Damos las gracias al personal de Anican (CRCL, Lyon) por su asistencia técnica con el cuidado animal. Este trabajo fue apoyado por el Institut National de la Santé et de la Recherche Médicale (Programa Avenir INSERM), la Ligue Nationale Contre le Cancer, por la Association pour la Recherche sur le Cancer, por el Instituto Nacional del Cáncer, y por becas de la Ligue Nationale Contre le Cancer (JG), del Instituto Nacional del Cáncer (JG), del Ministère de l&#39;Enseignement Supérieur et de la Recherche de Francia (RMP y DFV) y de la Association pour la Recherche sur le Cancer ( DFV).

Todos los procedimientos fueron aprobados por un comité de ética bajo reglamentación de la autoridad gubernamental (&quot;Comité d&#39;Evaluación Commun au Centre Léon Bérard, à l&#39;Animalerie de tránsito de l&#39;ENS, au PBES et au laboratoire P4&quot; (CECCAPP)). Los ratones fueron mantenidos en una instalación específica de animales libres de patógenos en el &quot;Plateforme Anican, Centro Léon Bérard&quot; (Lyon, Francia) y manipulen de conformidad con las directrices institucionales. Una representación esquemática del procedimiento se muestra en la Figura 1. 1. La disección del páncreas y dilaceración (día 0) Una disección muy rápido es crítico para un rendimiento óptimo de la extracción y para asegurar una buena viabilidad de las células en cultivo. Con el fin de reducir el tiempo necesario para el aislamiento del páncreas, todos los instrumentos y equipos deben estar preparados antes de la eutanasia del ratón. <ol><li><html> La eutanasia ratón por asfixia con CO2 o dislocación cervical. </li></ol> A partir de este paso, todos los procedimientos tienen que ser realizadas bajo una atmósfera estéril (cabina de seguridad microbiológica, el nivel II) con el equipo de disección estéril. <ol start="2"><li> Fijar el ratón y rociar el abdomen del ratón con etanol al 70%. Con las tijeras de disección y pinzas, hacer una incisión en forma de V en el área genital y continuarla hasta el diafragma para abrir completamente la cavidad abdominal. </li><li> Coloque los lóbulos del hígado contra el diafragma, sino que deben permanecer allí si la cavidad corporal está abierta lo suficiente. Tire del intestino y el colon fuera de la cavidad abdominal a su izquierda, y encontrar el recto. Con un par de pinzas curvas y tijeras de disección, agarre y la sección del recto. </li><li> Con el mismo par de pinzas, cuidadosamente desenrollar por completo el intestino desde el recto hasta el estómago tirando del intestino a la izquierda. </li></ol>ent &quot;&gt; En este paso, el páncreas se puede distinguir como una pequeña franja entre el estómago y el comienzo del intestino. Sus ligaciones con el bazo permanecen intactos. <ol start="5"><li> Uso de Noyes tijeras y un par de fórceps, cortar cuidadosamente el páncreas a lo largo del intestino y liberar con el bazo del resto del tracto digestivo. </li><li> Coge el bazo y el páncreas sección unido a él (Figura 2). </li></ol> En este paso, asegúrese de que no hay tejido grasa mesentérica y / u otro tejido adyacente (bazo, intestino, etc) pueden ser recogidos con el páncreas, para evitar la contaminación celular. Para el resto del procedimiento, todos los tampones deben estar preparados sin calcio de iones Ca 2 + quelantes para evitar la disociación completa del tejido pancreático exocrino en las células acinares individuales. <ol start="7"><li> Enjuague el páncreas dos veces en solución salina equilibrada de Hank (HBSS) 1x. </li></ol> En este paso, como el tejido de grasa flotará en contra de páncreas que se hundirá, es fácilmente posible para visualizar y eliminar rápidamente el contaminante tejido adiposo blanco todavía unido a páncreas. Si necesita el páncreas para ser transportado a las instalaciones de cultivo celular, se debe tener en hielo en HBSS 1x. <ol start="8"><li> Transferir el páncreas en una placa de Petri estéril que contiene 5 ml de HBSS 1x. Uso de Noyes tijeras y un bisturí, cortar el páncreas en pequeños trozos de 1 a 3 mm 3 (Figura 3A). </li></ol> 2. Disociaciones enzimáticas y mecánicos de Páncreas (día 0) <ol><li> Transferirlos a un tubo de polipropileno estéril de 50 ml. </li><li> Se centrifuga durante 2 minutos a 450 xg y 4 ° C. Aspirar y descartar el sobrenadante para eliminar los fragmentos celulares y las células sanguíneas. </li><li> Añadir 10 ml de solución de colagenasa IA (1x HBSS que contiene HEPES 10 mM, 200 U / ml de collagenaSE IA, y 0,25 mg / ml de inhibidor de tripsina) a las secciones de páncreas. Usando una pipeta de 25 serológica, transferirlos a un matraz de 25 cm 2. Incubar