Name: a customizable chamber for measuring cell migration
Uploaded: 2017-03-12T14:00:00.000+00:00
Duration: 7 min 33 s
Description: Watch this Scientific Journal Video about A Customizable Chamber for Measuring Cell Migration at JoVE.com

作者承认克莱姆森大学创意咨询程序和NSF CBET1254609为这个项目提供资金。

1，一种生产微通道室以产生浓度梯度<ol><li> 切割丙烯酸防霉片 <ol><li>获得一块所需宽度的丙烯酸。通常选用1/16英寸厚的压克力板材。较厚的片是难以切割好，非常薄的片材不具有必要的机械强度，使它们的过程中断裂或变形。 </li><li>创建与丙烯酸片的所需形状的CAD文件到聚二甲基硅氧烷（PDMS）内产生的空腔。调节通道的宽度和长度，以实现相关的个别实验方案所需的梯度。 <ol><li>在这里，用丙烯酸片尺寸如下:由0.127厘米宽的通道连接（0.254厘米长）两个正方形（0.254厘米点¯x0.254厘米）（ 图1）。 </li></ol></li><li>根据马努在激光切割机导入CAD文件到激光切割设备，然后将一块亚克力厂家的协议。 注:另外，3D打印从CAD设计作品。这种方法的优点是，替代材料，可用于在模具中;但是，分辨率和重复性，可以用3D打印相比，激光切割下降。 </li></ol></li></ol><img alt="图1" src="/files/ftp_upload/55264/55264fig1.jpg" /> 图1:微通道商会计算机辅助设计（CAD）表示。该图像描述了创建微室所需的亚克力插入的CAD图纸。 1 A）CAD出图，1B）CAD的侧视图英寸的尺寸图，1C）CAD的顶视图英寸的尺寸图的顶视图<a href="http://ecsource.jove.com/files/ftp_upload/55264/55264fig1large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <ol start="2"><l我&gt; 浇注聚二甲基硅氧烷（PDMS） <ol><li>在一个称量船（重量）商业弹性体基的（ 如的Sylgard 184）与1重量份的商业橡胶固化剂（ 如的Sylgard 194）的混合10份。通常情况下，混合10克橡胶基地到1g弹性体固化剂。 </li><li>搅用微量5分钟的基料和固化剂相结合。 </li><li>设立一个真空钟，并将与PDMS中称量船在真空30分钟以除去截留的空气气泡。 </li><li>关闭真空并取出称重舟。倒入一个小培养皿丙烯酸切出顶部的PDMS。确保PDMS完全覆盖刀片。 </li><li>允许PDMS固化过夜（至少18小时）在室温下。 </li></ol></li><li> 去除PDMS插入 <ol><li>固化的PDMS后，通过小心地切割PDMS周围的丙烯酸系片用手术刀取下刀片。 </li></ol&gt; </li></ol> 2.电镀细胞在微通道商会<ol><li> 灭菌电镀细胞室中。 注意:虽然PDMS可使用环氧乙烷或其他技术进行灭菌，快速UV处理是快速，廉价，并且足以用于细胞培养研究。短UV处理时间也没有损害的PDMS片的结构或机械完整性。 <ol><li>在标准细胞培养柜，完全覆盖，用70％的乙醇的腔室。 </li><li>把与所述盖子关闭层流罩的培养皿。 </li><li>关闭引擎盖打开紫外线灯。暴露于UV光1-2小时。 </li><li>打开层流罩，并等待15分钟重新建立流动。 </li><li>除去70％的乙醇。 </li><li>用无菌磷酸盐缓冲盐水（PBS）洗涤腔室的两倍。使用1毫升的PBS每次洗涤。除去PBS并允许室与盖子关干通宵。 注:此外，相反使用层流罩中，使用的UV腔室箱灭菌如果有一个可用。通过将UV光源在一个纸箱用铝箔衬里的背面使紫外线框。铝箔反射光，以允许甚至照明样品。 </li></ol></li><li> 在室内电镀细胞 <ol><li>使用计数台盼蓝和血球细胞。 <ol><li>加入100μL细胞悬液400微升0.4％台盼蓝，轻轻混匀。由玻璃盖玻片下轻轻填充两院应用100微升此解决方案血细胞计数仪。 </li><li>使用显微镜用10倍的目标集中在血球电网。用一只手式计数器，以一组的血球16平方内计数活的未染色细胞。 </li><li>在全体4套16平方的细胞。从4台16平方取平均细胞计数和由5×10 4繁殖。这个最后的数字是numbe在细胞悬浮液中细胞/ mL的河</li></ol></li><li>添加2500细胞一个孔在每个室中。这转化为的〜30,000 / cm 2的近汇合的细胞密度。 </li><li>允许细胞在腔室坐60分钟添加介质之前（Dulbecco氏改良的Eagle氏培养基，10％胎牛血清，1％青霉素 - 链霉素）。 </li></ol></li></ol> 3.浸泡葡聚糖琼脂糖块<ol><li> 创建琼脂糖块 <ol><li>琼脂糖倒3％到3D印刷模具（2毫米×2毫米×2mm）的。 </li><li>允许琼脂糖在真空室内固化30分钟，以除去任何气泡。 </li><li>除去从模具琼脂糖块。 注意:可替换地，琼脂糖倒3％成小培养皿在厚度为2毫米。切使用手术刀或者其它切割工具2毫米×2mm的×2mm的块。这不是作为模具系统那样精确，但是它可以更容易一点做。 </li></ol></li><li>完全submergE在趋化因子的所需浓度的溶液琼脂糖块。为了评估浓度梯度的形成，荧光右旋糖酐第一冲洗室。葡聚糖的浓度和分子量应匹配的可溶性因子或感兴趣的药物。 </li><li>浸泡琼脂糖块过夜。 </li></ol>葡聚糖扩散4.时间推移，评估可溶性因子浓度梯度<ol><li>将葡聚糖浸泡琼脂糖块在微室的小方井。 </li><li>放置所述微通道室中的细胞成像系统，或使用其他荧光显微镜与时间推移的能力。根据制造商的说明开始的时间推移。 注:显示的结果采取GFP过滤器，50％的光，pH值4/10和放大4倍。 </li></ol>细胞生长5.定时<ol><li>板中的细胞微通道室中的一端室。在这里，使用3T3成纤维细胞。添加2500细胞一个孔在所述微通道室中。在2.2.1中描述计数细胞。 <ol><li>允许细胞在腔室坐60分钟添加介质之前（Dulbecco氏改良的Eagle氏培养基，10％胎牛血清，1％青霉素 - 链霉素）。保持与铺板的细胞在微室中于37℃在5％CO 2。通过信道细胞迁移可以用显微镜观察。 </li><li>添加标记或使用在腔室作为标记的显微缺陷，以作为一个起点，以量化的距离的小区前移动。 </li></ol></li><li>通过将琼脂糖凝胶（8 mm 3的块）包含建立梯度浓缩生长因子，趋化引诱剂，药物，或者被测试其他因子（这里，40％的FBS被用作一个例子）在所述微通道的一端室。 </li><li>就拿移动单元前的时间推移图像。在此，结果表明，该细胞的图像来回核苷酸已采取使用成像系统每12小时。 </li><li>使用电池前的照片来量化细胞的生长速度。计算由第一利用MATLAB polyfit功能（roipoly）来创建由细胞前，通道壁，和参考标记限定的多边形掩模的增长率。该掩模的尺寸，得到由此计算平均电池锋速度前的迁移距离。其他研究已经利用类似的方法8。 注:在该相同的距离进行比较的细胞生长前面各帧的可溶因子的浓度上的边缘。可溶性因子的浓度梯度是使用一维扩散模型近似计算。 </li><li>采取用鬼笔环肽和DAPI染色的细胞的荧光图像。 <ol><li>与鬼笔环肽染色之前修复细胞。 <ol><li>在37℃的温水4％多聚甲醛。 </li><li>补充足够的4％多聚甲醛覆盖细胞。保持在细胞中多聚甲醛10分钟。 </li><li>用PBS冲洗两次，每次15分钟。使用足够的PBS覆盖细胞。 </li></ol></li><li>从细胞中除去PBS中并添加足够的透化溶液（0.1％的Triton X-100的PBS），以覆盖细胞。离开溶液10分钟。 </li><li>用PBS洗两次，每次5分钟。使用足够的PBS覆盖细胞。 </li><li>从细胞中除去PBS中并添加20分钟阻断溶液（在PBS中1％牛血清白蛋白）。 </li><li>除去阻断溶液并用PBS洗涤2次，每次5分钟。使用足够的PBS覆盖细胞。 </li><li>从这点来说，在不使用时保护细胞免受光用铝箔。从细胞中除去PBS中并在PBS中添加鬼笔环肽488 1:50稀释1小时。 </li><li>洗2次在PBS中5分钟，每次洗涤。使用足够的PBS覆盖细胞。然后从细胞中移除PBS。 </li><li>在PBS 1000细胞15分钟:加入DAPI（4&#39;，6-二脒基-2-苯基）稀释1。 </li><li>删除和DAPI洗涤细胞两次，用PBS进行5分钟，每次洗涤。 </li><li>删除最后一次洗涤和补充足够的PBS覆盖细胞。细胞现在可以用荧光显微镜成像。 </li></ol></li></ol>

