避免气泡或气泡的形成。如果泡沫形成，但仔细赶快试试要么破裂，或用枪头吸出来。轻轻将菜放回孵化器，让外胶聚合。 </li></ol></li><li>取从培养箱培养皿后聚合45分钟。凝胶应已相当固化，在这一点上，虽然它仍可以从底面脱落如果处理大致。 <ol><li>轻轻倾2毫升温热细胞培养基的菜（ 图1C）。确保该凝胶在培养基中完全浸没。刷新介质每2 - 3天整个培养的持续时间。 </li></ol></li></ol> 4。活细胞成像<ol><li>使用倒置共聚焦显微镜配备了长期活细胞成像能力进行成像。包括一个内置的孵育室的温度（37℃）和CO 2（5％）控制。接通显微镜和开始实验之前预热阶段至少1小时。 注意:使用物镜具有长工作距离，以优化细胞的观测和定位在3D凝胶。 </li><li>孵育在含有5微升荧光细胞追踪染料30分钟介质的凝胶，以允许细胞在3D系统的准确定位。随后，通过洗涤用1×PBS中去除未结合的染料三次。之后，加入细胞培养基的培养皿中。 </li><li>取菜从孵化器，并把它放在显微镜阶段（ 图1D）。 注:活细胞成像能够在原则外凝胶的聚合反应后立即启动。然而，在这点上，小区in中的内部凝胶尚未普及。要检查已侵入外硅胶，活细胞成像可以开始24小时培养开始后（取决于细胞类型）的第一个细胞的迁移。作为粗略导，约12 - 需要14天培养的大多数细胞中的内凝胶进入外凝胶。 </li><li>查看（两用粉饼的）的地区选择卷在内部凝胶周围外胶。后培养24小时，该细胞群体将已经扩散，越过内侧和外侧的凝胶之间的界面，并开始侵入外凝胶。 <ol><li>对于两用粉饼的，包括凝胶区域紧邻凝胶界面，中间区，和区域靠近外侧的凝胶7的郊区。排除的区域的距离小于50微米的底面和侧面，以及从该凝胶的顶部，以避免可能的边缘效应。每个两用粉饼一般测量647×647×100微米3（在X，y和z方向上，分 ​​别地），以在z -stack5μm的间隔。 </li></ol></li><li>确保成像模式，信道/过滤器，曝光时间，和图像分辨率被正确地选择。对于胶原蛋白网络的无标记成像，同时采用激光共聚焦显微镜反射时间推移活细胞成像过程。 </li><li>取样品的图像，画出了强度直方图，并调整增益和偏移来观察足够的信号，并避免饱和通过确保直方图在于零与最大强度之间。不整个实验的持续时间的任何更改变这些设置。 </li><li>取10分钟，8小时（或更长，如果必要的话）的选择的两用粉饼的时间推移的图像，以一时间间隔，ΔT，。 </li></ol> 5.细胞跟踪和数据分析<ol><li>使用appropria 的 Z -stack图像进行定量图像后处理<html>德的图像处理软件。 <ol><li>段的时间推移的图像来自动选择在（X，Y，Z，t）的三维细胞的位置。 </li><li>对于每一帧，手动检查的定位精度和除去由于细胞碎片和细胞突起可能已被误认为细胞误报。除去从分析和分割重叠或连接成细胞的不同对象增殖性细胞。 </li></ol></li><li>生成由按时间顺序连接的每个单元的位置，在前面的步骤中获得的细胞的坐标（X，Y，Z，t）的三维时间推移单元轨道。 </li><li>通过删除曲目大于阈轨道长度（通常为20分钟）较短消除随机和系统噪音。 </li><li>如果必要，通过减去总净位移的轨道校正样品漂移。 </li><li>计算单元的位移，尔斯/ ftp_upload / 52735 / 52735eq2.jpg"WIDTH ="80"/&gt;，和细胞迁移的距离， <img alt="公式3" fo:content-width="1in" src="/files/ftp_upload/52735/52735eq3.jpg" width="100" />从所观察到的细胞的轨迹，其中，是表示一个小区的时间的三维位置的矢量和是时间点的总数。 <ol><li>计算单元速度为S = D /（正•Δt），其中Δt是帧之间的时间间隔。计算使用P =量Δd/ D细胞迁移的方向性（或持久性）。持久性的这种简单措施意味着，对于P = 0的净位移是零和用于P = 1的轨迹是直线方向线。 </li></ol></li></ol>

