Este trabajo fue apoyado en parte por la National Science Foundation (CBET 0846453) y los Institutos Nacionales de Salud (1R01GM089999).

1. Fabricación de los maestros de silicio estándar con SU-8 fotolitografía 1 (no se muestra en este video). <ol><li> El uso estándar de SU-8 métodos de fotolitografía para crear un SU-8 &quot;maestros&quot; (SU-8 2050, MicroChem, Newton, MA) para la fabricación de los diferentes componentes (PDMS de diferente grosor, el co-cultivo de la cámara) en una sala limpia. Moldes como maestro se pueden fabricar en cualquier centro de microfabricación (por ejemplo, Stanford microfluídica de fundición; <a href="http://thebigone.stanford.edu/foundry/">http://thebigone.stanford.edu/foundry/</a> ). </li><li> Antes de la replicación PDMS, exponer el SU-8 maestros a fluorosilane vapor en un desecador conectado a una fuente de vacío para facilitar la liberación fácil de PDMS del maestro. Añadir una gota de fluorosilane a una toalla de papel y aplicar vacío durante un minuto. Retire el vacío y dejar 30 minutos para la deposición de fluorosilane. Mantenga el SU-8 maestro en un recipiente cerrado para su uso futuro. </li></ol> 2. Replica moldeo de PDMS de SU-8 master 2 <ol><li> La capa de fluido y la capa de control son realizadas por moldeo réplica de PDMS de SU-8 maestro. </li><li> Mezclar el PDMS pre-polímero y la cruz enlazador en un peso de 10:01. relación y desgasificar en un desecador durante una hora (o hasta que las burbujas de aire de la mezcla de PDMS). </li><li> Coloque el SU-8 maestro en una placa de Petri y con cuidado vierta la mezcla de PDMS en la parte superior de la SU-8 maestro. Desgasificar la PDMS nuevo. </li><li> Girar el molde a 1200 rpm durante un minuto para conseguir un espesor uniforme PDMS de alrededor de 100μm en el molde. </li><li> Calentar la cápsula de Petri que contiene el PDMS y el SU-8 molde maestro a 80 ° C durante una hora para curar el PDMS. </li><li> Quite la cubierta de curado PDMS SU-8 maestros de la placa. </li><li> Pelar el molde de PDMS del maestro SU-8. </li><li> Perfore cuatro agujeros en el molde de PDMS con un calibre 20 de extremo romo de la aguja dos entradas de bacterias, una toma de corriente y un puerto de neumáticos. </li></ol> 3. La unión de varias capas dispositivos PDMS: Este dispositivo se monta con tres capas: una capa superior que contiene el canal principal, una capa de neumático medio, y una capa inferior de bacterias que contiene los canales para las bacterias. Las tres capas se montan como se describe a continuación. <ol><li> Húmedas las membranas de PDMS con metanol y con cuidado la cáscara de la SU-8 molde maestro utilizando pinzas limpias. Coloque la membrana en una hoja de teflón. </li><li> Coloque la membrana de neumáticos y la capa de fluido en una cámara de plasma de oxígeno. A su vez en el plasma durante 40 segundos (la presión de oxígeno 10 sccm, RF 100W). </li><li> Después de la exposición al plasma, alinee (10 minutos) de la membrana en la parte inferior del canal PDMS el uso de fórceps. Adición de metanol en la membrana y ayuda a las capas PDMS libre circulación de las capas, y hace que la alineación sea más fácil. En este caso, el dispositivo se coloca en un trozo de gasa para permitir que los vapores de metanol para escapar. </li><li> Curar las capas alineadas PDMS sobre una placa a 80 ° C durante 8 h. </li><li> Después de la unión de la membrana neumática en la capa inferior, repita los pasos 3,2 a 3,4 para unir el canal superior en el dispositivo montado compuesto PDMS. </li></ol> 4. Montaje del dispositivo de PDMS 3,4 <ol><li> Taladrar agujeros en los puertos de entrada y salida utilizados para la siembra de las células eucariotas y las bacterias. </li><li> Conecte el tubo Tygon (0,01 pulgadas OD) a los puertos y operar la capa de neumáticos tirando con una jeringa de 20 ml. </li><li> Coloque el modelo de PDMS y un portaobjetos de vidrio pre-limpieza en una cámara de plasma de oxígeno. A su vez en el plasma durante 20 segundos (la presión de oxígeno 20 sccm, RF de 300 vatios), sin aplicar el vacío. </li><li> Con el fin de evitar el contacto entre la isla de la pared (PDMS) de unión a la lámina de vidrio, inmediatamente conecta una jeringa en el dispositivo y aplicar vacío tirando de la jeringa y manteniéndolo en su lugar. </li><li> Presione suavemente el modelo de PDMS sobre el vidrio (en 2 minutos). </li><li> Cure el dispositivo PDMS a 80 ° C durante 10 minutos. </li></ol> 5. El tratamiento de la superficie del vidrio con la fibronectina <ol><li> Esterilizar dispositivo bajo UV durante 15 minutos después de la conexión de tubos Tygon para cada puerto. </li><li> Llena y el flujo de la esterilización en el canal PBS para eliminar los desechos en el canal. Aplicar el flujo de 10 minutos para eliminar las burbujas de aire. Las burbujas de aire pueden escapar fácilmente el PDMS, debido a su permeabilidad a los gases. </li><li> Llene una solución de tinte de color en el canal de neumáticos para evitar la formación de burbujas de aire en el canal. </li><li> Para facilitar la siembra de células eucariotas en el cristal, introducir un 1 mg / ml de solución de fibronectina en el dispositivo y se incuba durante 40 min a 37 ° C. </li><li> Lavar el exceso de fibronectina con 1 ml de medio de crecimiento. </li></ol> 6. Formación y medición de los comensales E. coli en el biofilmislas <ol><li> Diluir las bacterias comensales (por ejemplo, E. coli BW25113/pCM18) en los medios de comunicación M9 mínimo de OD 600 ~ 1 para la formación de un biofilm comensales en la puerta cerrada / islas. </li><li> Cerca de la pared isla mediante la aplicación de presión positiva-líquido. Un casero de aire-líquido del convertidor de presión se utiliza para prevenir la formación de burbujas de aire en el canal. </li><li> Introducir el comensal E. coli en las islas con la válvula cerrada, y permitir que las bacterias para fijar durante 1 hora a 32 ° C. </li><li> Lavado de las bacterias sin ataduras y perfundir una suspensión diluida bacteriana (OD 600 ~ 