Name: a device for performing cell migration/wound healing in a 96-well plate
Uploaded: 2017-03-07T15:00:00.000+00:00
Duration: 5 min 46 s
Description: Watch this Scientific Journal Video about A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate at JoVE.com

笔者想感谢谭宝梁先生，王梓健先生和香港浸会大学理学院车间的技术人员，对他们的技术技能和建议，使这一wounder的雏形。

1.简介Wounder的部件（图1） <ol><li>通过在相等的距离固定伤人引脚准备针座。握住枪头，并上下移动调节伤人引脚调节尖端的高度。 </li><li>适合于不同品牌的96孔培养板中的可调节的引导杆，并确保该受伤区域是在一个固定的位置上。 </li><li>通过拧紧六角螺丝固定可调伤人引脚。通过拧紧两端的六角帽固定在适当位置的指导吧。 </li></ol> 2.设置销座的宽度（图2） <ol><li>松开六角帽用M5的内六角扳手。 </li><li>插入调节环到销保持器的两侧的一适当数，使得导向杆与96孔培养板的宽度伏贴。 注:康宁和磐城板块需要1对大环和1对小环。猎鹰和Nunc公司PL阿泰需要1对大环。 </li><li>拧紧六角帽固定的指导吧。 </li></ol> 3.调节导杆（图3） <ol><li>适合用无菌的10微升塑料吸管提示伤人引脚。 </li><li>松开六角帽用M5的内六角扳手。 </li><li>握住wounder和浸伤人针到96孔培养板的一列。 </li><li>调节导向杆的高度，直到所有的尖端刚好触及孔的底部。 </li><li>拧紧六角帽。确保wounder现在完全适合于培养板中，该销良好定位在孔的中间。 </li></ol> 4.引脚的校准<ol><li>按住wounder垂直于平坦的表面消毒（ 如培养皿），松开所有的M3六角扳手六角螺丝。 </li><li>点击wounder，直到所有的提示均匀地接触到平坦表面消毒。&lt;/ LI&gt; <li>再次拧紧六角螺钉位置锁定引脚。 </li><li>通过轻轻拍打wounder平无菌表面上检查每个尖端的均匀性。 </li></ol> 5.划痕细胞单层（图4） <ol><li>种子人脐静脉内皮细胞上的0.1％明胶包被的96孔细胞培养板。在199培养基培养细胞补充有20％热灭活的胎牛血清，1％青霉素/链霉素和0.09克/升的肝素。在使用前过夜保持在37ºC用5％二氧化碳的潮湿培养箱中的细胞。细胞伤人前应达到100％汇合。 </li><li>放置伤人提示在最左边的（或最右边的）培养板的同一列内的每个孔一侧。对面井的另一侧滑动wounder;确保伤人尖端接触井的底部。 </li><li>重复所有列的划伤（步骤5.2）。 </li><li>伤人后，丢弃Ť他中等每口井和更换新鲜含中测试化合物。 </li></ol> 6.数据采集和图像分析<ol><li>整个井与使用具有数码相机（ 例如 ，10X物镜）的低功率显微镜细胞单层的机械伤口后图像立即伤人（t = 0的小时）。 </li><li>添加试验化合物，如果需要的孵育时间的期望的长度。图像的整个井与所需孵育时间（t =ΔH）后伤口再次区域。 </li><li>使用图像分析软件，如ImageJ的（ <a href="https://imagej.nih.gov/ij/">https://imagej.nih.gov/ij/</a> ），测量使用手绘工具在图像上的伤员区域。 </li><li>计算伤口闭合的百分比: <img alt="方程" src="/files/ftp_upload/55411/55411eq1.jpg" /></li></ol>