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In this protocol we describe an in vitro assay to study the 3D migrational behavior of cells in matrix environments that topologically resemble ECMs encountered in vivo. There are three main strengths of this assay as compared to other currently available methods. First, this assay allows one to simultaneously examine the cell migration mechanisms at both population level and individual cell level. This opens up possibilities of studying collective cell migration13, which has to date been largely limited to 2D assays14, while at the same time observing the behavior of the individual cell within the population. For cells that do not naturally migrate in groups or clusters, the assay offers a way to analyze the migratory properties in detail from a large number of cells with diverse local microenvironment, which can provide important insights into the biophysical origins of migration efficiency and directional persistence7,15.
Second, by modifying the gel conditions of the inner and outer gels, one can directly study the effect of matrix mechanics and microstructure on 3D cell migration behavior10. This is especially important, as it is known that cell migration and cancer invasion are highly influenced by matrix mechanics and porosity16,17, and that soft tissues in our body have a very diverse topology18. Moreover, the method is readily adaptable for matrices other than collagen. This is advantageous, as not all cell lines can spread and migrate in collagen-based matrices. A critical step that must be noted is that, to ensure physical connectivity of the gel interface, the formation of the outer gel must be initiated before complete polymerization of the inner gel. Since this is strongly dependent on the specific polymerization kinetics of the gels (also for collagen prepared under different conditions, e.g., concentration, polymerization pH, temperature, cross-linking8), an optimal polymerization duration should first be systematically identified. Imperfections in the interfacial connectivity between the inner and outer gel usually results in a gap and collection of the spreading cell population in the interface. Note, however, that perfect interfacial connectivity can introduce a practical problem: the boundary between the inner and outer gels becomes less obvious when imaging. If necessary, sparse fiducial markers, such as inert beads, can be used to distinguish the inner and outer gels.
Third, the possibility of long-term 3D culture (up to 3 weeks in our laboratory) and live-cell imaging allows a wide application of the assay. It is now possible to obtain spatially-resolved viscoelastic measurements of the local matrices surrounding the cells using microrheological approaches19. Furthermore, our recent study10 reveals that the efficacy of various anti-migratory drugs (administered on the 7th day of the experiment) can be strongly affected by the architecture and stiffness of the matrix (i.e., outer gel). In the future, we plan to use these experimental frameworks to further understand how such effects can arise from the basic molecular mechanisms of cell migration (e.g., cytoskeletal contractility20, extracellular matrix reorganization21, and pericellular proteolysis22).
One limitation of this assay is the asymmetry of the mechanical support: the top surface of the gel is not attached to any solid surface, while the bottom surface adheres to the glass surface of the dish. While edge effects can be avoided simply by excluding cells near the edges, the lack of mechanical support at the top surface can potentially lead to in-plane preferential migration and therefore introduce artificial flattening of the 3D cell trajectories7. One way to solve this issue while still maintaining oxygen and nutrition supply to the cells is by topping the gel with holed glass plate23. Additionally, to further suppress any wall effect, a thin layer of collagen gel can be first made on the glass surfaces, before the formation of the inner and outer gels. This ensures complete geometrical encapsulation of the gels.
To obtain meaningful and accurate description of cell migration behavior, it is important to consider the time scale and the nature of migration, which can vary considerably between cell lines. Preliminary experiments must be systematically performed to understand these factors, and then the interval time between image captures and live-cell imaging duration can be adjusted. The interval time should be no larger than the characteristic time scale of cell turning. It is also important to consider that cell migration behavior may change both spatially and temporally, depending on the biophysical conditions of the local microenvironment7. It is always a good idea to simultaneously visualize the cells and the fibrous matrix, for example using multi-color confocal fluorescence or multimodal (e.g., reflectance, second harmonic generation) imaging. After imaging, post-processing of the time-lapse 3D image, construction of the cell tracks, and analysis of the trajectories can in principle be done with any image processing software.
In conclusion, the concentric gel assay described here provides a simple and useful experimental system for quantitatively studying 3D cell migration, mechanosensing and mechanotransduction in diverse microenvironments.