durante 20-30 min a 37 ° C. Durante este tiempo (cada 5 min), lleve a cabo una disociación mecánica por enérgicamente en movimiento hacia atrás y hacia adelante los fragmentos de páncreas alrededor de diez veces, en pipetas estériles de tamaño decreciente (25, 10, y 5 pipetas serológicas ml). </li></ol> En este paso, es esencial controlar con frecuencia la medida de la disociación enzimática de secciones pancreáticas. <ol start="4"><li> Cuando el tejido pancreático parece ser bien disociado (de acuerdo con la desaparición de los fragmentos pancreáticas y para el aumento de la turbidez de la solución) (Figura 3B), detener la reacción enzimática mediante la adición de 10 ml de solución de lavado fría tamponada (HBSS que contiene 1x 5 % de suero fetal bovino (FBS) y 10 mM de HEPES). </li><li> Traslado en un tubo de polipropileno estéril de 50 ml y centrifuge durante 2 minutos a 450 xg y 4 ° C. Con cuidado aspirar y descartar el sobrenadante para eliminar la solución de colagenasa IA. </li><li> Resuspender y lavar el precipitado con 10 ml de solución de lavado tamponada. Centrifugar durante 3 min a 450 xg y 4 º C. Con cuidado aspirar y descartar el sobrenadante. Repita este paso dos veces más. </li></ol> 3. La filtración y la siembra de Dispersed acinos (día 0) <ol><li> Resuspender el sedimento celular en 7 ml de medio de Waymouth que contiene 2,5% de FBS, 1% de penicilina-estreptomicina mezcla (PS), 0,25 mg / ml de inhibidor de tripsina, y 25 ng / ml de factor de crecimiento epidérmico recombinante humano (EGF). </li><li> El filtrado de la mezcla de células por permitiendo que pase a través de un filtro de 100 micras para retener los fragmentos no digeridos (conductos, vasos sanguíneos, y los islotes de Langerhans). Estructuras acinares pancreáticas (acinus de 10-15 células) pasar a través. </li><li> Enjuague el filtro con 6 ml de medio que contiene FBS al Waymouth, PS, tripsina inhibitor y EGF. </li></ol> Después de este paso, las células tienen que ser tratado con mucho cuidado, para evitar cualquier acinos disociación. <ol start="4"><li> Semilla los acinos aislado en una placa de cultivo de 6 pocillos (2 ml por pocillo) (Figura 3C). Cultura ellas a 37 ° C bajo 5% (v / v) de atmósfera de CO 2. </li></ol> Después de este paso, las células acinares se cultivan en suspensión. 4. Cultivo de células acinares (Día 1 a 10) <ol><li> Veinticuatro horas después, transferir los acinos (en suspensión) en una nueva placa de cultivo de 6 pocillos, para eliminar las células contaminantes y los restos celulares que se han adherido durante la noche (figura 4). </li></ol> Si necesita extenderse durante varios días o si las condiciones experimentales requieren células cultivadas en monocapa, se recomienda para la transferencia y la semilla de acinos en andamios de matriz del cultivo celular. <ol start="2"><li> El día antes de verding el soporte de la matriz (día 0), recubrir una placa de cultivo de 6 pocillos con colágeno tipo I (5 mg / cm 2). Añadir 1 ml de solución de colágeno tipo I (50 mg / ml en ácido acético 0,02 M, 0,2 m-filtrada) a cada pocillo y permitir que se adsorba pasivamente sobre plástico, durante 1 hora a 37 ° C (o durante la noche a 4 ° C ). </li><li> Aspirar la solución de colágeno tipo I y enjuagar el pozo cubierto dos veces con buffer fosfato salino-1x. </li><li> Deje que el pozo cubierto para secar (en una cabina de seguridad microbiológica) por lo menos 12 horas antes de su uso. </li><li> Transferir los acinos primaria aislado (obtenida en la etapa 4.1) en el colágeno de tipo I-recubierto placa de cultivo de 6 pocillos y la cultura en las mismas condiciones que las descritas anteriormente (a 37 ° C bajo 5% (v / v) de atmósfera de CO 2) . Las células se adhieren al sustrato de colágeno tipo I durante 2 días. </li><li> En el Día 3, cambiar el medio de cultivo para eliminar las células no viables que no se han adherido. Con el tiempo en cultivo, las células se progresivamente spleer en el soporte que contiene colágeno. Cambiar el medio de cultivo cada 3 días (Figura 5). </li></ol> Las células acinares aislados obtenidos se pueden contar, después de una disociación mecánica completa mediante el uso de una cámara de recuento de células Thoma. Tenga en cuenta que las células acinares aislados no pueden mantenerse en cultivo después. La calidad de la cultura acinares obtenido puede ser controlado por el control de la expresión de marcadores específicos acinares tales como tripsinógeno, Páncreas Factor de Transcripción 1 subunidad alfa, o carboxipeptidasa A1 (por inmunocitoquímica o experimentos de inmunofluorescencia).