<ol>
	<li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nat Rev Cancer. 3 (5), 362-374 (2003).</li><li>Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prespecification+and+plasticity:+shifting+mechanisms+of+cell+migration.">Prespecification and plasticity: shifting mechanisms of cell migration.</a> Curr Opin Cell Biol. 16 (1), 14-23 (2004).</li><li>Lampugnani, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+into+a+wounded+area+in+vitro.">Cell migration into a wounded area in vitro.</a> Methods Mol Biol. 96, 177-182 (1999).</li><li>Sholley, M. M., Gimbrone, M. A., Cotran, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+migration+and+replication+in+endothelial+regeneration:+a+study+using+irradiated+endothelial+cultures.">Cellular migration and replication in endothelial regeneration: a study using irradiated endothelial cultures.</a> Lab Invest. 36 (1), 18-25 (1977).</li><li>Gotlieb, A. I., Spector, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+into+an+in+vitro+experimental+wound:+a+comparison+of+porcine+aortic+endothelial+and+smooth+muscle+cells+and+the+effect+of+culture+irradiation.">Migration into an in vitro experimental wound: a comparison of porcine aortic endothelial and smooth muscle cells and the effect of culture irradiation.</a> Am J Pathol. 103 (2), 271-282 (1981).</li><li>Chen, Y. C., 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=Single-cell+migration+chip+for+chemotaxis-based+microfluidic+selection+of+heterogeneous+cell+populations.">Single-cell migration chip for chemotaxis-based microfluidic selection of heterogeneous cell populations.</a> Sci Rep. 18 (5), 9980 (2015).</li><li>Yarrow, J. C., Periman, Z. E., Westwood, N. J., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+cell+migration+assay+using+scratch+wound+healing,+a+comparsion+of+image-based+readout+methods.">A high-throughput cell migration assay using scratch wound healing, a comparsion of image-based readout methods.</a> BMC Biotechnol. 4, 21 (2004).</li><li>Lauder, H., Frost, E. E., Hiley, C. R., Fan, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+repair+process+involved+in+the+repair+of+a+cell+monolayer+using+an+in+vitro+model+of+mechanical+injury.">Quantification of the repair process involved in the repair of a cell monolayer using an in vitro model of mechanical injury.</a> Angiogenesis. 2 (1), 67-80 (1998).</li><li>Yue, P. Y. K., Leung, E. P. Y., Mak, N. K., Wong, R. N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+method+for+quantifying+cell+migration/+wound+healing+in+96-well+plates.">A simplified method for quantifying cell migration/ wound healing in 96-well plates.</a> J. Biomol Screen. 15 (4), 427-433 (2010).</li><li>Yue, P. Y., 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=Elucidation+of+the+mechanisms+underlying+the+angiogenic+effects+of+ginsenoside+Rg(1)+in+vivo+and+in+vitro.">Elucidation of the mechanisms underlying the angiogenic effects of ginsenoside Rg(1) in vivo and in vitro.</a> Angiogenesis. 8 (3), 205-216 (2005).</li><li>Kwok, H. H., Chan, L. S., Poon, P. Y., Yue, P. Y., Wong, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ginsenoside-Rg1+induces+angiogenesis+by+the+inverse+regulation+of+MET+tyrosine+kinase+receptor+expression+through+miR-23a.">Ginsenoside-Rg1 induces angiogenesis by the inverse regulation of MET tyrosine kinase receptor expression through miR-23a.</a> Toxicol Appl Pharmacol. 287 (3), 276-283 (2015).</li></ol>

作者宣称，他们没有竞争的经济利益。

我们的细胞wounder在解决传统的细胞迁移实验的问题，几个独特的功能。 8通道机械细胞wounder与可以通过热压灭菌寿命长采用优质不锈钢（304）的。几乎所有的商业品牌的市场上可用的96孔培养板可与该机械细胞wounder使用，因为可调节导向杆的。可调导向杆的设计也保证了划痕区域是在每个的中心孔。在井的中心位置上的伤口可以使用显微镜促进图像捕获。图像具有一些变型中，而在不同的时间点...

细胞迁移在细胞过程，如胚胎发生，神经发生，血管生成，伤口愈合，粘膜修复，在正常发育和疾病状况1上皮-间质跃迁中起重要作用。它是一个复杂的过程，涉及大量的间和细胞内的事件，包括信号分子相互作用，细胞极化，细胞骨架重组，基质重塑，膜突起和动态细胞-细胞粘连调制2的协调。通过研究细胞迁移，化学物质或生物分子导致细胞的运动和它的相关的生物化学途径的发现和验证可以被确定。这一基本过程可以有利地用作用于各种应用，如疾病的治疗和药物靶向的代理。 的伤人测定是细胞迁移测定法3中的一个。这里，一个细胞的个体的样品将通过机械装置被部分地去除，以产生一个裸露区，让细胞将迁移以覆盖面积。回收的在特定时间点的百分比将被监测。当处理大量的样品，如样品中实验成本和交叉污染的问题可能会出现问题。虽然许多商业工具和测定法可用于研究细胞迁移4，5，6，其中许多是需要昂贵和复杂的设备和仍需要改进。由于这些原因，一个8通道机械细胞wounder（ 图1）显影。 我们的8通道机械细胞wounder在解决上述问题已经将几个独特的功能。它提供了执行细胞迁移/在96孔培养板格式伤人测定的灵活性。可调区iding杆设计保证了刮开区是在每个中心位置很好。另外，导向杆的高度可以调整，使得wounder适用于各种品牌培养板。最后，可调伤人针设计能够与培养板表面实现同步和可重复伤人7,8的连枪头接触。