0,05) en 0.25mL/hr durante 12 horas. </li><li> Enjuague la biopelícula con los medios de cultivo DMEM fresco para eliminar las bacterias débilmente unidos y prepararse para la fijación de las células eucariotas. </li><li> La imagen de la biopelícula con una Leica TCS SP5 Confocal Laser Scanning Microscope (Bannockburn, IL), y visualizar la arquitectura de biofilm con el software Imaris 5. </li></ol> 7. La siembra de las células eucariotas alrededor de las islas bacteriana 6-8 <ol><li> Trypsinize células HeLa del frasco de cultivo de tejidos y resuspender en DMEM los medios de comunicación para una densidad de siembra de 5 x 10 5 células / ml. </li><li> Introducir células HeLa en el dispositivo con un caudal de 2 ml / min usando una bomba de jeringa Picoplus (Harvard Apparattus, MA). Asegúrese de que el muro de la isla es baja por lo que la biopelícula bacteriana es independiente de la región de células HeLa. Este paso se debe realizar en el escenario de un microscopio de luz para que la uniformidad de la siembra se puede determinar. </li><li> Abrazadera de la toma de células para reducir el movimiento de la suspensión de células en el canal y se incuba durante 6 horas a 37 ° C en un 5% de CO 2 incubadora. </li><li> Perfundir el dispositivo con el medio de crecimiento a 0,5 mL / h para eliminar las células sueltas. </li><li> Actualizar los medios de comunicación DMEM de HeLa y E. comensales coli, cada 6 horas. </li></ol> 8. Introducción de bacterias patógenas en la isla y la exposición a las células eucariotas <ol><li> Prepare E. coli O157: H7 (ECEH) para infectar las células HeLa a una multiplicidad de infección (MOI) de 100:1 200:1 (número de células HeLa por ECEH). </li><li> Enjuagar y lavar las bacterias comensales débilmente unidos. </li><li> Introducir una suspensión diluida EHEC (OD 600 ~ 0,1) en las islas. </li><li> Incubar dispositivo a 37 ° C en un 5% de CO 2 incubadora durante 6 horas sin flujo para permitir que ECEH de estar expuestos a las señales presentes en el biofilm comensales. </li><li> Levantar el muro de PDMS para exponer por ECEH en las islas comensales a las células HeLa durante 6 horas. </li><li> Enjuague el canal PBS y la mancha de células vivas y muertas utilizando el kit de Live / Dead (L3224, Invitrogen, CA). </li><li> Lugares de múltiples imágenes en un microscopio Zeiss Axiovert 200M fluorescencia y estimar el número de células vivas y muertas. </li></ol> 9. Los resultados representativos 9 La secuencia de eventos en las infecciones del tracto GI fue imitado por el desarrollo de una monocapa de células HeLa y E. comensales coli biofilm y la exposición de ECEH de la biopelícula comensales antes de la infección de células HeLa. Los pasos involucrados en la fabricación de la co-cultivo de microfluidos del modelo se muestra en la Figura 1. Figura 2A muestra una representación en 3 dimensiones del dispositivo de co-cultivo. Las dos regiones en el dispositivo de co-cultivo de la cámara de cultivo de células eucariotas y la isla de bacterias se muestran en la Figura 2B con tintes de diferentes colores (morado para las regiones eucariotas y verde para la isla de bacterias). La posibilidad de aislar a las regiones de cultivo bacteriano de la región circundante eucariotas se muestra en la Figura 2C el uso de tintes de color. Figura 3 muestra los resultados representante de co-cultivo de células epiteliales y bacterias. Figura 3A muestra la colonización del biofilm comensales (verde) por la isla ECEH (rojo). Figura 3B muestra la yuxtaposición de las células epiteliales y E. comensales coli con ECEH en la isla de bacterias. El PP expresa ECEH y las buenas prácticas agrarias expresar las bacterias comensales se localizan en la isla cuando la pared PDMS se reduce, lo que demuestra la viabilidad de aislar a la isla de bacterias de las células epiteliales. <img src="/files/ftp_upload/1749/1749fig1.jpg" alt="Figura 1" /> Figura 1: Esquema de la siembra celular en el modelo de co-cultivo. (1) El muro de PDMS se reduce para formar una isla, las bacterias comensales son introducidos en la isla, y fibronectina se corría alrededor de la isla. (2) células HeLa se siembran en las regiones que rodean la isla. (3) Después de llegar a las células HeLa confluencia y el biofilm comensales ha desarrollado, ECEH se introduce en la isla. (4) El muro de PDMS se levantapara exponer las células HeLa que rodean la isla de ECEH. El recuadro muestra los detalles de la operación de la válvula. <img src="/files/ftp_upload/1749/1749fig2a.jpg" alt="Figura 2a" /> <img src="/files/ftp_upload/1749/1749fig2bc.jpg" alt="Figura 2 AC" /> Figura 2: Modelo de microfluidos para el co-cultivo de células epiteliales y bacterias. (A) en tres dimensiones versión del dispositivo de co-cultivo que muestra neumático accionado por regiones de captura para la formación de islas de las infecciones en las células epiteliales. Cada isla bacteriana (1200 m de diámetro y 1.000 m de distancia) tiene una entrada independiente y una salida para proporcionar medios de cultivo y la eliminación de los residuos de la isla. (B) Micrografía del dispositivo de co-cultivo con tintes de colores que muestra las diferentes regiones (zona de las células epiteliales, las islas de bacterias). (C) La fidelidad del sistema de recogida neumática se muestra mediante la reducción de la pared PDMS (panel izquierdo), utilizando un canal neumática activa (azul), la introducción de tinte púrpura en la cerrada islas, y colorante amarillo que fluye alrededor de ella durante 48 h. Cuando el muro se levanta PDMS (panel derecho), la región insular está expuesta al medio circundante de color amarillo. Barra de escala representa a 500 micras. <img src="/files/ftp_upload/1749/1749fig3.jpg" alt="Figura 3" /> Figura 3: Co-cultivo de células HeLa y bacterias. (A) Imagen de fluorescencia de RFP-expresando ECEH y que expresan GFP E. coli BW25113 en la isla. (B) Superposición de transmisión, verde y rojo, imágenes de fluorescencia en el dispositivo. Barra de escala representa a 200 micras.

