Name: a customizable chamber for measuring cell migration
Uploaded: 2017-03-12T14:00:00
Description: Watch this Scientific Journal Video about A Customizable Chamber for Measuring Cell Migration Video 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><l...

<ol>
	<li>Sia, S. K., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+fabricated+in+poly+(dimethylsiloxane)+for+biological+studies.">Microfluidic devices fabricated in poly (dimethylsiloxane) for biological studies.</a> Electrophoresis. 24 (21), 3563-3576 (2003).</li><li>Lin, F., 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=Generation+of+dynamic+temporal+and+spatial+concentration+gradients+using+microfluidic+devices.">Generation of dynamic temporal and spatial concentration gradients using microfluidic devices.</a> Lab Chip. 4 (3), 164-167 (2004).</li><li>Darby, I., Hewitson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+Differentiation+in+Wound+Healing+and+Fibrosis.">Fibroblast Differentiation in Wound Healing and Fibrosis.</a> Int Rev Cytol. 257, 143-179 (2007).</li><li>Thampatty, B. P., Wang, J. H. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+study+fibroblast+migration.">A new approach to study fibroblast migration.</a> Cell Motil Cytoskel. 64, 1-5 (2007).</li><li>Discher, D., Mooney, D., Zandstra, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+Factors,+Matrices,+and+Forces+Combine+and+Control+Stem+Cells.">Growth Factors, Matrices, and Forces Combine and Control Stem Cells.</a> Science. 324, 1673-1677 (2009).</li><li>Zengel, 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=&#181;-Slide+Chemotaxis:+A+new+chamber+for+long-term+chemotaxis+studies.">&#181;-Slide Chemotaxis: A new chamber for long-term chemotaxis studies.</a> BMC Cell Biol. , (2011).</li><li>Chen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boyden+Chamber+Assay.">Boyden Chamber Assay.</a> Methods Molec Biol. 2, 15-22 (2005).</li><li>Safferling, K., 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=Wound+healing+revised:+A+novel+reepithelialization+mechanism+revealed+by+in+vitro+and+in+silico+models.">Wound healing revised: A novel reepithelialization mechanism revealed by in vitro and in silico models.</a> JCB. 203 (4), 691-709 (2013).</li><li>Saadi, W., 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=Generation+of+stable+concentration+gradients+in+2D+and+3D+environments+using+a+microfluidic+ladder+chamber.">Generation of stable concentration gradients in 2D and 3D environments using a microfluidic ladder chamber.</a> Biomed Microdevices. 9, 627-635 (2007).</li><li>Cammer, M., Cox, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotactic+Responses+by+Macrophages+to+a+Directional+Source+of+a+Cytokine+Delivered+by+a+Micropipette.">Chemotactic Responses by Macrophages to a Directional Source of a Cytokine Delivered by a Micropipette.</a> Methods Mol Biol. , 125-135 (2014).</li><li>Zicha, D., Dunn, G. A., Brown, 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=A+new+direct-viewing+chemotaxis+chamber.">A new direct-viewing chemotaxis chamber.</a> J Cell Sci. 99 (4), 769-775 (1991).</li><li>Wells, C. M., Ridley, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cell+migration+using+the+Dunn+chemotaxis+chamber+and+time-lapse+microscopy.">Analysis of cell migration using the Dunn chemotaxis chamber and time-lapse microscopy.</a> Methods Mol Biol. , 31-41 (2005).</li><li>Liang, C. C., Park, A. Y., Guan, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+scratch+assay:+a+convenient+and+inexpensive+method+for+analysis+of+cell+migration+in+vitro.">In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro.</a> Nat. Protoc. 2, 329-333 (2007).</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. 5, (2015).</li><li>Wright, G. A., 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=On-chip+open+microfluidic+devices+for+chemotaxis+studies.">On-chip open microfluidic devices for chemotaxis studies.</a> Microsc. Microanal. 18 (04), 816-828 (2012).</li><li>Adler, M., Polinkovsky, M., Gutierrez, E., Groisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+oxygen+gradients+with+arbitrary+shapes+in+a+microfluidic+device.">Generation of oxygen gradients with arbitrary shapes in a microfluidic device.</a> Lab Chip. 10 (3), 388-391 (2010).</li><li>Huang, Y., Agrawal, B., Sun, D., Kuo, J. S., Williams, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics-based+devices:+New+tools+for+studying+cancer+and+cancer+stem+cell+migration.">Microfluidics-based devices: New tools for studying cancer and cancer stem cell migration.</a> Biomicrofluidics. 5 (1), 013412 (2011).</li><li>Taylor, A. M., Rhee, S. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+chambers+for+cell+migration+and+neuroscience+research.">Microfluidic chambers for cell migration and neuroscience research.</a> Microfluid Tech: Rev Protoc. , 167-177 (2006).</li><li>Tang, S. K., Whitesides, G. M., Fainman, Y., Lee, L., Psaltis, D., Yang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basic microfluidic and soft lithographic techniques. Optofluidics: Fundamentals, Devices and Applications. , (2010).</li><li>Taylor, A. M., 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=Microfluidic+multicompartment+device+for+neuroscience+research).">Microfluidic multicompartment device for neuroscience research).</a> Langmuir. 19 (5), 1551-1556 (2003).</li><li>Aidelberg, G., Goldshmidt, Y., Nachman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Microfluidic+Device+for+Studying+Multiple+Distinct+Strains.">A Microfluidic Device for Studying Multiple Distinct Strains.</a> JoVE. (69), (2012).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</td>
			<td>Falcon&#160;</td>
			<td>25373-041</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate&#8211;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&#39;s Modified Eagle&#39;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.&#160;</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 Video at JoVE.com

A Customizable Chamber for Measuring Cell Migration Video (Video) | JoVE

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 ...

Research

JoVE Journal

Biology

A Customizable Chamber for Measuring Cell Migration

Measuring Exocytosis in Neurons Using FM Labeling

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测量中使用FM标签的神经元的胞外分泌

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

科研

生物学

Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber

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脑片的刺激，利用微流控网络和标准灌注腔

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The ...

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

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住在果蝇眼睛成虫光盘胶质细胞迁移成像

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Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

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测量细菌细胞的抗弯刚度，使用光学陷阱

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Measuring Peptide Translocation into Large Unilamellar Vesicles

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测量肽转运到大Unilamellar囊泡

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration Video (Video) | JoVE

在鼠标提睾肌为外周干细胞迁移分析的微循环活体显微镜

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Single-cell Microinjection for Cell Communication Analysis

Single-cell Microinjection for Cell Communication Analysis Video (Video) | JoVE

Cell的传播分析单细胞显微注射

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

In vitro Cell Migration and Invasion Assays

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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 ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy Video (Video) | JoVE

2维直接随机光学重建显微成像中间丝和微管

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera Video (Video) | JoVE

用自动光学相机进行细胞迁移的体外试验优化划痕试验

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for ...