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+of+secretagogues+on+a+new+preparation+of+functionally+intact,+isolated+pancreatic+acini.">Action of secretagogues on a new preparation of functionally intact, isolated pancreatic acini.</a> Am. J. Physiol. 235, 517-524 (1978).</li><li>Logsdon, C. D., Williams, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+binding+and+biologic+effects+on+mouse+pancreatic+acini.">Epidermal growth factor binding and biologic effects on mouse pancreatic acini.</a> Gastroenterology. 85, 339-345 (1983).</li><li>Han, B., Logsdon, C. 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F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tif1gamma+suppresses+murine+pancreatic+tumoral+transformation+by+a+smad4-independent+pathway.">Tif1gamma suppresses murine pancreatic tumoral transformation by a smad4-independent pathway.</a> Am. J. Pathol. 180, 2214-2221 (2012).</li><li>Vincent, D. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+TIF1gamma+cooperates+with+Kras+to+induce+cystic+tumors+of+the+pancreas.">Inactivation of TIF1gamma cooperates with Kras to induce cystic tumors of the pancreas.</a> PLoS Genet. 5, e1000575 (2009).</li><li>Dorrell, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mouse+pancreatic+alpha,+beta,+duct+and+acinar+populations+with+cell+surface+markers.">Isolation of mouse pancreatic alpha, beta, duct and acinar populations with cell surface markers.</a> Mol. Cell Endocrinol. 339, 144-150 (2011).</li><li>Case, R. 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Los autores declaran que no tienen intereses financieros en competencia.

En este protocolo, se describe un procedimiento para el aislamiento de células acinares pancreáticas. Este método hace posible aislar más de 20 x 10 6 células acinares por animal en menos de 1 hora. Gracias a su rápida y fácil aplicación (hasta 10 páncreas se pueden procesar de forma independiente por experimento en paralelo), este protocolo aparece como un buen compromiso entre los métodos de aislamiento existentes 3-5,9-12,17. Los pasos críticos / Local...

