Este trabajo fue apoyado por la American Heart Asociación de becas predoctorales para JLI y los Institutos Nacionales de Salud de subvención R01AR056624.

A. único polímero electrospinning <ol><li> Antes de preparar la solución de electrospinning, hacer una solución de peso de 0,5% del fotoiniciador, Irgacure 2959 (I2959), en agua destilada por la disolución a 37 ° C durante varios días. Este paso no es necesario si un polímero fotorreactivos no se está utilizando. </li><li> Combinar el ácido hialurónico methacrylated (MEHA, ver Burdick et al. Para la síntesis), el poli (óxido de etileno) (PEO, 900 kDa), y I2959 en agua destilada para preparar una solución con una concentración final de 2% en peso MEHA, 3% en peso PEO, y 0,05% en peso I2959. Use un remolino para mezclar la solución hasta que esté claro. El tipo de polímero y concentración, así como el solvente usado puede ser modificada en este paso en función de las propiedades deseadas del andamio. </li><li> La transferencia de la solución en una jeringa y fijar un calibre 18, 6 pulgadas de largo, aguja de punta roma hasta el final. </li><li> Programa de una jeringa de la bomba para expulsar a una tasa de 1,2 ml / hr. Inserte la jeringa y la aguja en el dispositivo. </li><li> Conecte el cable a tierra de una fuente de alimentación de alta tensión al aparato de colección. Para recoger al azar las fibras distribuidas utilizan una placa de metal, o el uso de un mandril que gira a unos 10 m / s para recoger las fibras alineadas. Conecte el cable de carga positiva a la aguja. En la figura 1 una representación esquemática del aparato de electrospinning. </li><li> Ajuste la aguja o dispositivo de recogida, de tal manera que hay una distancia de 15 cm entre los dos. </li><li> Iniciar el flujo de la bomba de jeringa. Cuando el líquido se visualiza en la punta de la aguja, a su vez la fuente de alimentación y ajustar la tensión de 22 kV. </li><li> Después de la colección es completa (a partir de varios minutos de películas delgadas, de hasta 24 horas por una alfombra gruesa), retire el andamio desde el aparato de recogida y almacenarlo bajo vacío durante la noche para asegurar la eliminación completa del disolvente. </li><li> Visualizar la morfología de las fibras mediante microscopía electrónica de barrido (SEM), representante de un andamio se muestra en la Figura 1, tanto para las estructuras alineados y no alineados. </li></ol> Nota: La tasa de flujo de la muestra, la distancia al dispositivo de recolección, y la tensión dependen de la combinación de polímero y solvente, y debe ser optimizado para cada sistema, por lo general mediante la observación de la morfología del andamio con SEM. B. Photocrosslinking y Photopatterning <ol><li> Cortar las muestras de 5mm x 5mm de la estera del andamio. </li><li> Preparar para la reticulación mediante la colocación de cada andamio en una lámina portaobjetos de vidrio cubierta, la colocación de la fotomáscara directamente en el andamio, que cubre con un portaobjetos de vidrio limpio y el recorte de los dos extremos con clips de la carpeta. Nota: cuatro muestras se puede hacer a la vez, y una transparencia sin un patrón se puede utilizar para la reticulación, si un patrón no deseado. </li><li> Purga andamio instalado en una cámara de nitrógeno. Es importante mantener la construcción libre de oxígeno, que puede inhibir la reticulación. </li><li> Lugar de instalación de andamios en la cámara de nitrógeno a ~ 10 mW / cm 2 de luz de 365 nm con un adaptador de colimación durante 5 minutos. Vea el esquema de la Figura 2. </li><li> Retire cada andamio y colocar en una placa de 12 también. </li><li> Añadir 2 ml de agua destilada en cada pozo, la placa de parafina para evitar la evaporación del agua y el lugar a 37 ° C durante 24 horas. Cambiar el agua tres veces. </li><li> Visualizar la formación de poros bajo microscopio de luz (ver ejemplo en Figura 2). Los andamios ya están listos para su uso. </li></ol> Nota: Photocrosslinking no es necesario para muchos tipos de polímeros, sin embargo photopatterning sólo se puede utilizar con polímeros fotorreactivos. C. Doble polímero fluorescente electrospinning con la visualización de fibra <ol><li> Prepare una solución al 5% en peso de PEO (200 kDa) en el 90% de etanol. Revuelva a 700 rpm a 50 ° C durante al menos 2 horas antes de electrospinning. </li><li> Transferir la solución a una jeringa de OPE y añadir DAPI para una concentración final de 10 mg / mL de concentración en la solución de electrospinning. Envuelva la jeringa en papel de aluminio para protegerlo de la luz. </li><li> Preparar la misma solución que se describe en el paso A2, con la excepción también se suman methacryloxyethyl tiocarbamoil rodamina B (MeRho, 683,24 g / mol) para una concentración final de 25 mM en la solución de electrospinning. Envuelva la jeringa en papel de aluminio para protegerlo de la luz. </li><li> Cuidadosamente el uso de cinta adhesiva para asegurar methacrylated cubreobjetos de vidrio (véase Khademhosseini et al.) A la superficie del mandril. Nota: las fibras se convertirá atados a cubreobjetos de vidrio methacrylated. </li><li> Conecte un extremo de una pieza de 5 mm de longitud de tubo de silicona con archivos adjuntos Luer a una de las jeringas y conecte el otro extremo a una aguja. Inserte la jeringa en la bomba de jeringa y poner la aguja a través del agujero en el agricultor. Repita con la otra jeringa y agricultor en el lado opuesto del mandril. Los agricultores traducir el length del mandril y