<ol>
	<li>McDonald, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prototyping+of+microfluidic+devices+in+poly(dimethylsiloxane)+using+solid-object+printing.">Prototyping of microfluidic devices in poly(dimethylsiloxane) using solid-object printing.</a> Anal Chem. 74, 1537-1545 (2002).</li><li>Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+fabrication+of+integrated+passive+valves+and+pumps+for+flexible+polymer+3-dimensional+microfluidic+systems.">Design and fabrication of integrated passive valves and pumps for flexible polymer 3-dimensional microfluidic systems.</a> Biomed Microdevices. 4, 117-121 (2002).</li><li>Baek, J. Y., Park, J. Y., Ju, J. I., Lee, T. S., Lee, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pneumatically+controllable+flexible+and+polymeric+microfluidic+valve+fabricated+via+in+situ+development.">A pneumatically controllable flexible and polymeric microfluidic valve fabricated via in situ development.</a> J Micromech Microeng. 15, 1015-1020 (2005).</li><li>Grover, W. H., Ivester, R. H., Jensen, E. C., Mathies, R. A., A, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+multiplexed+control+of+latching+pneumatic+valves+using+microfluidic+logical+structures.">Development and multiplexed control of latching pneumatic valves using microfluidic logical structures.</a> Lab Chip. 6, 623-631 (2006).</li><li>Lee, J., Jayaraman, A., Wood, T. K., K, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indole+is+an+inter-species+biofilm+signal+mediated+by+SdiA.">Indole is an inter-species biofilm signal mediated by SdiA.</a> BMC Microbiol. 7, 42-42 (2007).</li><li>Hsu, C. H., Folch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+with+tunable+topographies.">Microfluidic devices with tunable topographies.</a> Appl Phys Lett. 86, 023508-023508 (2005).</li><li>Hui, E. E., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromechanical+control+of+cell-cell+interactions.">Micromechanical control of cell-cell interactions.</a> Proc Natl Acad Sci USA. 104, 5722-5726 (2007).</li><li>Lee, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+sensing+hydrogel+microstructures+into+micropatterned+hepatocellular+cocultures.">Integrating sensing hydrogel microstructures into micropatterned hepatocellular cocultures.</a> Langmuir. 25, 3880-3886 (2009).</li><li>Kim, J., Hegde, M., Jayaraman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-culture+of+epithelial+cells+and+bacteria+for+investigating+host-pathogen+interactions.">Co-culture of epithelial cells and bacteria for investigating host-pathogen interactions.</a> Lab-on-Chip. , (2009).</li></ol>

