Los autores agradecen enormemente la plataforma PICT IBISA en el Institut Curie (CNRS UMR144). Este trabajo fue financiado por becas de: el Consejo Europeo de Investigación para AM.LD (Strapacemi 243.103), el Nationale pour la Recherche Asociación (ANR-09-Piri-0027-PCVI), la fundación InnaBiosanté (Micemico) a MP y AM. LD y el joven investigador subvención ERC Strapacemi a AM.LD.

Nota importante: Este protocolo supone que el molde que contiene la forma de los microcanales deseados ya se ha hecho. Más información sobre la preparación del molde ha sido ya publicada 10. Este protocolo también asume que la cultura los DC de la médula ósea se conoce. 1. Fabricación de chips <ol><li> Mezclar el aceite PDMS y agente de curado en una relación en peso de 10:01 en un vaso de plástico. Mezclar ambos compuestos a fondo. </li><li> Fundido la mezcla en los microcanales de cojinete de molde. La altura total debe estar entre 0,5 a 1 cm. </li><li> Elimine las burbujas de aire en una campana de vacío durante 1 hora. </li><li> PDMS endurecer en el molde mediante la colocación de este último en un horno durante 2 horas a 65 ° C. </li><li> Una vez que el PDMS es a temperatura ambiente, se cortó un pedazo grande alrededor de la estructura con una cuchilla quirúrgica y despegarlo del molde (Figura 1A). </li><li> Perforar agujeros donde se inyectan células (normalmente 2 mm) y cambiar el tamaño del PDMScon una hoja de bisturí para ajustarse al tamaño de los platos con fondo de cristal en el que la migración se evaluará (Figura 1B). Antes de continuar, quitar el polvo residual de la cápsula, utilizando papel de limpieza de lentes. Esto favorece la unión de PDMS de vidrio se describe en el paso 1.10. </li><li> Limpie el chip PDMS redimensionada que contiene los canales por pegar y despegar la cinta adhesiva en los lados de la estructura. </li><li> Sonicar las piezas de PDMS 30 segundos en etanol al 70% para eliminar el polvo y las pequeñas partículas de PDMS. Seque rápidamente después por soplado de aire limpio. </li><li> Activar el PDMS (estructuras hacia arriba) y placas de cultivo por el aire (u oxígeno) de tratamiento con plasma durante 30 segundos a 300 mTorr. </li><li> Coloque ambas superficies activadas en contacto a pegarse permanentemente el PDMS al sustrato. Si es necesario, el uso de fórceps metálicos para presionar ligeramente en la parte superior de los PDMS para forzar el contacto entre el polímero y el vidrio del plato (Figura 1C). </li><li> Incubar el chip en el horno a 65 ° C FOr 1 hora de fortalecer la unión. </li></ol> 2. Recubrimiento de microcanales <ol><li> Activar toda la estructura por plasma de aire a 300 mTorr durante 1 min. Esto promoverá la entrada de líquido en los canales en el siguiente paso. </li><li> Llene rápidamente los orificios de entrada del chip con fibronectina a 10 g / ml en agua. Otros sustratos tales como PEG se pueden usar para modificar la adherencia de células a las paredes del canal. Preste atención a que el líquido se extiende a lo largo de toda la estructura. Esto se puede comprobar fácilmente a simple vista bajo luz normal o el uso de un microscopio de campo claro regular. NOTA: En estructuras muy pequeñas, en las que la difusión es más difícil, la entrada de líquido en los canales puede ser forzado mediante la colocación de la estructura en una campana de vacío durante al menos 15 min. Para comprobar la eficacia del recubrimiento sustratos fluorescentes se pueden utilizar (Figura 2). </li><li> Incubar 1 hora a temperatura