Los autores agradecen a Ronald F. Renzi y Michael S. Bartsch por sus contribuciones al diseño y desarrollo de dispositivos de DMF y el cubo DMF. Esta investigación fue plenamente apoyada por el Programa de Desarrollo de Laboratorio Director de Investigación y en Sandia National Laboratories. Sandia es un laboratorio multi-programa administrado y operado por Sandia Corporation, una subsidiaria de propiedad total de Lockheed Martin Corporation, para el Departamento de Administración de Seguridad Nuclear Nacional de Energía de los EE.UU. bajo contrato DE-AC04-94AL85000.

Este protocolo general está diseñada para soportar la aplicación de la plataforma a una amplia variedad de estudios; aspectos específicos para el estudio representativo se describe en este informe se separan entre paréntesis Figura 2 ilustra un estudio representativo llevado a cabo utilizando el protocolo.. Tenga en cuenta que en el protocolo, todos los comandos de &quot;instruir&quot; se automatizan con el uso de scripts predeterminados. Tenga en cuenta también que el paso 2 puede llevarse a cabo en paralelo con el Paso 1 (por ejemplo, durante los pasos 1.7 y 1.8), y que el paso 2.1 es a menudo ignoradas (porque las placas de dispositivos DMF modelado / recubiertos se pueden lavar y volver a utilizarse; ver Paso 5.2) . 1. Ensamble y rellenar las cámaras de perfusión celular <ol><li> Escudo las superficies interiores de esterilizado (autoclave) microcapilares (sílice fundida; 679 micras OD, 534 Identificación del micras; 15 cm de largo) con fibronectina, dando instrucciones a la bomba de jeringa para extraer una solución de fibronectina estéril (30 l de 50 mg / ml) en cada unomicrocapilar, incubando (37 ° C durante 2 hr), dando instrucciones a la bomba de jeringa para extraer la solución, y dejar que el microcapilares seco (temperatura ambiente (TA) durante 6 horas). </li><li> Conectar cada microcapilar recubiertos de fibronectina (cámara de perfusión) a un tubo de policarbonato (500 micras OD, 100 Identificación del micras) y el tubo a la bomba de jeringa multi-puerto, el uso de accesorios de CapTite (6 cámaras de perfusión, 8-puerto de la bomba de jeringa). </li><li> Con cinta, asegure el cuerpo de cada cámara de perfusión a un bloque de calor establecidas en la temperatura deseada (37 º C). </li><li> Sumergir los extremos abiertos de las cámaras de perfusión en un depósito (tubo de microcentifuge o placa de microtitulación de) células que contienen (P388D.1 macrófagos murinos) suspendidas en medio de crecimiento fresco (RPMI-1640, 10% de FBS, 500 unidades / ml de penicilina, 500 mg / ml de estreptomicina) a una concentración deseada (10 6 células / ml). </li><li> Instruir a la bomba de jeringa para extraer células (10 l, 10 4 células) en cada cámara de perfusión seguido por suficiente air para mover el tapón de líquido (~ 4,5 cm) a la sección asegurada al bloque de calor. </li><li> Permitir que las células se adhieran a las superficies interiores recubiertas de fibronectina de las cámaras de perfusión (37 ° C durante 1 - 2 h). Mientras tanto, la transferencia de los extremos abiertos de las cámaras de perfusión desde el depósito de las células a un nuevo depósito que contiene medio de cultivo fresco. </li><li> Después del periodo de cumplimiento, instruir a la bomba de jeringa para enviar los tapones de líquido (medio condicionado y las células no unidas) a los residuos, retirar un nuevo medio (10 l), y seguir con el aire suficiente para colocar las nuevas bujías de líquido (medio fresco) sobre el adherido las células. </li><li> Continuar llevando a cabo intercambios media (es decir, repita el Paso 1.7) a intervalos regulares (cada 2 horas) hasta las poblaciones de células son suficientemente equilibrada y amplia (16 - 24 h de cultivo a microescala generados poblaciones de ~ 1.000 células / cámara). </li></ol> 2. Fabrique el dispositivo DMF y Monte la Arquitectura Hub <ol><li&gt; Como se describió previamente 14,29, patrón de la placa inferior del dispositivo de DMF con cuarenta y seis de óxido de indio y estaño (ITO) electrodos (conductores de gota accionamiento) por fotolitografía y el grabado, y la capa con 5 micras de parileno-C y 50 nm de teflón AF. Formar la placa superior del dispositivo de DMF por un sustrato de vidrio de revestimiento no pautada ITO con teflón AF, como anteriormente. </li><li> El uso de fotogramas de compresión de metal, fijar las placas de DMF en un molde de polímero 14,30 con rebajes que mantienen una separación de 400 m entre las