细胞迁移起着各种生理反应，如胚胎发育，止血，和免疫反应，以及在病理过程，如血管疾病，炎症，癌症和1的关键作用。解剖底层细胞迁移的生物化学和生物物理因素，因此从根本上重要的是不仅要了解的细胞功能的基本原则，又能促进各种生物医学应用，例如在组织工程，抗转移和抗炎药物开发。由于体内观察是技术上的挑战，有很多的努力一直专注于体外再演细胞迁移。 体外方法来研究细胞迁移已经基本上被设计为检测在二维（2D）表面上，最显着的划伤或伤口愈合测定法2。这些试验提供了简单的实验装置，方便在线 -细胞成像，并提供有益的见解底层细胞迁移的各种生化机制。但是，这些测定法不占外基质（ECM）架构和重塑，这是在理解体内迁移关键方面。最近，人们已越来越多地认识到，3D培养模型，常常在基于胶原的基质3中，提供了更好的类似于体内情况的一个平台。事实上，细胞表现出的迁徙动力学是从那些在2D表面明显，特别是由于不同的维数的环境4。此外，基体的生物物理和机械性能灵敏影响细胞迁移5，包括在肿瘤细胞侵袭6的上下文中。 在这里，我们提出研究三维细胞迁移行为的ECM与生物物理特性，可以轻松地用不同的制备条件的方法。细胞是接种在"内凝胶"和被允许逃逸到并侵入最初无细胞"外凝胶"。该方法依赖于细胞的能力来识别的存在，并倾向成，无细胞迁移区域中的外凝胶，这是密切相关的细胞mechanosensing 7。在这项研究中，我们采用的胶原蛋白网络，通过高度侵袭性​​乳腺癌细胞，MD-MBA-231入侵的ECM。机械性能和两者的内部和外部的凝胶的微结构可以被调谐8和9，其特征实现生理学相关的条件。重建单元轨道和分析允许在两个群体水平和个体细胞水平的时空迁移行为的详细的定量检查。重要的是，同心凝胶系统的设置模仿面临迁移细胞，尤其是侵入癌细胞，从而提供了重要的见解的体内组织的拓扑对细胞迁移和转移的物理机制。

<table><tbody><tr><td>Cell culture incubator</td><td>Fisher Scientific Pte Ltd</td><td>Model: 371, S/No 318854-6055</td><td></td></tr><tr><td>Confocal microscope</td><td>Nikon A1R</td><td></td><td>Inverted confocal laser scanning microscope equipped with incubator chamber</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Life Technologies</td><td>11965-092</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>10082-147</td><td></td></tr><tr><td>Fluorescent CellTracker dye CMTMR</td><td>Life Technologies</td><td>C2927</td><td></td></tr><tr><td>Glass-bottom dish</td><td>IWAKI Cell Biology</td><td>3931-035</td><td>35 mm diameter dish with 12 mm diameter glass-bottom well</td></tr><tr><td>Hemocytometer</td><td>iN CYTO</td><td>DHC-N01 (Neubauer Improved)</td><td></td></tr><tr><td>Microprocessor pH meter</td><td>Hanna Instruments</td><td>pH 211</td><td></td></tr><tr><td>Nutragen Collagen</td><td>Advanced BioMatrix</td><td>#5010-D</td><td>Acid-solubilized bovine collagen type I (stock pH ~ 2)</td></tr><tr><td>Objective lens</td><td>Nikon</td><td></td><td>CFI Super Plan Fluor ELWD ADM 20XC, W.D. 8.2-6.9mm, NA 0.45.</td></tr><tr><td>Penicillin-Streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>pH meter</td><td>Sartorius</td><td>S/No 29153352</td><td>Basic pH Meter PB-11</td></tr><tr><td>Trypsin-EDTA</td><td>Life Technologies</td><td>15400-054</td><td></td></tr></tbody></table>

concentric gel system to study the biophysical role of matrix microenvironment on 3d cell migration