ambiente para permitir la adsorción de la fibronectina o cualquier otro recubrimiento substtasa a las paredes de los canales. </li><li> Lavar el estructuras 3 veces con PBS para eliminar el sustrato no unido. </li><li> Después del lavado, proceda al Protocolo de 3 o almacenar el chip a 4 ° C para un uso posterior (24 horas máximo). </li></ol> 3. Celular Loading <ol><li> Retire la PBS de la placa y llenar el microsistema con medio celular. Deje incubar durante 1 hora para saturar los canales con el medio. NOTA: Para los experimentos con drogas como los inhibidores de moléculas, se aconseja preincubar los canales con un medio que contiene el fármaco en la concentración correcta. Además, algunos medicamentos tienden a solubilizar en la estructura de PDMS que reduce la concentración efectiva. Se han propuesto algunas soluciones para contrarrestar este problema 11-12. </li><li> Retire flotantes centros de datos y recuperar las células semi-adherentes mediante lavado con medio de cultivo. Contar las células utilizando un hemocitómetro. NOTA: Para imágenes núcleo, una tinción Hoechst 33342 se puede lograr de antemano por encubating 2 x 10 6 células durante 30 min con el colorante a 200 ng / ml en medio completo. Lavar dos veces por centrifugación para eliminar el exceso de tinte antes de seguir el protocolo. </li><li> Centrifugar las células a 300 xg durante 5 min (tiempo y velocidad pueden ser diferentes dependiendo del tipo de célula) y se elimina el medio. Diluir la pastilla para alcanzar una concentración de 20 x 10 6 células / ml. </li><li> Retire el exceso de medio de la placa y vaciar los orificios de entrada en la estructura de PDMS con una micropipeta. Rellene las entradas con 5 l de solución de células para alcanzar una cantidad de 1 x 10 5 células en cada orificio de entrada. ATENCIÓN: se necesita alta densidad celular para favorecer el contacto de las células con los canales. Densidad celular baja puede resultar en un bajo número de células dentro de los canales y el fracaso del experimento. </li><li> Incubar el microchip durante 30 min a 37 ° C en la incubadora. Añadir 2 ml de medio completo al plato experimento. NOTA: La estructura PDMS conjunto debe be cubierto con el fin de evitar el secado de las células durante el experimento. Si 2 ml no es suficiente, agregue más medio para cubrir completamente la estructura de PDMS. </li></ol> 4. Imaging <ol><li> Limpiar la superficie inferior externa de los platos con el tejido de limpieza de lentes antes de colocar las placas bajo el microscopio. </li><li> Para analizar la migración en gran número de células, utilizar un aumento de 10X y amplia iluminación de campo en un CO 2 y la temperatura y equipado de vídeo-microscopio (Figura 3). Para facilitar el seguimiento de la célula, Hoechst tinción ya la luz UV puede ser utilizado (véase el paso 3.2). La migración puede ser también analizado utilizando microscopía confocal con el fin de obtener una mayor resolución de las estructuras celulares durante la migración. </li><li> Para la microscopía de lapso de tiempo a 10 veces, elegir una frecuencia de tiempo de acuerdo a la velocidad de células esperada (normalmente 2 min para las células dendríticas migran a una velocidad de 5 m / min). Nota: El protocolo descrito aquí hace nOT incluye el análisis de los parámetros de la migración celular. Algunos puntos se enumeran en la discusión para aconsejar a los lectores sobre cómo proceder para extraer información de películas a intervalos. </li></ol>