se utilizan para garantizar una distribución equitativa de los polímeros en los que resulta andamio. Vea el esquema de la Figura 3. </li><li> Ajustar los parámetros descritos en la sección A apropiadamente de acuerdo a la Tabla 1. Asegúrese de que las puntas de aguja se centran en el mandril. Tabla 1. Doble polímero electrospinning Parámetros <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Jeringa </td><td> Agricultor a punta de la aguja (cm) </td><td> Punta de la aguja de Mandril (cm) </td><td> Flujo (ml / h) </td><td> Aplica tensión (kV) </td></tr><tr><td> MEHA </td><td> 6 </td><td> 15 </td><td> 1.2 </td><td> 22 </td></tr><tr><td> OPE </td><td> 6 </td><td> 10 </td><td> 1.2 </td><td> 15 </td></tr></tbody></table> </li><li> Una vez que todo esté alineado correctamente, apague las luces y encender el mandril y las bombas de jeringa. Retire el papel aluminio de las jeringas. Cuando el líquido es visible en las puntas de ambas agujas, simultáneamente a su vez en las fuentes de alimentación y conecte el los agricultores. </li><li> Cuando la colección está completa, apagar la fuente de alimentación y el mandril y desenchufe el los agricultores. Retire con cuidado el cubreobjetos y la cinta con una cuchilla de afeitar. </li><li> Visualizar las fibras utilizando un microscopio de fluorescencia equipado con filtros de rodamina y DAPI. Véase el ejemplo en la figura 3. Nota: las fibras fluorescentes son más claros a la vista cuando electrospun por períodos cortos de tiempo (&lt;3 minutos), sin embargo, este proceso puede ser prorrogado por una cantidad ilimitada de tiempo a fin de crear más gruesas las construcciones, especialmente sin el uso de la cubreobjetos de vidrio . </li></ol> D. Las células de siembra en los andamiajes <ol><li> Coloque cada cubreobjetos con electrospun polímero unido a un individuo así de una placa y de tamaño adecuado. Nota: las interacciones celulares con las fibras son fácilmente visibles por electrospinning en cubreobjetos de vidrio methacrylated. Además, MeRho nuevo se puede utilizar para la visualización de la fibra. </li><li> Incubar los andamios en PBS durante la noche para asegurar la completa eliminación de solventes y cualquier lixiviable posibles subproductos. </li><li> Retire la PBS de los pozos. </li><li> Para esterilizar los andamios, los coloca bajo una lámpara germicida en una campana de flujo laminar durante 30 minutos. Si una completa estructura es ser cabeza de serie, dar la vuelta al cadalso y colóquelo debajo de una lámpara germicida por otros 30 minutos. </li><li> Realizar el cultivo celular estándar y preparar una suspensión celular se concentró en la densidad celular deseada (por ejemplo, 6.000 células cm -2). Por ejemplo, utilizar una de 100 ml de suspensión celular de una de 22 mm X 22 mm cubreobjetos. Lugar en la incubadora durante 1 hora. </li><li> Añadir la cantidad apropiada de medios de cultivo celular a cada pocillo. Coloque los andamios de nuevo en la incubadora. </li><li> Tinción de las células utilizando un kit disponible comercialmente Live / Dead. Nota: otros tipos de tinción de células, como DAPI (núcleos de las células) y la etiqueta fluorescente phalloidin (fibras de actina estrés) puede ser aplicado para visualizar las células. </li><li> Visualizar las células y las fibras utilizando un microscopio de fluorescencia equipado con filtros para FITC y TRITC. Véase el ejemplo en la Figura 4. </li></ol> Los resultados representativos: <img src="/files/ftp_upload/1589/1589figure1tmb.jpg" border="0" alt="Figura 1" /> Figura 1. Esquema que ilustra la instalación de dispositivos para los países no alineados formación de andamios (arriba) y se alinea la formación de andamios (abajo). Ejemplo de digitalización de imágenes de microscopía electrónica de cada tipo de andamio se muestran. Barra de escala = 5μm. Por favor, <a href="https://www.jove.com/files/ftp_upload/1589/1589figure1.jpg">haga clic aquí</a> para ver una versión ampliada de la figura 1. <img src="/files/ftp_upload/1589/1589figure2tmb.jpg" border="0" alt="Figura 2" /> Figura 2. Esquema del método de los patrones. Patrones se forman mediante la colocación de una fotomáscara entre la fuente de luz y andamio durante photocrosslinking y luego lavando polímero sin reaccionar. Fotomáscara SEM y la imagen de los andamios después de la formación de poros y la liofilización. Barra de escala = 100 micras. Por favor, <a href="https://www.jove.com/files/ftp_upload/1589/1589figure2.jpg">haga clic aquí</a> para ver una versión ampliada de la figura 2. <img src="/files/ftp_upload/1589/1589figure3tmb.jpg" alt="Figura 3" border="0" /> Figura 3 Esquema de la configuración multi-polímero electrospinning y los parámetros necesarios de procesamiento, así como una imagen representativa de fluorescencia de una mezcla de dos poblaciones de fibra (por ejemplo: rojo MEHA y PEO azul). Que eran al mismo tiempo electrospun para formar un andamio de varios polímeros . Barra de escala = 100 micras. Por favor, <a href="https://www.jove.com/files/ftp_upload/1589/1589figure3.jpg">haga clic aquí</a> para veruna versión más grande de la figura 3. <img src="/files/ftp_upload/1589/1589figure4tmb.jpg" border="0" alt="Figura 4" /> Figura 4. Un ejemplo Live / Dead tinción imagen de células madre mesenquimales humanas y sus interacciones con las fibras electrospun. Barra de escala = 100 micras. Por favor, <a href="https://www.jove.com/files/ftp_upload/1589/1589figure4.jpg">haga clic aquí</a> para ver una versión ampliada de la figura 4.