placas; esta separación, combinado con el tamaño del electrodo de accionamiento (2.5 mm 2), define el volumen de la gotita (2,5 l por electrodo). Tenga en cuenta que el conjunto de DMF se puede lograr por medios más simples 24. </li><li> Insertar transferencia revestido de teflón microcapilares (360 micras OD, 100 Identificación del micras; 3.5 - 4.0 cm de largo) en el espacio entre las placas de DMF, posicionar cada uno para extenderse hasta el borde de su electrodo de accionamiento cognado. </li><li> Para la linea de frented de cada transferencia colocar microcapillary un accesorio CapTite. </li><li> Enganche los conectores eléctricos para suministrar voltaje a los electrodos de ITO. Secuencias de activación de electrodos están automatizadas a través del uso de un interfaz electrónico controlado por ordenador ejecutar scripts predeterminados. </li><li> Opcional: Mover el cubo DMF montada en la etapa de traslación de un microscopio (SZ-6145TR (Olympus, Japón)) equipado con un dispositivo de carga acoplada (CCD) de la cámara (DCR-HC96 (Sony, Japón)) para facilitar el seguimiento de las gotitas 31. </li></ol> 3. Conecte las cámaras de perfusión al Hub DMF y estimular las poblaciones celulares <ol><li> Retire los extremos abiertos de las cámaras de perfusión del depósito de medio (consulte el Paso 1.6) e insertarlos en los conectores CapTite fijados a los extremos de la transferencia microcapilares del cubo DMF (ver Paso 2.4). </li><li> Instruir a la bomba de jeringa para enviar los tapones de líquido (medio acondicionado) en las cámaras de perfusión que perder. </li><li> InstRUCT el cubo de DMF para elaborar estímulo (E. coli biopartículas a 100 g / ml en medio fresco con 0,1% de Pluronic F127 w / v) de un depósito de a bordo y entregar una gota de tamaño apropiado (10 l) a cada transferencia microcapilar . Los reservorios de a bordo se componen de compartimentos con forma cilíndrica (1,5 x 1,5 cm cada una) y conectados al concentrador DMF a través de tubos. </li><li> Instruir a la bomba de jeringa para extraer los tapones de estímulo a la transferencia microcapilares y siga con el aire suficiente para colocar los tapones en las poblaciones de células en las cámaras de perfusión. </li><li> Se incuban las células con el estímulo (37 ° C durante 1 - 4 horas). Opcional: Cambio de un nuevo impulso (es decir, repita los pasos 3.2 a 3.4) a intervalos regulares. </li></ol> 4. Terminar estimulación y recuperación lisados ​​de células para el análisis <ol><li> Opcional: Si se requiere un período posterior a la estimulación, intercambiar los tapones de líquidos que contienen estímulo para el medio fresco (es decir, repita los pasos 3,2-30.4, sustituyendo el medio fresco para el estímulo), y continuar a incubar e intercambiar según sea necesario. </li><li> Cambio de los tapones de líquido en las cámaras de perfusión de tampón de lavado (preludio directo Lisis Módulo de Tampón B) (es decir, repita los pasos 3.2 a 3.4, sustituyendo el tampón de lavado para el estímulo), y se incuba según sea necesario (5 min). </li><li> Cambio de los tapones de líquido (tampón de lavado) para la solución de lisis (preludio directo Tampón de Lisis Módulo A con 0,1% de Pluronic F127 w / v) (es decir, repita los pasos 3.2 a 3.4, sustituyendo solución de lisis para el estímulo), y se incuba según sea necesario (5 min) . </li><li> Instrucciones a la bomba de jeringa para enviar los tapones de líquido (lisados ​​celulares) al cubo DMF. </li><li> Desmontar el eje DMF, retire la placa superior del dispositivo DMF (teniendo cuidado de no inclinar el plato de lado, ya que esto puede dar lugar a gota la mezcla), y recoger los lisados ​​utilizando un Pipetman o jeringa para análisis fuera de la plataforma (perfiles de expresión génica mediante qPCR). </li></ol> 5. Plataformas de hardware puro paraReutilización <ol><li> Sumergir la transferencia microcapilares y accesorios CapTite en lejía al 10% (v / v en agua) durante 10 min a TA, enjuague bien con agua desionizada, y el uso de gas nitrógeno seco. </li><li> Sumergir las placas de dispositivos DMF en 10% de lejía por 10 min a TA, enjuague bien con isopropanol seguido por agua desionizada, y el uso de gas nitrógeno seco seguido de cocción a 160 ° C durante 10 min. </li><li> El tubo de policarbonato de la bomba de jeringa, y el polímero fundido del dispositivo de DMF y el marco de la compresión, se puede volver a utilizar sin limpieza. La cámara de perfusión microcapilares se desechan después de cada experimento. </li></ol>