<table><tbody><tr><td>96-well cell culture plate</td><td>Nunc</td><td>167008</td><td>Other brands of 96-well cell culture plate can also be used</td></tr><tr><td>P10 pipette tips</td><td>Axygen</td><td>301-03-051</td><td>Short P10 pipette tip is more easy to create a clear wound</td></tr><tr><td>Wounder</td><td>R&amp;amp;P Technology Limited</td><td></td><td></td></tr><tr><td>Medium 199</td><td>Sigma</td><td>M2520-1L</td><td>For cell culture of human umbilical vein endothelial cells.&lt;br/&gt; Use appropirate culture medium and condition for other type of cells.</td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>26140079</td><td>For cell culture of human umbilical vein endothelial cells.&lt;br/&gt; Use appropirate culture medium and condition for other type of cells.</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140122</td><td>For cell culture of human umbilical vein endothelial cells.&lt;br/&gt; Use appropirate culture medium and condition for other type of cells.</td></tr><tr><td>Heparin sodium salt from porcine intestinal mucosa</td><td>Sigma</td><td>H3393</td><td>For cell culture of human umbilical vein endothelial cells.&lt;br/&gt; Use appropirate culture medium and condition for other type of cells.</td></tr><tr><td>Gelatin from bovine skin</td><td>Sigma</td><td>G9391</td><td>For cell culture of human umbilical vein endothelial cells.&lt;br/&gt; Use appropirate culture medium and condition for other type of cells.</td></tr></tbody></table>

a device for performing cell migration/wound healing in a 96-well plate

细胞迁移/伤人测定法是一种常用的方法来研究细胞迁移和其它生物过程，如血管生成和肿瘤转移。在该试验中，细胞生长，以形成汇合单层，并通过与一个设备刮擦创建的机械伤口。然后朝裸露区域的细胞的迁移速率可以通过成像来监测。我们的8通道机械wounder旨在解决大部分与细胞迁移测定相关的问题。首先，我们的wounder可以容易地通过高压灭菌或与常用消毒剂消毒。其次，个人可调节销允许甚至与细胞培养板，使得可以创建锐利和可再现的伤口接触。第三，关于wounder两侧的导杆确保在每一致伤人位置很好。为伤人还可以提供更好的处理wounder的以及以最小化交叉铅污染的使用一次性塑料吸头离子。总之，我们的细胞wounder可以使用标准的96孔培养板进行细胞迁移测定法提供与用户友好和可重现装置的研究人员。

该8通道机械wounder被构造以执行小区迁移测定划伤的细胞单层。这是一个用户友好的设备，可以在不到一分钟进行96孔板细胞划伤实验无需特殊培训。此wounder可以在细胞单层引入伤口面积为约600微米的均匀的宽度，并与锋利的边缘（ 图5和6）。伤口的宽度取决于品牌枪头的和用户的性能。伤口宽度的范围适用于普通图像的低功率放大下捕获（ <stron...

Watch this Scientific Journal Video about A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate at JoVE.com

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

在这里，我们提出了一个协议，以在96孔细胞培养板进行半高通量细胞迁移测定。该协议是建立在一个细胞单层一致划伤伤口快速，简便，经济的方法。

一种在96孔板表演细胞迁移/伤口愈合设备

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

a-device-for-performing-cell-migrationwound-healing-in-a-96-well-plate

Research

JoVE Journal

Developmental Biology

10.4K 次观看.  Hong Kong Baptist University. 在这里，我们提出了一个协议，以在96孔细胞培养板进行半高通量细胞迁移测定。该协议是建立在一个细胞单层一致划伤伤口快速，简便，经济的方法。 

视频: A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

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 ...

