Name: study of cell migration in microfabricated channels
Uploaded: 2014-02-21T14:00:00
Description: Watch this Scientific Journal Video about Study of Cell Migration in Microfabricated Channels at JoVE.com

作者大大承认PICT IBiSA平台在居里研究所（CNRS UMR144）。这项工作是由来自补贴：欧洲研究理事会AM.LD（Strapacemi 243103），该协会国立倒拉RECHERCHE（ANR-09-PIRI-0027-PCVI），该InnaBiosanté基金会（Micemico）到MP和PM。 LD和紧急救济协调员Strapacemi年轻的研究者授予AM.LD.

重要提示：本协议假定已经被制成含形状所需的微模具。在模具的制备方法的进一步信息已经被发表10。该协议还假定骨髓DCs培养是已知的。 1。芯片制造<ol><li>混合的PDMS油和固化剂在塑料杯中的重量比为10:1。调匀两种化合物。 </li><li>投组合在模具轴承微通道。的总高度应该为0.5-1厘米之间。 </li><li>中1小时，在真空罐钟除去气泡。 </li><li>在模具中硬?...

<ol>
	<li>Lauffenburger, D. A., Horwitz, A. F. <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:+a+physically+integrated+molecular+process.">Cell migration: a physically integrated molecular process.</a> Cell. 84 (3), 359-369 (1996).</li><li>L&#228;mmermann, T., 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=Rapid+leukocyte+migration+by+integrin-independent+flowing+and+squeezing.">Rapid leukocyte migration by integrin-independent flowing and squeezing.</a> Nature. 453 (7191), 51-55 (2008).</li><li>Schor, S. L., Allen, T. D., Harrison, C. J. <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+through+three-dimensional+gels+of+native+collagen+fibres:+collagenolytic+activity+is+not+required+for+the+migration+of+two+permanent+cell+lines.">Cell migration through three-dimensional gels of native collagen fibres: collagenolytic activity is not required for the migration of two permanent cell lines.</a> J. Cell. Sci. 41, 159-175 (1980).</li><li>Steinman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decisions+about+dendritic+cells:+past,+present,+and+future.">Decisions about dendritic cells: past, present, and future.</a> Annu. Rev. Immunol. 30, 1-22 (2012).</li><li>Faure-Andr&#233;, G., 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=Regulation+of+dendritic+cell+migration+by+CD74,+the+MHC+class+II-associated+invariant+chain.">Regulation of dendritic cell migration by CD74, the MHC class II-associated invariant chain.</a> Science. 322 (5908), 1705-1710 (2008).</li><li>Renkawitz, J., 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=Adaptive+force+transmission+in+amoeboid+cell+migration.">Adaptive force transmission in amoeboid cell migration.</a> Nat. Cell Biol. 11 (12), 1438-1443 (2008).</li><li>Jacobelli, J., 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=Confinement-optimized+three-dimensional+T+cell+amoeboid+motility+is+modulated+via+myosin+IIA-regulated+adhesions.">Confinement-optimized three-dimensional T cell amoeboid motility is modulated via myosin IIA-regulated adhesions.</a> Nat. Immunol. 11 (10), 953-961 (2010).</li><li>Irimia, D., Charras, G., Agrawal, N., Mitchinson, T., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polar+stimulation+and+constrained+cell+migration+in+microfluidic+channels.">Polar stimulation and constrained cell migration in microfluidic channels.</a> Lab Chip. 7 (12), 1783-1790 (2007).</li><li>Moreau, H., 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=Dynamic+in+situ+cytometry+uncovers+T+cell+receptor+signaling+during+immunological+synapses+and+kinapses+in+vivo.">Dynamic in situ cytometry uncovers T cell receptor signaling during immunological synapses and kinapses in vivo.</a> Immunity. 37 (2), 351-363 (2012).</li><li>Heuz&#233;, M. L., Collin, O., Terriac, E., Lennon-Dum&#233;nil, A. M., Piel, M. <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+in+confinement:+a+micro-channel-based+assay.">Cell migration in confinement: a micro-channel-based assay.</a> Methods Mol. Biol. 769, 415-434 (2011).</li><li>Ren, K., Zhao, Y., Su, J., Ryan, D., Wu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convenient+method+for+modifying+poly(dimethylsiloxane)+to+be+airtight+and+resistive+against+absorption+of+small+molecules.">Convenient method for modifying poly(dimethylsiloxane) to be airtight and resistive against absorption of small molecules.</a> Anal. Chem. 82 (14), 5965-5971 (2010).</li><li>Ren, K., Dai, W., Zhou, J., Su, J., Wu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-Teflon+microfluidic+chips.">Whole-Teflon microfluidic chips.</a> PNAS. 108 (20), 8162-8166 (2011).</li><li>Maiuri, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+World+Cell+Race.">The first World Cell Race.</a> Curr Biol. 22 (17), 673-675 (2012).</li></ol>

这里，我们描述了微通道构成，以研究大量的细胞中单个实验的迁移特性的方法的设备。该实验系统由模拟内源性游走细胞在组织中发现的密闭环境的制约。然而，通过强制迁移在单个维度，它有利于细胞自动跟踪和衡量指标的提取（ 图5）。我们还表明，我们的设备是用荧光显微镜兼容的，因此可以适用于细胞运动的不同阶段（ 图4）来研究细胞器的定位。 <p class="jo...