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H., Oliver, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarrays,+deep+sequencing+and+the+true+measure+of+the+transcriptome.">Microarrays, deep sequencing and the true measure of the transcriptome.</a> BMC Biology. 9, (2011).</li><li>Wu, 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=Microfluidically-unified+cell+culture,+sample+preparation,+imaging+and+flow+cytometry+for+measurement+of+cell+signaling+pathways+with+single+cell+resolution.">Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution.</a> Lab on a Chip. 12, 2823-2831 (2012).</li><li>Srivastava, N., 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=Fully+integrated+microfluidic+platform+enabling+automated+phosphoprofiling+of+macrophage+response.">Fully integrated microfluidic platform enabling automated phosphoprofiling of macrophage response.</a> Analytical Chemistry. 81, 3261-3269 (2009).</li><li>Herr, A. E., 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+immunoassays+as+rapid+saliva-based+clinical+diagnostics.">Microfluidic immunoassays as rapid saliva-based clinical diagnostics.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 5268-5273 (2007).</li></ol>

Los autores declaran que no tienen intereses financieros en competencia.

Hemos desarrollado una plataforma automatizada simple y versátil para el cultivo celular micro-escala y experimentos de estimulación. La plataforma nos permite trabajar con pequeñas cantidades de cultivos y poblaciones de células (1 - 20 ly 100 - 2000 células por cámara); tamaños cultura podría reducirse aún más mediante el uso de microcapilares de menor diámetro. Trabajar en estas escalas reduce el costo de estudios de rutina y hace que los estudios viables que requieren el uso de reactivos precioso...