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我们的微腔可用于目的，包括确定细胞迁移速率响应于生长因子和趋化因子以及从聚合物基质测定药物的扩散速率的多种。它可以利用我们的微腔室生长细胞并放置一个趋化在腔室的一端。细胞生长响应于趋化和细胞迁移可以通过取可在为了确定细胞迁移率分析的时间推移的图像进行量化。这种方法的代表性结果示于图3，其中被确定的增长率是使用MATLAB polyfit函数13.4微米/小时。 <...

In order for vital processes such as wound healing, immune responses, and embryonic development to occur, cell migration must take place. Cell migration involves the interaction between cells and neighboring cells, the extracellular matrix, and soluble chemical cues (attractants or repellants). As an example, in the process of wound healing, fibroblasts play an integral role in fibrogenesis and wound contraction, where the cells are recruited to the site of injury to synthesize collagen in order to form the extracellular matrix3. Numerous mechanisms behind the migration of fibroblasts to a wound site have been studied, and they include different mechanical, physical, electrical, and chemotactic factors4. Fibroblasts respond especially well to different concentration gradients of growth factors. These different growth factors work together to optimize tissue regeneration5. While observing the chemotactic response of fibroblasts to growth factor concentrations, one can study the pattern of directional migration of fibroblasts and how they orient themselves around physical obstacles in order to reach their destination. Therefore, the goals of this study were to first develop a system in which fibroblast growth could be tracked under guidance by physical barriers and to secondly model the growth of fibroblasts as they navigate through the system.
Currently, the Boyden chamber assay is the most widely used system to measure the migration of cells6. The Boyden chamber consists of a two-chamber multi-well plate where each well may contain medium with or without chemoattractants7. A filter membrane provides a porous interface between the two chambers in each well; this creates a barrier so that cells cannot pass through unless it is by active migration. Typically, for the Boyden chamber, a chemoattractant is added to the lower chamber, and the system is allowed to equilibrate to form a gradient between the upper and lower wells4. One problem with the Boyden chamber assay is that steep gradients end up forming along a single axis perpendicular with the surface of the membrane. This causes the difference in the chemoattractant concentration between the upper and lower wells to be a lower than what was originally expected. Due to this constraint, the Boyden chamber assay makes it hard to correlate specific cell responses with particular gradient characteristics, such as the slope and the concentration difference. Without these measurements, it is hard to study multi-gradient signal integration.
To address some of the constraints of the traditional Boyden chamber assay, microfluidic assays have been developed to form customizable concentration gradients1. Standard methods for creating microfluidic systems require clean rooms for lithographic techniques. These techniques can be difficult to learn especially in a standard classroom setting. Thus, we have designed a chamber system for measuring cell migration that can be made without using a clean room. Using our system, the wells of the assay can be adjusted to a preferred size, and a linear concentration gradient of customized slope can be produced. This allows for accurate measurement of chemotaxis from random movement. The design is an inexpensive and easy-to-use system to model cell growth in response to different chemical stimuli.