Un problema frecuente para los laboratorios de investigación que trabajan en tejido pancreático exocrino es la dificultad de cultivar las células acinares in vitro durante un periodo de tiempo suficientemente largo para permitir que un experimento a largo plazo. Un factor que impide el desarrollo de estos sistemas de cultivo es la sensibilidad intrínseca del tejido pancreático a la manipulación experimental, debido al alto contenido de enzimas glucolíticas, proteolítica y lipolítica, que literalmente digerir el tejido pancreático cuando se liberan durante el aislamiento de las células pancreáticas. Un segundo factor es la notable en la plasticidad in vitro de las células acinares, que tienden a perder sus características de secreción y transdifferentiate a otras células maduras, tales como células ductales pancreáticas o células de hepatocitos como 1. In vitro, esta plasticidad de las células varía con las condiciones experimentales (tales como composición del medio de cultivo) 2 </sup&gt; e introduce un grado de complejidad en el diseño de las condiciones de cultivo apropiados para las células pancreáticas exocrinas 1. Varios métodos han sido desarrollados para el aislamiento y cultivo de células acinares, primero desde el páncreas conejillo de indias 3-5. Inicialmente, los protocolos involucrados digestión de tejido pancreático con colagenasa, quimotripsina, y un cóctel de proteasa, con el aislamiento final por disociación mecánica vigorosa. Las células pancreáticas aisladas de esta manera muestran características estructurales y funcionales anormales, en particular una pérdida de estructuras apicales y daños significativos a sus receptores de membrana. Las células aisladas se mantuvieron viables sólo 1 ó 2 días. Preparación de acinos dispersa mantiene su arquitectura intra-e intercelular, la preservación de las membranas celulares, lo que limita el daño a receptores de la superficie, y por lo tanto la mejora de la secreción exocrina en respuesta a secretagogos de 6-8. Como resuLT, este método ofrece la importante ventaja de extender la viabilidad celular acinar a 7-10 días in vitro. Por otra parte, actualmente se prefiere este método de aislamiento de células acinares 9-12 porque el mantenimiento de los contactos intercelulares, incluyendo acoplamiento celular por uniones, es un determinante esencial de la exocrina pancreática acinar fenotipo celular 13. A medida que la desdiferenciación de las células acinares y su transdiferenciación de células ductales es uno de los mecanismos propuestos para la génesis de los cánceres de páncreas exocrino agresivos 14, el modelo de acinos dispersa es también un sistema adecuado para estudiar la plasticidad pancreática y sus mecanismos moleculares subsiguientes. Además, en combinación con el uso de animales modificados genéticamente 15,16 y el desarrollo de técnicas de transferencia de genes (adenoviral 2 o transducción lentiviral, el uso de las nanopartículas, etc), en este modelo de células acinares primaria in vitro puedeser muy útil en la determinación de cómo diversas disfunciones genéticas afectan la regulación de la diferenciación de células acinares o desdiferenciación y deberían proporcionar una mejor comprensión de los eventos moleculares responsables de la aparición de la pancreatitis, lesiones precancerosas, y los cambios en la plasticidad de las células. El aislamiento de los acinos dispersa es el enfoque que usamos en nuestro laboratorio con las células acinares pancreáticas cultura. Se describen y discuten el método utilizado. Se trata de la disociación enzimática de tejido pancreático (con una colagenasa bacteriana) acoplado a la disrupción mecánica sin la disociación de las células acinares. Aunque la mayoría de protocolos implican el cultivo de la acinos, en suspensión o en sustratos de plástico especialmente tratadas, les crecen en suspensión sólo brevemente (durante 24 horas), sembrando en ellos después andamios matriz si se requiere cultivo celular prolongado. Este protocolo permite el aislamiento rápido (en menos de 1 hora) de la dispersión de páncreas de corriente alternaini, sostenible desde hace más de una semana en la cultura. Se permite el aislamiento de más de 20 x 10 6 células acinares por páncreas de ratón. Su simplicidad hace que sea posible procesar de forma independiente un máximo de 10 páncreas en paralelo. Mediante el mantenimiento de la arquitectura intra e intercelular de los acinos y por lo tanto el fenotipo de las células acinares primarias aisladas, este modelo constituye un sistema de elección para el estudio de los mecanismos de transdiferenciación, como todos los otros modelos pancreáticas exocrinas actualmente disponibles se derivan de los tumores pancreáticos se presentan muchos genética alteraciones que conduce a la transformación celular.