细胞迁移的能力是在各种各样的细胞功能，在整个从胚胎发育寿命和伤口愈合肿瘤和肿瘤转移的关键。尽管强烈的研究努力，细胞迁移的基本生物化学和生物物理原理仍然没有完全了解，特别是在生理相关的三维（3D）的微环境。在这里，我们描述了在体外试验设计允许的三维细胞迁移行为定量检查。该方法利用了细胞的mechanosensing能力和倾向迁移到先前未被占据的细胞外基质（ECM）。我们使用高度侵袭性​​乳腺癌细胞的侵袭，MDA-MB-231，在胶原凝胶作为模型系统。细胞群的传播和单个细胞的迁移动力学数周的文化可以用活细胞成像进行监测和分析，以提取spatiotemporally分辨数据。此外中，该方法很容易适应于不同的细胞外基质，从而提供了一种简单而功能强大的方法来研究的生物物理因素对细胞迁移的微环境中的作用。

使用高度侵袭性乳腺癌细胞进行这里提出的同心凝胶测定中，MDA-MB-231，用2.4毫克/毫升的内胶原凝胶和= 2×10 6个细胞/ ml的细胞接种密度，作为一个例子。 如图2中 ，典型地经过培养几天，细胞违反内外凝胶接口，并开始侵入外凝胶。该细胞群主要传播径向向外。 外凝胶的聚合条件可被修改以研究对细胞迁移特性的矩阵的密度和机械性能的作用。 图3示出了单个细胞中的1.5毫克外凝胶的移动/毫升，2.4毫克/毫升，和4.0毫克/毫升的胶原，聚合在pH 7.4。分别获得在内层和外层的凝胶之间的边界8小时的共聚焦时间推移的图像，并且将细胞在三个不同的胶原concen位移trations显示在白色箭头。取决于细胞接种密度和增殖率，〜200各个细胞的轨迹通常可以被提取并在每个样品中进行分析。 我们定量分析而言平均净位移，行驶距离，定向持久性在3种不同浓度的胶原蛋白的细胞的轨迹，和平均车速超过成像的8小时（ 图4）。据观察，平均在58微米和141.5微米，分别位移和距离分别为最高为4.0毫克/毫升的浓度，虽然没有在小区之间的位移和距离无显著差异在2.4毫克/毫升和4.0毫克/毫升的凝胶。在1.5毫克/毫升凝胶，平均位移和距离分别为最小。这一观察可能会出现矛盾，显示了入侵的效率在降低凝胶随着胶原蛋白的浓度11,12最近的报告。但是，请注意，位移和在我们的检测距离是所有单个细胞曲目主要是径向向外迁移人口内测量。这些参数，因此，以最快的移动细胞亚群小得多敏感并且不被随机运动完全支配。 在所有情况下，盖（ 图4B）的总距离大于净位移（ 图4A）。限定持久只是以距离位移之间的比，我们获得了在所有凝胶的条件（ 图4C）的0.4〜持久性。这相对低的持久性反映了细胞在我们的测定法中固有弱定向迁移。我们预计，定向趋化刺激或生物化学梯度的存在将增加的持久性，从而可能在胶原浓度依赖性。此外，我们还观察到细胞迁移的平均速度没有显著改变Wi第胶原浓度（ 图4D），很可能是因为矩阵刚度（其与胶原蛋白的浓度增加）和孔径（这降低了与胶原浓度）10，以及迁移特征的细胞群7中的时空变化之间的相互影响的。 31微米/小时和37.5微米/小时之间的平均速度变化，与为7μm/ hr的最小单个电池的速度和114微米/ hr的最大个体细胞的速度。 <img alt="图1" src="/files/ftp_upload/52735/52735fig1.jpg" /> 图1.原理的参与建立同心凝胶3D迁移实验步骤。（A）内的胶原蛋白凝胶在井的玻璃底菜中心的形成，含有细胞。（B）的形成（无细胞）外胶原凝胶封装内凝胶（℃）在细胞培养基中的凝胶浸没。然后将细胞使其平衡并连接到周围的纤维状基质。此步骤可能需要几个小时1天凝胶聚合开始后，（D）的活细胞成像，监测细胞群的扩展到外凝胶。 <img alt="图2" src="/files/ftp_upload/52735/52735fig2.jpg" /> 图2.传播细胞群。 （A）中经过12天的培养中，MDA-MB-231细胞已经违反内外凝胶接口（绿色虚线）和侵入向外成原本非细胞外的凝胶（比例尺= 200微米）。较亮的区域都被细胞所占用的区域，而较暗的区域都在这一阶段对比度图像无细胞的区域。黄色矩形表示的两用粉饼的兴趣，其中该三维癌细胞侵袭进行监测。