<table><tbody><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Sigma-Aldrich</td><td>761036</td><td>poly(dimethylsiloxane) 2-part kit including silicone elastomer base and silicone elastomer curing agent</td></tr><tr><td>Acrylic Sheets</td><td>US Plastic</td><td>44200</td><td></td></tr><tr><td>Disposable Petri Dishes&amp;nbsp;</td><td>Falcon&amp;nbsp;</td><td>25373-041</td><td></td></tr><tr><td>Fluorescein isothiocyanate&amp;ndash;dextran</td><td>Sigma-Aldrich</td><td>FD20s-100MG</td><td></td></tr><tr><td>Agarose, Type I, Low EEO</td><td>Sigma-Aldrich</td><td>A6013-100G</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium</td><td>Fisher Scientific</td><td>11965092</td><td>Cell media components</td></tr><tr><td>Fetal Bovine Serium</td><td>Fisher Scientific</td><td>16000036</td><td>Cell media components</td></tr><tr><td>Penicillin-streptomycin</td><td>Fisher Scientific</td><td>15140148</td><td>Cell media components</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Fisher Scientific</td><td>BP24384</td><td></td></tr><tr><td>EVOS XL Cell Imaging System</td><td>Thermo Fisher Scientific</td><td>AME3300</td><td>Instrument used for taking time-lapse images</td></tr><tr><td>Versa Laser</td><td>Universal Laser Systems, Inc.&amp;nbsp;</td><td>Model number VLS2.30</td><td>Laser cutter used for cutting plastic</td></tr></tbody></table>

a customizable chamber for measuring cell migration

细胞迁移是免疫反应，生长和伤口愈合的重要组成部分。细胞迁移是一个复杂的过程，涉及到细胞，细胞外基质，和可溶的和不可溶的化学因素（ 例如 ，化学引诱物）之间的相互作用。标准方法测量细胞，如Boyden小室测定法中，通过在除法器的任一侧细胞计数工作的迁移。这些技术很容易使用;然而，他们提供针对不同应用的小几何修改。与此相反，微流体装置可用于观察与可溶性因子1,2定制浓度梯度细胞迁移。然而，用于制造基于微流体分析的方法是很困难的学习。 这里，我们描述用于创建细胞培养室，测量响应于化学浓度梯度细胞迁移的简单方法。我们的细胞英里格雷申室方法可以为了研究细胞迁移对于各种应用创建不同的线性浓度梯度。该方法是相对容易使用，并且通常由大学生进行。 所述微通道室通过将在最后的微腔的形状的丙烯酸插入井到培养皿创建。在此之后，聚（二甲基硅氧烷）（PDMS）倒在刀片的顶部。将PDMS被允许硬化，然后除去插入件。这允许以任何所需的形状或尺寸的创作孔中。细胞可以随后添加到微室中，和可溶性剂可以通过在所需的剂浸泡琼脂糖块被加入到孔中的一个。琼脂糖块加入到孔中的一个，和时间推移的图片可以量化细胞迁移可采取微通道室。变化，以这种方法可以用于给定的应用进行，使得这种方法HIG溶血素可定制的。

图2示出横跨通道的细胞前的响应于放置在信道的从细胞接种，其中相对端胎牛血清的梯度的运动。小区前示出在48小时（ 图2A），72小时（ 图2B），96小时（ 图2C），和120小时（ 图2D）交电镀。小区前的运动用这些时间推移图象跟踪，和迁移距离和生长率分别由MATLAB polyfit函数计算。由此看来，平均气泡锋速?...