<table border="1" fo:keep-together.within-page="always">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>0.2 &mu;m filter</td>
			<td>Dutscher</td>
			<td>146560</td>
			<td></td>
		 </tr>
		 <tr>
			<td>10 ml serological pipettes</td>
			<td>Beckton Dickinson</td>
			<td>357551</td>
			<td></td>
		 </tr>
		 <tr>
			<td>100 &mu;m filter</td>
			<td>Beckton Dickinson</td>
			<td>352360</td>
			<td></td>
		 </tr>
		 <tr>
			<td>100 mm Petri dish</td>
			<td>Beckton Dickinson</td>
			<td>353003</td>
			<td></td>
		 </tr>
		 <tr>
			<td>1000 &mu;l filter tips</td>
			<td>Starlab</td>
			<td>S1122-1830</td>
			<td></td>
		 </tr>
		 <tr>
			<td>20 &mu;l filter tips</td>
			<td>Starlab</td>
			<td>S1120-1810</td>
			<td></td>
		 </tr>
		 <tr>
			<td>200 &mu;l filter tips</td>
			<td>Starlab</td>
			<td>S1120-8810</td>
			<td></td>
		 </tr>
		 <tr>
			<td>25 ml serological pipettes</td>
			<td>Beckton Dickinson</td>
			<td>357535</td>
			<td></td>
		 </tr>
		 <tr>
			<td>5 ml serological pipettes</td>
			<td>Beckton Dickinson</td>
			<td>357543</td>
			<td></td>
		 </tr>
		 <tr>
			<td>50 ml polypropylene tube</td>
			<td>Beckton Dickinson</td>
			<td>352070</td>
			<td></td>
		 </tr>
		 <tr>
			<td>6-well plate</td>
			<td>Beckton Dickinson</td>
			<td>353046</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Acetic acid 100%</td>
			<td>VWR BDH Prolabo</td>
			<td>20104.298</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Collagenase IA</td>
			<td>Sigma-Aldrich</td>
			<td>C2676</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Curved forceps, Dumont #7</td>
			<td>World Precision Instruments</td>
			<td>14188</td>
			<td>To sterilize before use</td>
		 </tr>
		 <tr>
			<td>Dissecting scissors, straight</td>
			<td>World Precision Instruments</td>
			<td>14393</td>
			<td>To sterilize before use</td>
		 </tr>
		 <tr>
			<td>Epidermal Growth Factor, human</td>
			<td>Promokine</td>
			<td>C-60180</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ethanol absolute (AnalaR Normapur)</td>
			<td>VWR BDH Prolabo</td>
			<td>20821.310</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fetal Bovine Serum</td>
			<td>Lonza</td>
			<td>14-801F</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Forceps, Dumont #5</td>
			<td>World Precision Instruments</td>
			<td>14098</td>
			<td>To sterilize before use</td>
		 </tr>
		 <tr>
			<td>Hank's Balanced Salt Solution 1x </td>
			<td>Gibco</td>
			<td>14025050</td>
			<td></td>
		 </tr>
		 <tr>
			<td>HEPES 1 M (pH 6.98-7.30)</td>
			<td>Lonza</td>
			<td>17-737F</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Incubator O2/CO2</td>
			<td>Sanyo</td>
			<td>MCO-19M</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse TS100</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Matrigel</td>
			<td>Beckton Dickinson</td>
			<td>356234</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Microbiological Safety Cabinet, level II</td>
			<td>Faster</td>
			<td>SafeFast Elite 212 S</td>
		 </tr>
		 <tr>
			<td>Noyes scissors, sharp/sharp tips, German</td>
			<td>World Precision Instruments</td>
			<td>500228-G</td>
			<td>To sterilize before use</td>
		 </tr>
		 <tr>
			<td>Penicillin-Streptomycin mixture</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Phosphate Buffer Saline 10x </td>
			<td>Gibco</td>
			<td>14200067</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pipet-Aid</td>
			<td>Drummond Scientific Company</td>
			<td>Pipet-Aid XP</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pipetman P1000</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pipetman P20</td>
			<td>Gilson</td>
			<td>F123600</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pipetman P200</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Refrigerated centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Scalpel</td>
			<td>Paramount Surgimed Ltd.</td>
			<td>Disposable Scalpel Size 23</td>
		 </tr>
		 <tr>
			<td>T25 flask, 25 cm2</td>
			<td>Sigma-Aldrich</td>
			<td>Z707481</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trypsin inhibitor, from Glycine Max</td>
			<td>Sigma-Aldrich</td>
			<td>T6522</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Type I collagen</td>
			<td>Beckton Dickinson</td>
			<td>354236</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Waymouth's medium</td>
			<td>Gibco</td>
			<td>31220-023</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

isolation and culture of mouse primary pancreatic acinar cells

Este protocolo permite el aislamiento rápido (en menos de 1 hora) de acinos pancreáticos murino, por lo que es posible mantener en cultivo durante más de una semana. 10 6 células acinares Más de 20 x puede obtenerse a partir de un único páncreas murino. Este protocolo ofrece la posibilidad de procesar de forma independiente hasta 10 páncreas en paralelo. Debido a que preserva la arquitectura acinar, este modelo es muy adecuado para el estudio de la fisiología del páncreas exocrino in vitro en contraste con las líneas celulares establecidas a partir de tumores de páncreas, que muestran muchas alteraciones genéticas que resultan en la pérdida parcial o total de su diferenciación acinar.