El estudio de las células se mantuvieron en cultivo (análisis in vitro) ha proporcionado valiosa información sobre los principios fundamentales y los mecanismos moleculares que regulan los complejos sistemas biológicos y la salud humana. Los métodos convencionales para el cultivo, la estimulación, y colección de las células para el análisis, que utilizan placas de Petri y las placas de microtitulación, se han diseñado para el estudio de grandes poblaciones de células (≥ 10 5) en volúmenes de cultivo escala ≥ mililitro (0,1 ml). Sin embargo, hay muchos casos en los que sólo cantidades limitadas de células están disponibles (por ejemplo, células primarias), o pequeñas poblaciones de células son deseables (por ejemplo, para reducir la variabilidad de célula a célula a través de la población), o reactivos requeridos son difíciles de obtener o prohibitivamente caro (por ejemplo, factores secretados por células purificadas). Estos temas pueden ser abordados con éxito por la escala hasta el tamaño cultura, que tiene el beneficio adicional de reducir el consumo de todos losreactivos necesarios para el análisis en 1,2 vitro. Desafortunadamente, el equipo y los métodos convencionales no soportan la manipulación precisa y reproducible de las culturas micro-escala y los sistemas automatizados de microfluidos basadas actualmente disponibles 3-11 son demasiado complejos y especializados para el uso habitual en la mayoría de los laboratorios. En este reporte se describe el montaje y el uso de una plataforma de tecnología simple y versátil para el cultivo automatizado, el estímulo y la recuperación de las pequeñas poblaciones de células (100 - 2000 células) en los volúmenes de micro-escala (1-20 l). La arquitectura de la plataforma (Figura 1) es de diseño modular: un conjunto de fibronectina recubierto microcapilares (&quot;cámaras de perfusión de células&quot; módulo) sirve como el sitio para el establecimiento, el mantenimiento, y la estimulación de cultivos de micro-escala, y un microfluídica digital (DMF ) 12,13 14,15 dispositivo equipado con células de rutas &quot;transferencia&quot; microcapilares (módulo central &quot;hub&quot;)y reactivos hacia y desde las cámaras de perfusión. DMF permite al usuario resolver individualmente varias gotas al mismo tiempo y cambiar o volver a ordenar las manipulaciones (es decir, volver a configurar los trenes de procesamiento de la muestra) sin alterar el hardware del dispositivo. Su gran flexibilidad es evidente en su reciente aparición como una tecnología clave en una amplia gama de aplicaciones, incluido el cultivo celular 16,17, 18,19 ensayos enzimáticos, inmunoensayos 20,21, 22,23 análisis de ADN, el procesamiento de proteínas, 24,25 y procesamiento de la muestra clínica. 26,27 Nuestro cubo central toma ventaja de la flexibilidad inherente a los dispositivos de DMF, y se mejora aún más mediante la adición de interfaces de microcapilares, lo que proporciona la oportunidad de llevar a cabo un subconjunto de manipulaciones (por ejemplo, cultivo celular) en periférica especializada módulos, en lugar de en el dispositivo de DMF en sí. La compartimentación de los trenes de procesamiento de esta manera también simplifica el diseño de la plaArquitectura TForm (sin necesidad de construir un dispositivo de DMF que pueden llevar a cabo todas las etapas de elaboración) y facilita su evolución como nuevas funciones se requieren (simplemente integrar nuevos módulos periféricos, según sea necesario). Transporte de las células y los reactivos dentro del cubo central es accionado por electrohumectación fuerzas generadas por la activación secuencial de electrodos dentro del dispositivo de DMF 13,28; transporte hacia, desde y dentro de las cámaras de perfusión es impulsado por los cambios de presión generados por una jeringa de alta precisión bomba. Todos estos movimientos fluidos son controlados a través de una interfaz electrónica simple