科研

癌症研究

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of cyclic nucleotides. By modulating intracellular cyclic nucleotide concentration, MRP4 can regulate multiple cyclic nucleotide-dependent cellular events including cell migration. Previously, we demonstrated that in the absence of MRP4, fibroblast cells contain higher levels of intracellular cyclic nucleotides and can migrate faster. To understand the underlying mechanisms of this finding, we adopted a direct yet multifaceted approach. First, we isolated potential interacting protein complexes of MRP4 from a MRP4 over-expression cell system using immunoprecipitation followed by mass-spectrometry. After identifying unique proteins in the MRP4 interactome, we utilized Ingenuity Pathway Analysis (IPA) to explore the role of these protein-protein interactions in the context of signal transduction. We elucidated the potential role of the MRP4 protein complex in cell migration and identified F-actin as a major mediator of the effect of MRP4 on cell migration. This study also emphasized the role of cAMP and cGMP as key players in the migratory phenomena. Using high-content microscopy, we performed cell-migration assays and observed that the effect of MRP4 on fibroblast migration is completely abolished by disruption of the actin cytoskeleton or inhibition of cAMP-dependent kinase A (PKA). To visualize signaling modulations in a migrating cell in real time, we utilized a FRET-based sensor for measuring PKA activity and found, the presence of more polarized PKA activity near the leading edge of migrating Mrp4-/- fibroblast, compared to Mrp4+/+fibroblasts. This in turn increased cortical actin formation and augmented the process of migration. Our approach enables identification of the proteins acting downstream to MRP4 and provides us with an overview of the mechanism involved in MRP4-dependent regulation of fibroblast migration.

MRP4 regulates various cyclic nucleotide-dependent signaling events including a recently elucidated role in cell migration. We describe a direct, but multifaceted approach to unravel the downstream molecular targets of MRP4 resulting in identification of a unique MRP4 interactome that plays key roles in the fine-tuned regulation of fibroblast migration.

方法在成纤维细胞迁移的规律研究大分子复合物含MRP4，

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of ...

发育生物学

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 ...

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 ...

生物工程

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

一种新的体外创面愈合试验评价细胞迁移

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

免疫与感染

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

以显微镜为基础的方法评估上皮细胞迁移过程中的体外创面愈合

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

医学

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

在体外细胞迁移和侵袭实验

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

生物学

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

划痕迁移分析和背皮折叠室的体外和在Vivo分析伤口愈合

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

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 ...

An Innovative 3D-Printed Insert Designed to Enable Straightforward 2D and 3D Cell Cultures

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of conditioned media remains time-consuming and far from physiological and pathological conditions. Although a few models of co-culture are commercially available, they remain restricted to specific assays and are mostly for two types of cells. 
Here, 3D-printed inserts are used that are compatible with numerous functional assays. The insert allows the separation of one well of a 6-well plate into four compartments. A wide range of combinations can be set. Moreover, windows are designed in each wall of the compartments so that potential intercellular communication between every compartment is possible in the culture medium in a volume-dependent manner. For example, paracrine intercellular communication can be studied between four cell types in monolayer, in 3D (spheroids), or by combining both. In addition, a mix of different cell types can be seeded in the same compartment in 2D or 3D (organoids) format. The absence of a bottom in the 3D-printed inserts allows the usual culture conditions on the plate, possible coating on the plate containing the insert, and direct visualization by optical microscopy. The multiple compartments provide the possibility to collect different cell types independently or to use, in each compartment, different reagents for RNA or protein extraction. In this study, a detailed methodology is provided to use the new 3D-printed insert as a co-culture system. To demonstrate several capacities of this flexible and simple model, previously published functional assays of cell communication were performed in the new 3D-printed inserts and were demonstrated to be reproducible. The 3D-printed inserts and the conventional cell culture using conditioned media led to similar results. In conclusion, the 3D-printed insert is a simple device that can be adapted to numerous models of co-cultures with adherent cell types.

In this paper, a newly designed 3D-printed insert is presented as a model of co-culture and validated through the study of the paracrine intercellular communication between endothelial cells and keratinocytes.

创新的 3D 打印插入物，旨在实现简单的 2D 和 3D 细胞培养

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of ...