行政长官LLS迁移主要沿径向向外，由如箭头所示迁移的MDA-MB-231细胞的共聚焦荧光图像（红色;当与绿色伪彩色胶原覆盖黄色）的（B）的叠加。胶原基质的和反射图像（绿色）。箭头指向单个细胞内的5小时的时间跨度迁移的方向。比例尺= 50微米。（c）从所述的时间推移的图像，能够获得的细胞的磁迹。这里一个单元轨道是颜色编码为时间，由彩条（时间刻度= 8小时）所指示的。从Sun 等 7,10适应。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52735/52735fig2large.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/52735/52735fig3.jpg" /> 图3.细胞运动在不同的胶原凝胶密度。 </st荣&gt; MDA-MB-231细胞在外侧的凝胶用三种不同外胶原浓度的运动:1.5毫克/毫升，2.4毫克/毫升和4.0毫克/毫升。细胞用活细胞跟踪CMTMR（红色）。白色箭头指示单个细胞内8小时成像的净位移。比例尺= 100微米。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52735/52735fig3large.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/52735/52735fig4.jpg" /> 图分析细胞迁移轨迹4.个别MD-MBA-231细胞超过8小时，进行量化的（A）净位移，（B）距离计算的迁移轨迹，（C）的持久性，以及（D）的意思速度不同的外层凝胶胶原浓度:1.5毫克/毫升，2.4毫克/毫升和4.0毫克/毫升。内层和外层的凝胶的界面附近被收购的时间推移的图像。数据平均值±200多个细胞轨迹在每个条件3个独立的实验中获得的平均值（SEM）的标准误差。星号（*）表示统计学显著差异（p值&lt;0.01）。

Watch this Scientific Journal Video about Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration at JoVE.com

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

机械性能和细胞外基质的组织强烈地影响细胞的三维迁移。 的体外方法来研究在生物物理变量的环境中，在人口和单个细胞水平的时空细胞迁移行为，进行说明。

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 ...

concentric-gel-system-to-study-biophysical-role-matrix

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

8.5K 次观看.  FOM Institute AMOLF. 机械性能和细胞外基质的组织强烈地影响细胞的三维迁移。 的体外方法来研究在生物物理变量的环境中，在人口和单个细胞水平的时空细胞迁移行为，进行说明。 

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

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

活细胞成像的移植细胞在一个三维矩阵表示荧光标记的蛋白质

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

科研

生物工程

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

研究微加工通道细胞迁移的

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

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 ...