Ensayos convencionales para la fijación y la colonización de patógenos utilizar una monocapa de células eucariotas en placas de cultivo de tejido en el que los agentes patógenos se agregan. Estos modelos no son fisiológicamente relevantes, ya que no incorporan un biofilm de bacterias comensales desarrollado en las células eucariotas. Simple adición de un cultivo de bacterias pre-convertido en células eucariotas es improbable que lleve a esta conformación, como los biofilms son muy organizadas las estructuras q...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
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<td>SU-8 2050</td>
<td>&nbsp;</td>
<td>Microchem Corp, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>high-resolution (16,256 dpi) photolithography mask</td>
<td>&nbsp;</td>
<td>Fineline-Imaging Inc, CO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Trichloro(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)silane</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>77279</td>
<td>&nbsp;</td>
<tr>
<td>PDMS</td>
<td>&nbsp;</td>
<td>Dow Corning, WI</td>
<td>184 SIL ELAST KIT 0.5KG</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>SH30002.02</td>
<td>&nbsp;</td>
<tr>
<td>Programmable spin coater</td>
<td>&nbsp;</td>
<td>Laurell Tech Corp</td>
<td>WS0650S</td>
<td>&nbsp;</td>
<tr>
<td>Mask aligner</td>
<td>&nbsp;</td>
<td>Neutronix-Quintel, PA</td>
<td>Q4000</td>
<td>&nbsp;</td>
<tr>
<td>Oxygen plasma etcher</td>
<td>&nbsp;</td>
<td>March Plasma System, CA</td>
<td>CS-1701</td>
<td>&nbsp;</td>
<tr>
<td>Syringe pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Live/Dead Viability/Cytotoxicity Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>L-3224</td>
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microfluidic co-culture of epithelial cells and bacteria for investigating soluble signal-mediated interactions