迁移是一个复杂的细胞功能，是许多生理过程中的多细胞生物体，包括发育，免疫应答，和组织再生很重要。此外，某些病理情况下，如肿瘤的侵袭和转移依赖于细胞运动1。由于这些原因，细胞迁移已成为研究的一个主要领域中的基本和平移研究的背景下， 在体内 ，大部分组织的特征在于丰富的细胞外基质和高细胞密度。细胞迁移，因此，在生理条件下，发生在一个复杂的密闭环境中。古典的，很有可能是由于历史的原因和技术的限制，细胞迁移进行了研究，2D平面系统不重现许多组织，如坐月子中发现的环境属性。此外，因素如细胞粘附，这对于运动中的2D必不可少的，最近已表明，以不necessarily需要在体内或内部凝胶迁移，这表明该规则的运动细胞在二维和在其他环境中的机制是不同的2。几个系统已经发展到模仿的组织，最有名的是胶原蛋白凝胶，在扼要的细胞外基质成分3的性质，目的的复杂性。在这里，我们提出了微通道作为一个简单的补充方法，它允许一个密闭的环境下，在一个维度研究细胞迁移。 在这个系统中的细胞沿微通道进入，他们自发地进入迁移。迁移细胞然后获得的信道的形状，采用管状的几何形状，最有可能增强其极性。该细胞在信道的线性运动使自动跟踪单元和从实验定量参数的提取。从技术角度来看，这个系统很容易和灵活。该金属 -克的通道壁可以被操纵，所述通道的尺寸和形状可以适应，并有大量的细胞可以在单一实验中进行分析。该系统还可以按比例放大进行参与细胞运动的分子中程屏幕分析。这里所描述的协议已经使用的树突状细胞（DC）作为细胞模型已经标准化。这些细胞是关键的免疫系统，因为它们参与的特异性免疫反应的引发和维持4。 在体外 ，议会已经显示自发迁移在密闭的环境中，因此，研究细胞运动中的微通道5,6的良好模型。重要的是，该系统可以扩展到分析的任何其他能动的细胞类型如T淋巴细胞，中性粒细胞，或肿瘤细胞7-9的迁移。

<table border="0" cellpadding="0" cellspacing="0" width="947">
	<colgroup>
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:273px;">PolyDimethylSiloxane (PDMS)</td>
			<td style="width:131px;">GE Silicones</td>
			<td style="width:139px;">RTV615</td>
			<td style="width:405px;">Package of 90% silicone base and 10% curing agent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Core sample cutter</td>
			<td>Ted Pella Int.</td>
			<td>Harris Uni-Core&#160;</td>
			<td>Diameter 2,5 mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass-bottom dish</td>
			<td>WPI</td>
			<td>Fluorodish FD 35-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrasonic cleaner</td>
			<td>Branson Ultrasonics</td>
			<td>Branson 200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC 32 G</td>
			<td>For small samples (35 dishes). A bigger version is also available</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin from bovine plasma</td>
			<td>Sigma Aldrich</td>
			<td>F0895</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PolyLysine grafted PEG (Pll-g-PEG)</td>
			<td>Susos</td>
			<td>PLL(20)-g[3.5]-PEG(5)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hoechst 33342</td>
			<td>Sigma Aldrich</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y27632</td>
			<td>TOCRIS</td>
			<td>1254</td>
			<td></td>
		</tr>
	</tbody>
</table>

study of cell migration in microfabricated channels

这里所描述的方法允许在一个维度在封闭条件下细胞迁移的研究。它是基于使用微加工渠道，通过物理约束施加极化表型的细胞。一旦进入通道，细胞只有两种可能：向前或向后移动。这个简化的迁移，其中方向性限制有助于细胞的自动跟踪和量化参数来描述小区移动的提取。这些参数包括运动过程中细胞的速度，改变方向，并暂停。微通道也与使用荧光标记兼容，因此适合在高分辨率细胞迁移过程中，研究细胞内的细胞器和结构的定位。最后，该通道的表面可以用不同的基底进行官能化，允许的信道或haptotaxis的研究的粘合性能的控制。总之，本系统HERE的描述是为了分析大细胞数目的迁移中，其中两个的几何形状和对环境的生化性质受到控制的条件下，方便的独立实验的正常化和可重复性。

在每个实验中的PDMS的表面涂有适于研究的感兴趣的分子， 图2示出了通道涂覆有荧光分子，PLL-G-PEG，前和洗涤（步骤2.4）之后。这样的实验可以在通道中的涂层的均匀性的控制。 后细胞加载，视频显微镜可以执行遵循细胞迁移。 图3A示出的微通道的DC迁移至适当的浓度来跟踪小区的例子。既，相衬和Hoechst染色可以用于此目的（见讨论）。该技术也用?...

Watch this Scientific Journal Video about Study of Cell Migration in Microfabricated Channels at JoVE.com

Study of Cell Migration in Microfabricated Channels

研究细胞的自发迁移的一维密闭微环境的定量方法。此方法采用微加工通道的优势，并可以用来在单个实验中在不同条件下研究了大量的细胞迁移。

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 ...

Research

JoVE Journal

Bioengineering

Microfabricated Platforms for Mechanically Dynamic Cell Culture

微机械动态细胞培养平台

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

科研

生物工程

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

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

电场控制的二维和三维环境中的神经祖细胞定向迁移

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

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

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 ...

Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion

同时捕获实时图像在两个发射通道采用了双摄像头发射分光系统：应用到细胞粘附

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

使用微流体芯片实时成像和损伤研究的响应<em&gt;果蝇</em&gt;幼虫

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

在一个三维胶原蛋白矩阵分析细胞迁移

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

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

同心凝胶系统来研究基质微环境的三维细胞迁移的生物物理作用

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

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

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

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 ...

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

细胞迁移研究在垂直化学和氧梯度聚二甲基硅氧烷的聚碳酸酯微流体装置

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...