<ol>
	<li>Burdick, J. A., Chung, C., Jia, X., Randolf, M. A., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+degradation+and+mechanical+behavior+of+photopolymerized+hyaluronic+acid+networks.">Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks.</a> Biomacromolecules. 6, 386-391 (2005).</li><li>Baker, B. M., Gee, A. O., Metter, R. B., Nathan, A. S., Marklein, R. A., Burdick, J. A., Mauck, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+to+improve+cell+infiltration+in+composite+fiber-aligned+electrospun+scaffolds+by+the+selective+removal+of+sacrificial+fibers.">The potential to improve cell infiltration in composite fiber-aligned electrospun scaffolds by the selective removal of sacrificial fibers.</a> Biomaterials. 29, 2348-2358 (2008).</li><li>Ifkovits, J. L., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Photopolymerizable+and+degradable+biomaterials+for+tissue+engineering+applications.">Review: Photopolymerizable and degradable biomaterials for tissue engineering applications.</a> Tissue Engineering. 13, 2369-2385 (2007).</li><li>Khademhosseini, A., Eng, G., Yeh, J., Fukuda, J., Blumling, J., Langer, R., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromolding+of+photocrosslinkable+hyaluronic+acid+for+cell+encapsulation+and+entrapment.">Micromolding of photocrosslinkable hyaluronic acid for cell encapsulation and entrapment.</a> J. Biomed Mater Res A. 79A, 522-532 (2006).</li><li>Mauck, R. L., Baker, B. M., Nerurkar, N. L., Burdick, J. A., Li, W. J., Tuan, R. S., Elliott, D. M., M, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+on+the+Straight+and+Narrow:+The+Mechanics+of+Nanofibrous+Assemblies+for+Fiber-Reinforced+Tissue+Regeneration.">Engineering on the Straight and Narrow: The Mechanics of Nanofibrous Assemblies for Fiber-Reinforced Tissue Regeneration.</a> Tissue Engineering B. 15, 171-193 (2009).</li><li>Sill, T. J., Von Recum, H. a. v. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning:+applications+in+drug+delivery+and+tissue+engineering.">Electrospinning: applications in drug delivery and tissue engineering.</a> Biomaterials. 29, 1989-2006 (2008).</li><li>Sundararaghavan, H. G., Metter, R. B., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+fibrous+scaffolds+with+multi-scale+and+photopatterned+porosity.">Electrospun fibrous scaffolds with multi-scale and photopatterned porosity.</a> , (2009).</li></ol>

