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

Os autores reconhecem Programa criativo Inquérito da Universidade de Clemson e NSF CBET1254609 por fornecer financiamento para este projeto.

1. Produzir uma Câmara MicroChannel criar um gradiente de concentração <ol><li> Cortando uma peça de molde de acrílico <ol><li> Obter um pedaço de acrílico com a largura desejada. Normalmente, utilizam 1 placas de acrílico /-16 polegadas de espessura. folhas mais espessas são difíceis de cortar bem e muito finas folhas não têm a resistência mecânica, causando-lhes a quebrar ou deformar durante o processo. </li><li> Criar um ficheiro CAD com a forma desejada da peça de acrílico para pro...

<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>

A nossa câmara de microcanal pode ser utilizado para uma variedade de fins, incluindo a determinação da taxa de migração celular em resposta a factores de crescimento e quimioatractores e medindo a taxa de difusão de um fármaco a partir de uma matriz de polímero. É possível utilizar a câmara de microcanais para crescer as células e colocar um quimioatractor numa extremidade da câmara. As células crescem em resposta ao quimioatraente, e a migração de células pode ser quantificada tomando imagens de lapso...

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

A migração celular é uma parte vital da resposta imune, crescimento e cicatrização de feridas. A migração celular é um processo complexo que envolve interacções entre células, a matriz extracelular e factores químicos solúveis e não solúveis (por exemplo, quimioatractores). Métodos padrão para medir a migração de células, tais como o ensaio de câmara de Boyden, trabalho por contagem de células em ambos os lados de uma divisória. Estas técnicas são fáceis de utilizar; no entanto, eles oferecem pouca modificação geométrica para diferentes aplicações. Em contraste, os dispositivos microfluidicos podem ser utilizados para observar a migração de células com gradientes de concentração personalização de factores solúveis 1, 2. No entanto, os métodos para fazer os ensaios com base microfluidos pode ser difícil de aprender. Aqui, nós descrevemos um método fácil para criar câmaras de cultura de células para medir a migração celular em resposta a gradientes de concentração química. Nossos mi celularesmétodo câmara gração pode criar diferentes gradientes de concentração linear de modo a estudar a migração de células para uma variedade de aplicações. Este método é relativamente fácil de usar e é normalmente realizada por alunos de graduação. A câmara de microcanais foi criado por colocação de uma inserção de acrílico na forma da câmara de microcanais finais bem em uma placa de Petri. Depois disso, o poli (dimetilsiloxano) (PDMS) foi deitada no topo da inserção. O PDMS foi deixada endurecer e, em seguida, a inserção foi removida. Isto permitiu a criação de cavidades em qualquer forma ou tamanho desejado. As células podem ser posteriormente adicionado à câmara de microcanais, e agentes solúveis podem ser adicionados a um dos poços por imersão de um bloco de agarose, o agente desejado. O bloco de agarose é adicionado a um dos poços, e as imagens de lapso de tempo pode ser feita da câmara de microcanal, a fim de quantificar a migração celular. Variações deste método pode ser feita para uma dada aplicação, tornando este método highly personalizável.

A Figura 2 mostra o movimento da parte dianteira da célula através do canal em resposta a um gradiente de soro fetal de bovino colocado na extremidade oposta do canal, de onde as células são plaqueadas. A parte dianteira da célula é mostrada em 48 h (Figura 2A), 72 h (Figura 2B), 96 h (Figura 2C), e 120 h (Figura 2D) pós chapeamento. O movimento da frente de celular foi rastreado com estas imagens...

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

Este protocolo detalha um método personalizada para medir a migração celular em resposta a quimioatractores, que também podem ser utilizados para determinar a taxa de difusão de um fármaco a partir de uma matriz de polímero.

A Câmara customizáveis ​​para a migração celular de medição

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

Measuring Exocytosis in Neurons Using FM Labeling Video (Video) | JoVE

Medição Exocitose em Neurônios Usando Labeling FM

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 ...

Biologia

Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber

Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber Video (Video) | JoVE

Estimulação cerebral Slice Usando uma Rede microfluídicos e da Câmara de Perfusão padrão

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

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc Video (Video) | JoVE

Imagens ao vivo da migração das células da glia no Disco Drosophila Eye Imaginal

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap Video (Video) | JoVE

Medir a rigidez à flexão de células bacterianas Usando uma armadilha óptica

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Measuring Peptide Translocation into Large Unilamellar Vesicles

Measuring Peptide Translocation into Large Unilamellar Vesicles Video (Video) | JoVE

Medição Translocação Peptide em grandes vesículas Unilamelares

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

Microscopia intravital da Microcirculação no músculo Rato Cremaster de Análise de Migração Stem Peripheral celular

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

Microinjeção de uma única célula para análise da comunicação celular

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

In vitro Cell Migration and Invasion Assays Video (Video) | JoVE

Na migração celular in vitro e invasão Ensaios

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

Filamentos intermediários e microtúbulos com microscopia 2-dimensional direta estocástico reconstrução óptico de imagem

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

Otimizado zero ensaio para In Vitro testes de migração celular com uma câmera óptica automatizada

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