Revealing the Cytoskeletal Organization of Invasive Cancer Cells in 3D

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration in more appropriate 3D environments, which better resemble the dimensionality of the physiological processes in question. Migratory cells can substantially differ in their morphology and mode of migration depending on whether they are moving on 2D or 3D substrates. Due to technical difficulties and incompatibilities with most standard protocols, structural and functional analysis of cells embedded within 3D matrices still remains uncommon. This article describes methods for preparation and imaging of 3D cancer cell cultures, either as single cells or spheroids. As an appropriate ECM substrate for cancer cell migration, we use nonpepsinized rat tail collagen I polymerized at room-temperature and fluorescently labeled to facilitate visualization using standard confocal microscopes. This work also includes a protocol for 3D immunofluorescent labeling of endogenous cell cytoskeleton. Using these protocols we hope to contribute to a better description of the molecular composition, localization, and functions of cellular structures in 3D.

This article presents a method to fluorescently label collagen that can be further used for both fix and live imaging of 3D cell cultures. We also provide an optimized protocol to visualize endogenous cytoskeletal proteins of cells cultured in 3D environments.

在3D揭示侵入性癌症细胞骨架组织

Cell migration has traditionally been studied in 2D substrates. However, it has become increasingly evident that there is a need to study cell migration ...

医学

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.

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 ...

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 ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

表征细胞迁移在三维体外伤口环境

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

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 ...

Quantifying Three-Dimensional Cell Migration Within and Into Granular Hydrogel Biomaterials

Granular hydrogel scaffolds hold significant potential in regenerative medicine, functioning either as carriers for cell delivery or as interfaces for tissue integration. This article introduces two novel approaches for quantifying cell migration within and into granular hydrogels, highlighting the distinct applications of these scaffolds. First, a cell monolayer interface assay that simulates tissue growth into granular hydrogels for integration purposes is presented. Second, a spheroid-based assay is described, designed to track cell movement within the hydrogel matrix, specifically suited for applications involving cell delivery. Both methods enable precise and controlled measurements of cell migration, providing a comprehensive toolkit for researchers utilizing granular hydrogel scaffolds. The motivation for these methods stems from the need for tailored control over cell migration within the scaffold to align with specific applications. By optimizing and standardizing these quantification techniques, researchers can iteratively refine granular hydrogel properties, ensuring their effectiveness in diverse regenerative medicine contexts. This robust set of quantitative tools offers new opportunities to enhance granular hydrogel scaffolds, advancing their use in both cell delivery and tissue integration applications.

A protocol for the quantitative evaluation of 3D cell migration within and into the interface of granular hydrogels is presented here.

量化颗粒水凝胶生物材料内部和颗粒状水凝胶生物材料中的三维细胞迁移

Granular hydrogel scaffolds hold significant potential in regenerative medicine, functioning either as carriers for cell delivery or as interfaces for ...

Preparation of a User-Defined Peptide Gel for Controlled 3D Culture Models of Cancer and Disease

There is a growing awareness that cells grown in 3D better model in vivo behavior than those grown in 2D. In this protocol, we describe a simple and tunable 3D hydrogel, suitable for culturing cells and tissue in a setting that matches their native environment. This is particularly important for researchers investigating the initiation, growth, and treatment of cancer where the interaction between cells and their local extracellular matrix is a fundamental part of the model. Moving to 3D culture can be challenging and is often associated with a lack of reproducibility due to high batch-to-batch variation in animal-derived 3D culture matrices. Similarly, handling issues can limit the usefulness of synthetic hydrogels. In response to this need, we have optimized a simple self-assembling peptide gel, to enable the culture of relevant cell line models of cancer and disease, as well as patient-derived tissue/cells. The gel itself is free from matrix components, apart from those added during encapsulation or deposited into the gel by the encapsulated cells. The mechanical properties of the hydrogels can also be altered independent of matrix addition. It, therefore, acts as a &#8216;blank slate&#8217; allowing researchers to build a 3D culture environment that reflects the target tissue of interest and to dissect the influences of mechanical forces and/or biochemical control of cell behavior independently.

We present a method for creating a 3D cell culture environment, which can be used to investigate the importance of cell/matrix interactions in cancer progression. Using a simple self-assembling octapeptide, the matrix surrounding encapsulated cells can be controlled, with independent regulation of mechanical and biochemical cues.

There is a growing awareness that cells grown in 3D better model in vivo behavior than those grown in 2D. In this protocol, we describe a simple and ...