Electrospinning se utilizó para preparar los andamios fibra a partir de polímeros. Photocrosslinkable andamios a base de ácido hialurónico se utiliza como un ejemplo ilustrativo, donde se requiere exposición a la luz para la reticulación. Con el uso de macrómeros reactivos, como MEHA, los canales que previamente han demostrado una mayor distribución de celulares fueron incorporados en los andamios, con el uso de una máscara durante photocrosslinking para formar andamios de macro y micro poroso. Por otra parte, ...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DAPI</td>
<td> Reagent</td>
<td>Invitrogen</td>
<td>D1306</td>
<td>&nbsp;</td>
<tr>
<td>I2959</td>
<td>Reagent</td>
<td>Ciba Specialty Chemicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PEO 200 kDa</td>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>17503</td>
<td>&nbsp;</td>
<tr>
<td>PEO 900 kDa</td>
<td>Reagent</td>
<td>Sigma</td>
<td>189456</td>
<td>&nbsp;</td>
<tr>
<td>Methacryloxethyl thiocarbamoyl rhodamine B</td>
<td>Reagent</td>
<td>Polysciences</td>
<td>23591-100</td>
<td>Prepare stock solution in DMSO</td>
</tr>
<tr>
<td>Live/Dead Stain Kit</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>L3224</td>
<td>Contains Calcein (stains live cells green) and ethidium homodime (stains red dead cells)</td>
</tr>
<tr>
<td>Syringe Pump</td>
<td>Equipment</td>
<td>KD Scientific</td>
<td>KDS100</td>
<td>Two are needed for dual polymer spinning</td>
</tr>
<tr>
<td>Power Source</td>
<td>Equipment</td>
<td>Gamma High Voltage</td>
<td>ES30P-5W</td>
<td>Two are needed for dual polymer spinning</td>
</tr>
<tr>
<td>Motor</td>
<td>Equipment</td>
<td>Triem Electric Motors, Inc</td>
<td>0132022-15</td>
<td>Must attach to a custom built mandrel</td>
</tr>
<tr>
<td>Tachometer</td>
<td>Equipment</td>
<td>Network Tool Warehouse</td>
<td>ESI-330</td>
<td>Use to monitor mandrel speed</td>
</tr>
<tr>
<td>Omnicure UV Spot Cure System with collimating adapter</td>
<td>Equipment</td>
<td>Exfo Life Sciences Division</td>
<td>S1000</td>
<td>&nbsp;</td>
<tr>
<td>Silicone Tubing</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51135K151</td>
<td>&nbsp;</td>
<tr>
<td>Luer Lock Female Adapter</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51525K293</td>
<td>&nbsp;</td>
<tr>
<td>Luer Lock Male Adapter</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51525K143</td>
<td>&nbsp;</td>
<tr>
<td>Needles</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>14-825-16H</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips</td>
<td>Equipment</td>
<td>Corning</td>
<td>2875-22</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

electrospinning fibrous polymer scaffolds for tissue engineering and cell culture