El ser humano gastrointestinal (GI) es un entorno único en el que las células epiteliales intestinales y no patógenas (comensales), las bacterias conviven. Se ha propuesto que el microambiente que el patógeno se encuentra en la capa de comensales es importante para determinar la extensión de la colonización. Los métodos actuales de cultivo para la investigación de la colonización de patógenos no son muy adecuadas para la investigación de esta hipótesis, ya que no permiten el co-cultivo de bacterias y células epiteliales de una forma que imita el microambiente del tracto gastrointestinal. Aquí se describe un co-cultivo de microfluidos modelo que permite a la cultura independiente de las células eucariotas y las bacterias, y probar el efecto del microambiente comensales en la colonización de patógenos. El modelo de co-cultivo se demuestra mediante el desarrollo de un comensal<em&gt; Escherichia coli</em&gt; Biofilm entre las células HeLa, seguido por la introducción de enterohemorrágica<em&gt; E. coli</em&gt; (EHEC) en la isla de comensales, en una secuencia que reproduce la secuencia de eventos en infección del tracto GI.

Watch this Scientific Journal Video about Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions at JoVE.com

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

Este protocolo describe un co-cultivo de microfluidos modelo de cultivo simultáneo y localizada de células epiteliales y bacterias. Este modelo se puede utilizar para investigar el papel de las diferentes señales moleculares solubles en la patogénesis, así como la pantalla de la supuesta eficacia de cepas de bacterias probióticas.

Microfluidos Co-cultivo de células epiteliales y bacterias para la Investigación de la señal mediada Soluble Interacciones

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

microfluidic-co-culture-epithelial-cells-bacteria-for-investigating

Research

JoVE Journal

Biology

14.0K vistas.  Texas A&M University. Este protocolo describe un co-cultivo de microfluidos modelo de cultivo simultáneo y localizada de células epiteliales y bacterias. Este modelo se puede utilizar para investigar el papel de las diferentes señales moleculares solubles en la patogénesis, así como la pantalla de la supuesta eficacia de cepas de bacterias probióticas.

Video: Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

A neuronales y astrocitos Co-cultivo de ensayo para el análisis de alto contenido de neurotoxicidad

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Investigación

Biología

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

Un compartimiento de Multi-CNS neuronas-glia Co-cultivo de microfluidos Plataforma

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Aislamiento de células epiteliales mamarias de tridimensional mixto de células Esferoide Co-cultura

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Inducción y Análisis de epitelial a mesenquimal de transición

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

La investigación de las interacciones proteína-proteína en las células vivas utilizando la transferencia de energía de resonancia de bioluminiscencia

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Las mediciones del calcio citosólico en las células epiteliales renales mediante citometría de flujo

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

Método cryosectioning para microdisección de murino mucosa colónica

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

El uso de dispositivos de microfluidos para Medir Lifespan y Celular fenotipos en células individuales levadura en ciernes

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

Monitoreo in situ de conjuntos de chaperona molecular formadas transitoriamente en bacterias, levaduras y células humanas

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor &#945;) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.

Modelo alveolar humano multicelular compuesto por células epiteliales y células inmunes primarias para la evaluación de riesgos

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...