Watch this Scientific Journal Video about A Customizable Chamber for Measuring Cell Migration at JoVE.com

A Customizable Chamber for Measuring Cell Migration

该协议细节来测量响应于也可用于确定药物的扩散速度出聚合物基质的化学引诱物细胞迁移可定制方法。

定制室测定细胞迁移

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between ...

a-customizable-chamber-for-measuring-cell-migration

Research

JoVE Journal

Biology

10.5K 次观看.  Clemson University. 该协议细节来测量响应于也可用于确定药物的扩散速度出聚合物基质的化学引诱物细胞迁移可定制方法。 

视频: A Customizable Chamber for Measuring Cell Migration

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels &#8211; either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention.
We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

内皮细胞迁移评估暴露于有毒化学品后，

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

科研

发育生物学

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an &#34;in-incubator&#34; video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

细胞侵袭和迁移过程的评价: 视频 Microscope-based 刮伤法与博伊登室法的比较

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

癌症研究

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

癌症干细胞迁移的使用进行划分的微流控芯片和活细胞成像的评价

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

医学

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

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 ...

生物工程

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

使用自动活单元成像仪的简单迁移/入侵工作流

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

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 ...

Real-Time Quantitative Measurement of Tumor Cell Migration and Invasion Following Synthetic mRNA Transfection

Tumor cells are highly motile and invasive and display altered gene expression patterns. Knowledge of how changes in gene expression regulate tumor cell migration and invasion is essential for understanding the mechanisms of tumor cell infiltration into neighboring healthy tissues and metastasis. Previously, it was demonstrated that gene knockdown followed by the impedance-based real-time measurement of tumor cell migration and invasion enables the identification of the genes required for tumor cell migration and invasion. Recently, the mRNA vaccines against SARS-CoV-2 have increased interest in using synthetic mRNA for therapeutic purposes. Here, the method using synthetic mRNA was revised to study the effect of gene overexpression on tumor cell migration and invasion. This study demonstrates that elevated gene expression with synthetic mRNA transfection followed by impedance-based real-time measurement may help identify the genes that stimulate tumor cell migration and invasion. This method paper provides important details on the procedures for examining the effect of altered gene expression on tumor cell migration and invasion.

Many upregulated genes stimulate tumor cell migration and invasion, leading to poor prognosis. Determining which genes regulate tumor cell migration and invasion is critical. This protocol presents a method for investigating the effects of the increased expression of a gene on the migration and invasion of tumor cells in real time.

合成mRNA转染后肿瘤细胞迁移和侵袭的实时定量测量

Tumor cells are highly motile and invasive and display altered gene expression patterns. Knowledge of how changes in gene expression regulate tumor cell ...

Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells

The physiological electric field serves specific biological functions, such as directing cell migration in embryo development, neuronal outgrowth and epithelial wound healing. Applying a direct current electric field to cultured cells in vitro induces directional cell migration, or galvanotaxis. The 2-dimensional galvanotaxis method we demonstrate here is modified with custom-made poly(vinyl chloride) (PVC) chambers, glass surface, platinum electrodes and the use of a motorized stage on which the cells are imaged. The PVC chambers and platinum electrodes exhibit low cytotoxicity and are affordable and re-useable. The glass surface and the motorized microscope stage improve quality of images and allow possible modifications to the glass surface and treatments to the cells. We filmed the galvanotaxis of two non-tumorigenic, SV40-immortalized prostate cell lines, pRNS-1-1 and PNT2. These two cell lines show similar migration speeds and both migrate toward the cathode, but they do show a different degree of directionality in galvanotaxis. The results obtained via this protocol suggest that the pRNS-1-1 and the PNT2 cell lines may have different intrinsic features that govern their directional migratory responses.

We present a method to apply a physiological electric field to migrating, immortalized prostate cells in a custom-made galvanotaxis chamber. Using this method, we demonstrate that 2 lines of non-tumorigenic prostate cells demonstrate different degrees of migration directionality in the field.

利用定制设计的趋电钱伯斯研究前列腺细胞定向迁移

The physiological electric field serves specific biological functions, such as directing cell migration in embryo development, neuronal outgrowth and ...

生物学

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 ...