Como el campo de la ingeniería de tejidos evoluciona, hay una enorme demanda para producir materiales más adecuados y las técnicas de procesamiento con el fin de atender las necesidades (por ejemplo, la mecánica y la vascularización) de los órganos más complejos y los tejidos. Electrospinning es una técnica popular para crear andamios fibroso que imitan la arquitectura y la escala de tamaño de la matriz extracelular nativa. Estos andamios fibra también son útiles como sustratos de cultivo celular ya que las fibras se pueden utilizar para dirigir el comportamiento celular, incluyendo la diferenciación de células madre (ver comentarios extensos por Mauck<em&gt; Et al.</em&gt; Y el umbral<em&gt; Et al.</em&gt; Para más información). En este artículo se describe el proceso general de electrospinning polímeros y como ejemplo, un ácido hialurónico electrospin reactivo capaz de reticular con la exposición a la luz (ver Ifkovits<em&gt; Et al.</em&gt; Para una revisión de los materiales photocrosslinkable). También introducir capacidades adicionales de procesamiento como photopatterning y multi-polímero de formación de andamios. Photopatterning se puede utilizar para crear los andamios con los canales y la porosidad multi-escala para aumentar la infiltración celular y distribución en los tejidos. Multi-polímero andamios son útiles para ajustar mejor las propiedades (mecánicas y degradación) de un andamio, como medida de porosidad de la infiltración celular. Además, estas técnicas se puede ampliar para incluir una amplia gama de polímeros y macrómeros reactivos para crear andamios complejos que proporcionan las señales necesarias para el desarrollo de las construcciones de ingeniería tisular con éxito.

Watch this Scientific Journal Video about Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture at JoVE.com

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

El proceso de electrospinning polímeros de ingeniería de tejidos y cultivos celulares se trata en este artículo. En concreto, el electrospinning de macrómeros fotorreactivos con capacidades de procesamiento adicional de photopatterning y electrospinning multi-polímero se describe.

Electrospinning andamios fibroso polímero para ingeniería de tejidos y cultivos celulares

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

electrospinning-fibrous-polymer-scaffolds-for-tissue-engineering-cell

Research

JoVE Journal

Biology

21.4K vistas.  University of Pennsylvania. El proceso de electrospinning polímeros de ingeniería de tejidos y cultivos celulares se trata en este artículo. En concreto, el electrospinning de macrómeros fotorreactivos con capacidades de procesamiento adicional de photopatterning y electrospinning multi-polímero se describe.

Video: Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

Experimental Approaches to Tissue Engineering

Métodos experimentales para la Ingeniería de Tejidos

Investigación

Biología

Micro-scale Engineering for Cell Biology

Micro-escala de Ingeniería de Biología Celular

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Actina Co-sedimentación de ensayo, pues el análisis de proteína de unión a la F-actina

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Microfabricación de chip del tamaño de andamios para el cultivo de células en tres dimensiones

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research

A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.

In the past many in vitro culture systems -- mainly monolayer cultures -- often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.

Entrevista: biorreactores y en superficie modificada andamios 3D para la Investigación de Células Madre

A Nature Editorial in 2003 asked the question  "Good-bye, flat biology?"  What does this question imply? In the past, many in vitro culture systems,  ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

La encapsulación celular en hidrogeles 3D para ingeniería de tejidos

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

Ingeniería penetran en las células de proteínas

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Un sistema para el cultivo de células epiteliales del pigmento del iris para el Estudio de Regeneración del objetivo de Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

Transducción de las células humanas con polímeros complejos Lentivirus ecotropic de Mayor Seguridad de la Biotecnología

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Inducción y prueba de hipoxia en el cultivo celular

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...