y automatizado a través del uso de secuencias de comandos predeterminados. Como ejemplo representativo, se demuestra el uso de la plataforma para el estudio de las respuestas transcripcionales provocado en las células inmunes al reto con bacterias (Figura 2). La realización de estos experimentos en la plataforma nos permitió trabajar con un pequeño número de células ~ 1000 por la experimentación (condición de tal), minimizar la variabilidad experimento a experimento, reactivos conservar y volver a dirigir las manos-sobre el trabajo. Dadas las ventajas que ésta confiere, así como su accesibilidad y versatilidad, esta plataforma debe encontrar uso en una amplia variedad de laboratorios y aplicaciones y resultar especialmente útil para facilitar el análisis de las células y los estímulos que están disponibles sólo en cantidades limitadas.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Prelude Direct Lysis Module</td>
 <td>NuGEN</td>
 <td>1400-24</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypan Blue (0.4% w/v)</td>
 <td>GIBCO</td>
 <td>15250-061</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell Stripper</td>
 <td>Cellgro</td>
 <td>25-056-C1</td>
 <td></td>
 </tr>
 <tr>
 <td>Ovation PicoSL WTA</td>
 <td>NuGEN</td>
 <td>3310-048</td>
 <td></td>
 </tr>
 <tr>
 <td>Agencourt RNAClean XP</td>
 <td>Beckman Coulter Genomics</td>
 <td>A63987</td>
 <td></td>
 </tr>
 <tr>
 <td>pHrodo BioParticles</td>
 <td>Invitrogen</td>
 <td>P35361</td>
 <td></td>
 </tr>
 <tr>
 <td>CCL4 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00443111_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>CCL5 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm01302428_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>PTGS2 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00478374_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>TNF TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00443258_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>GAPDH TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm99999915_g1</td>
 <td></td>
 </tr>
 <tr>
 <td>Pluronic F127</td>
 <td>Sigma Chemical</td>
 <td>2594628</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluorinert FC-40</td>
 <td>Sigma Chemical</td>
 <td>51142-49-5</td>
 <td></td>
 </tr>
 <tr>
 <td>Parylene C dimer</td>
 <td>Specialty Coating Systems</td>
 <td>28804-46-8</td>
 <td></td>
 </tr>
 <tr>
 <td>Teflon-AF</td>
 <td>DuPont</td>
 <td>AF1600</td>
 <td></td>
 </tr>
 <tr>
 <td>Polyimide tape</td>
 <td>ULINE</td>
 <td>S-11928</td>
 <td></td>
 </tr>
 <tr>
 <td>Indium tin oxide (ITO) coated glass substrates </td>
 <td>Delta Technologies </td>
 <td>CB-40IN-1107</td>
 <td></td>
 </tr>
 <tr>
 <td>DMF hub Teflon-coated fused-silica microcapillaries</td>
 <td>Polymicro Technologies</td>
 <td>TSU100375</td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber microcapillaries</td>
 <td>Polymicro Technologies</td>
 <td>TSP530700</td>
 <td></td>
 </tr>
 <tr>
 <td>Tubing and microcapillary fittings</td>
 <td>Sandia National Laboratories</td>
 <td>&nbsp;</td>
 <td></td>
 </tr>
 <tr>
 <td>Polycarbonate tubing</td>
 <td>Paradigm Optics</td>
 <td>CTPC100-500-5</td>
 <td></td>
 </tr>
 <tr>
 <td>8-port precision syringe pump equipped with 30 mm (500 &mu;l capacity) syringes</td>
 <td>Hamilton Company</td>
 <td>54848-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Parylene-C vapor deposition instrument</td>
 <td>Specialty Coating Systems</td>
 <td>PDS 2010 Labcoter 2</td>
 <td></td>
 </tr>
 <tr>
 <td>High-voltage function generator</td>
 <td>Trek</td>
 <td>615A-1 615-3</td>
 <td></td>
 </tr>
 <tr>
 <td>MVX10 microscope</td>
 <td>Olympus</td>
 <td></td>
 <td>Optional (facilitates tracking of droplets on DMF hub)</td>
 </tr>
 <tr>
 <td>QIClick digital CCD camera</td>
 <td>QImaging</td>
 <td>QIClick-F-CLR-12</td>
 <td>Optional (facilitates tracking of droplets on DMF hub)</td>
 </tr>
</tbody>
</table>