<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>

Los autores no tienen nada que revelar.

Aquí se describe un dispositivo compuesto de microcanales como un método para estudiar las propiedades migratorias de gran número de células en los experimentos individuales. Este sistema experimental imita las limitaciones ambientales confinados que se encuentran en los tejidos por las células migratorias endógenos. Sin embargo, al forzar la migración en una sola dimensión, que facilita el seguimiento automático de células y la extracción de los mensurables (Figura 5). También mostramos que...

La migración es una función celular complejo que es importante para muchos procesos fisiológicos en los organismos multicelulares, incluyendo el desarrollo, las respuestas inmunes, y la regeneración de tejidos. Además, determinadas situaciones patológicas tales como la invasión tumoral y la metástasis dependen de la motilidad celular 1. Por estas razones, la migración celular se ha convertido en un importante campo de estudio en el contexto de la investigación fundamental y traslacional. In vivo, la mayoría de los tejidos se caracterizan por una rica matriz extracelular y de alta densidad celular. La migración celular, por lo tanto, en condiciones fisiológicas, se produce en un ambiente confinado complejo. Clásicamente, muy probablemente debido a razones históricas y límites técnicos, la migración celular se ha estudiado en sistemas 2D planas que no se reproducen muchas de las propiedades de entorno que se encuentran en los tejidos, tales como el confinamiento. Por otra parte, factores como la adhesión celular, que son esenciales para la motilidad en 2D, recientemente se han mostrado no ser necessariamente necesarios para la migración en vivo o dentro de un gel, lo que sugiere que los mecanismos que gobiernan la locomoción celular en 2D y en otros ambientes son distintos 2. Varios sistemas se han desarrollado para imitar las complejas propiedades de los tejidos, los más famosos geles de colágeno bienestar, cuyo objetivo es la recapitulación de las propiedades de la composición de la matriz extracelular 3. Aquí proponemos microcanales como un método complementario simple que permite la migración de células de estudio en una dimensión en un ambiente confinado. En este sistema las células migran a lo largo de microcanales en la que entran espontáneamente. Células migratorias entonces adquieren la forma de los canales, la adopción de una geometría tubular que lo más probable refuerza su polaridad. El movimiento lineal de las células en los canales permite el seguimiento automático de células y la extracción de parámetros cuantitativos de los experimentos. Desde el punto de vista técnico, este sistema es fácil y flexible. El revestimieg de las paredes del canal se puede manipular, el tamaño y la forma de los canales pueden ser adaptados, y un gran número de células puede ser analizado en los experimentos individuales. Este sistema puede ser también ampliadas al realizar el análisis de la pantalla de medio alcance de moléculas implicadas en la motilidad celular. El protocolo descrito aquí se ha estandarizado el uso de células dendríticas (DC) como un modelo celular. Estas células son clave para el sistema inmune a medida que participan en la iniciación y el mantenimiento de las respuestas inmunes específicas 4. In vitro, países en desarrollo se ha demostrado que migrar de forma espontánea en ambientes confinados y son por lo tanto un buen modelo para estudiar la motilidad celular en microcanales 5,6 . Es importante destacar que este sistema se puede ampliar para analizar la migración de cualquier otro tipo de células móviles como linfocitos T, neutrófilos, o células tumorales 7-9.

<table border="0" cellpadding="0" cellspacing="0" width="947">
	<colgroup>
		<col />
		<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

El método aquí descrito permite el estudio de la migración de células en condiciones de confinamiento en una dimensión. Se basa en el uso de canales microfabricados, que imponen un fenotipo polarizado a las células por las limitaciones físicas. Una vez dentro de los canales, las células tienen sólo dos posibilidades: seguir adelante o hacia atrás. Esta migración simplificado en el que la direccionalidad está restringida facilita el seguimiento automático de las células y la extracción de parámetros cuantitativos para describir el movimiento celular. Estos parámetros incluyen la velocidad de célula, los cambios de dirección, y las pausas durante el movimiento. Los microcanales son también compatibles con el uso de marcadores fluorescentes y son por lo tanto adecuados para estudiar la localización de los orgánulos y las estructuras intracelulares durante la migración celular en alta resolución. Por último, la superficie de los canales se puede funcionalizar con diferentes sustratos, lo que permite el control de las propiedades adhesivas de los canales o el estudio de Haptotaxis. En resumen, el sistema de hERE descrito está destinado a analizar la migración de grandes números de células en condiciones en las que se controlan tanto la geometría y de la naturaleza bioquímica del medio ambiente, lo que facilita la normalización y reproducibilidad de los experimentos independientes.

En cada experimento la superficie de la PDMS se recubre con una molécula adaptada a los intereses del estudio. La figura 2 muestra los canales revestidos con una molécula fluorescente, PLL-g-PEG, antes y después de lavar (paso 2.4). Tal experimento permite el control de la homogeneidad del recubrimiento en los canales. Después de la carga de células, microscopía de vídeo se puede realizar para seguir la migración celular. Figura 3A muestra un ejemplo...

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

Study of Cell Migration in Microfabricated Channels

Un método cuantitativo para estudiar la migración espontánea de las células en un microambiente confinado unidimensional se describe. Este método tiene la ventaja de canales microfabricados y se puede utilizar para estudiar la migración de gran número de células bajo diferentes condiciones en los experimentos individuales.

Estudio de la Migración celular en Canales Microfabricated

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 ...

study-of-cell-migration-in-microfabricated-channels

Research

JoVE Journal

Bioengineering

11.8K vistas.  Institut Curie.  Un método cuantitativo para estudiar la migración espontánea de las células en un microambiente confinado unidimensional se describe. Este método tiene la ventaja de canales microfabricados y se puede utilizar para estudiar la migración de gran número de células bajo diferentes condiciones en los experimentos individuales. 

Video: Study of Cell Migration in Microfabricated Channels

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

Plataformas microfabricated para el cultivo celular mecánico dinámico

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

Investigación

Bioingeniería

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

La creación de dos dimensiones con dibujos sustratos para el confinamiento de proteínas y células

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

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Campo eléctrico controlado por migración dirigida de células progenitoras neurales en entornos 2D y 3D

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

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.

Creación de degradados adhesivas y soluble para la migración celular con imágenes de microscopía de fluorescencia

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

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to investigate mechanisms governing cell adhesion under dynamic flow conditions. For instance, cancer cells labeled with multiple fluorophores can be perfused over a potentially reactive substrate to model mechanisms of cancer metastasis. However, multi-channel single camera systems and color cameras exhibit shortcomings in image acquisition for real-time live cell analysis. To overcome these limitations, we used a dual camera emission splitting system to simultaneously capture real-time image sequences of fluorescently labeled cells in the flow chamber. Dual camera emission splitting systems filter defined wavelength ranges into two monochrome CCD cameras, thereby simultaneously capturing two spatially identical but fluorophore-specific images. Subsequently, psuedocolored one-channel images are combined into a single real-time merged sequence that can reveal multiple target molecules on cells moving rapidly across a region of interest.

Dual camera emission splitting systems for two-color fluorescence microscopy generate real-time image sequences with exceptional optical and temporal resolution, a requirement of certain live cell assays including parallel plate flow chamber adhesion assays. When software is employed to merge images from simultaneously acquired emission channels, pseudocolored image sequences are produced.

La captura simultánea imágenes en tiempo real en los dos canales de emisión mediante un sistema de división de doble cámara de emisiones: Las solicitudes de adhesión de células

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

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

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

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

El uso de chips de microfluídica para imágenes en vivo y de estudio de las respuestas de lesiones en<em&gt; Drosophila</em&gt; Las larvas

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

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Análisis de la migración de células dentro de una matriz de colágeno tridimensional

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

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 tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell&#8217;s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

Gel System Concentric para estudiar el papel de Biofísica de Matrix Microambiente en la migración de células en 3D

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 ...

Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not occur. With conventional templates, the absence of internal micro-channels results in the lack of cell infiltration, distribution, and inhabitance deep inside the templates. Hence, a highly porous and uniformly interconnected trabecular-bone-like template with micro-channels (biogenic microenvironment template; BMT) has been developed to address these obstacles. The novel BMT was created by innovative concepts (capillary action) and fabricated with a sponge-template coating technique. The BMT consists of several structural components: inter-connected primary-pores (300-400 &#181;m) that mimic pores in trabecular bone, micro-channels (25-70 &#181;m) within each trabecula, and nanopores (100-400 nm) on the surface to allow cells to anchor. Moreover, the BMT has been documented by mechanical test study to have similar mechanical strength properties to those of human trabecular bone (~3.8 MPa)12.
The BMT exhibited high absorption, retention, and habitation of cells throughout the bridge-shaped (&#928;) templates (3 cm height and 4 cm length). The cells that were initially seeded into one end of the templates immediately mobilized to the other end (10 cm distance) by capillary action of the BMT on the cell media. After 4 hr, the cells homogenously occupied the entire BMT and exhibited normal cellular behavior. The capillary action accounted for the infiltration of the cells suspended in the media and the distribution (active migration) throughout the BMT. Having observed these capabilities of the BMT, we project that BMTs will absorb bone marrow cells, growth factors, and nutrients from the periphery under physiological conditions.
The BMT may resolve current limitations via rapid infiltration, homogenous distribution and inhabitance of cells in large, volumetric templates to repair massive skeletal defects.

A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels.

Plantillas distintivo acción capilar por micro-canales en hueso como puede mejorar el reclutamiento de células para la Restauración del Gran Bony Defecto

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not ...

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

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

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

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparación de geles de colágeno en 3D y microcanales para el estudio de las interacciones 3D<em&gt; En Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...