a versatile automated platform for micro-scale cell stimulation experiments

Estudio de las células en cultivo (análisis in vitro) ha proporcionado datos importantes en los sistemas biológicos complejos. Métodos y equipos convencionales para el análisis in vitro son muy adecuadas para el estudio de un gran número de células (≥ 10 5) en volúmenes de escala ≥ mililitro (0,1 ml). Sin embargo, hay muchos casos en los que es necesario o deseable que reducir el tamaño cultura para reducir el consumo de las células de interés y / o reactivos necesarios para su cultura, estimulación, o de procesamiento. Desafortunadamente, los enfoques convencionales no soportan una manipulación precisa y reproducible de cultivos de micro-escala, y los sistemas automatizados de microfluidos basados ​​actualmente disponibles son demasiado complejas y especializadas para el uso rutinario por la mayoría de los laboratorios. Para hacer frente a este problema, hemos desarrollado una plataforma de tecnología simple y versátil para el cultivo automatizado, estimulación, y la recuperación de pequeñas poblaciones de células (100 - 2000 células) en el volumen de micro-escalas (1 - 20 l). La plataforma consta de un conjunto de microcapilares recubiertos de fibronectina (&quot;cámaras de perfusión de células&quot;), dentro de la cual se establecieron cultivos de micro-escala, mantienen, y se estimularon; un dispositivo digital de microfluidos (DMF) equipado con &quot;transferencia&quot; microcapilares (&quot;cubo central &quot;), el cual las células rutas y reactivos hacia y desde las cámaras de perfusión; una bomba de jeringa de alta precisión, que alimenta el transporte de materiales entre las cámaras de perfusión y el eje central, y una interfaz electrónica que proporciona control sobre el transporte de materiales, que es coordinado y automatizado a través de scripts predeterminados. Como un ejemplo, se utilizó la plataforma para facilitar el estudio de las respuestas transcripcionales provocados en las células inmunes al reto con bacterias. El uso de la plataforma nos ha permitido reducir el consumo de las células y los reactivos, minimizar la variabilidad experimento a experimento, y re-directos práctica laboral. Dadas las ventajas que ésta confiere, así como su accesibilidad y versatilidad, Oplataforma ur debe encontrar uso en una amplia variedad de laboratorios y aplicaciones, y ser especialmente útil para facilitar el análisis de las células y los estímulos que están disponibles sólo en cantidades limitadas.

Como una demostración de la plataforma automatizada, lo usamos para llevar a cabo un estudio en el que pequeñas poblaciones de células inmunes se cultivaron en cultivos de micro-escala, desafió con bacterias, y zadas para el análisis fuera de la plataforma de las respuestas pro-inflamatorias (Figura 2 ). Cada una de las seis cámaras de perfusión de células se sembró con 10 4 células inmunes (P388D.1 macrófagos murinos) se resuspendieron en 10 l de medio...

Watch this Scientific Journal Video about A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments at JoVE.com

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Hemos desarrollado un cultivo de células automatizado y la plataforma de interrogación para experimentos de estimulación de células micro-escala. La plataforma ofrece un control sencillo, versátil y precisa en el cultivo y estimular las pequeñas poblaciones de células, y la recuperación de lisados ​​de análisis moleculares. La plataforma se adapta bien a los estudios que utilizan las células preciosos y / o reactivos.

Una plataforma automatizada versátil para Micro escala experimentos de estimulación celular

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

a-versatile-automated-platform-for-micro-scale-cell-stimulation

Research

JoVE Journal

Bioengineering

10.5K vistas.  Sandia National Laboratories. Hemos desarrollado un cultivo de células automatizado y la plataforma de interrogación para experimentos de estimulación de células micro-escala. La plataforma ofrece un control sencillo, versátil y precisa en el cultivo y estimular las pequeñas poblaciones de células, y la recuperación de lisados ​​de análisis moleculares. La plataforma se adapta bien a los estudios que utilizan las células preciosos y / o reactivos.

Video: A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

El colágeno organotípicos I del ensayo: Una plataforma maleable para evaluar el comportamiento celular en un contexto de 3 dimensiones

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Investigación

Bioingeniería

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
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GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.

GENPLAT: una plataforma automatizada para el descubrimiento de enzimas de la biomasa y la optimización Cocktail

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Exprimir la célula como un robusto, Microfluidic intracelular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

Una Plataforma Novela Estiramiento para Aplicaciones en Celular y Tisular Mecanobiología

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Una plataforma microfluídica para el aislamiento de gran procesamiento de celda única y Cultura

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Las construcciones en capas de alginato: Una plataforma para el co-cultivo de poblaciones celulares heterogéneas

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

Una de alto rendimiento plataforma de microarrays de células para análisis correlativo de la diferenciación celular y las fuerzas de tracción

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Un método versátil de Patterning proteínas y células

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Nanotubos de ADN como una herramienta versátil para el estudio de polímeros Semiflexible

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

Una plataforma microfluídica multicapa para la conducción de la expresión génica sin células prolongada

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...