La figura 1 esquematiza el método acinos &quot;dispersado&quot; para las células acinares aislamiento primario. Los pasos críticos, que tienen que ser estrictamente respetado durante el protocolo, se describen en la parte de discusión. Para facilitar su retirada, el páncreas tiene que ser recogida desde el abdomen junto con el bazo adjunto (Figura 2). Ambos órganos tienen que ser cortados a pedazos, y el tejido graso residual que podría ser aún en el...

Watch this Scientific Journal Video about Isolation and Culture of Mouse Primary Pancreatic Acinar Cells at JoVE.com

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

En esta publicación, se describe un procedimiento rápido y conveniente para el aislamiento y cultivo de las células acinares pancreáticas primarias del páncreas murino. Este método constituye un enfoque valioso para el estudio de la fisiología de las células pancreáticas exocrinas normales / no transformada primarios frescos.

Aislamiento y cultivo de ratón primarias células acinares pancreáticas

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

isolation-and-culture-of-mouse-primary-pancreatic-acinar-cells

Research

JoVE Journal

Biology

35.4K vistas.  Centre de Recherche en Cancérologie de Lyon.  En esta publicación, se describe un procedimiento rápido y conveniente para el aislamiento y cultivo de las células acinares pancreáticas primarias del páncreas murino. Este método constituye un enfoque valioso para el estudio de la fisiología de las células pancreáticas exocrinas normales / no transformada primarios frescos. 

Video: Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

El aislamiento de células primarias de ratón trofoblasto y ensayo de invasión trofoblasto

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

Investigación

Biología

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Aislamiento y cultivo primario de células hepáticas de ratas

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Assessing the Secretory Capacity of Pancreatic Acinar Cells

Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work demonstrates how the secretory capacity of these cells can be assessed by culture of isolated acini. The setup is advantageous since isolated acini, which retain many characteristics of the intact exocrine pancreas can be manipulated and monitored more readily than in the whole animal. Proper isolation of pancreatic acini is a key requirement so that the ex&#160;vivo culture will represent the in&#160;vivo nature of the acini. The protocol demonstrates how to isolate intact acini from the mouse pancreas. Subsequently, two complementary methods for evaluating pancreatic secretion are presented. The amylase secretion assay serves as a global measure, while direct imaging of pancreatic secretion allows the characterization of secretion at a sub-cellular resolution. Collectively, the techniques presented here enable a broad spectrum of experiments to study exocrine secretion.

Isolated pancreatic acini retain their in&#160;vivo morphology and activity and offer powerful ways for monitoring and manipulating secretion. This work demonstrates how acini can be isolated from the mouse pancreas, and how their secretory capacities can be assessed.

La evaluación de la capacidad secretora de las células pancreáticas acinares

Pancreatic acinar cells produce and secrete digestive enzymes. These cells are organized as a cluster which forms and shares a joint lumen. This work ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Aislamiento y cultivo primario de células endoteliales aórticas de ratón

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Aislamiento y cultivo de queratinocitos de ratón primario de piel de ratón neonatal y adulto

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (&#60;10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% &#177; 1.2%, 94.4% &#177; 1.1%, and 92.4% &#177; 1.7% (mean &#177; SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

An effective enzymatic method for isolation of primary porcine aortic endothelial cells (pAECs) from miniature pigs is described. The isolated primary pAECs can be used to investigate the immune and coagulation response in xenotransplantation.

Aislamiento y cultivo de células endoteliales aórticas primarias de cerdos en miniatura

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Disección eficiente y cultivo de células epiteliales de pigmento retiniano de ratón primario

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

Aislamiento y cultivo de queratinocitos orales primarios del paladar del ratón adulto

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Aislamiento y caracterización de células endoteliales sinusoidales sinusoidales hepáticas primarias de ratón

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

Aislamiento de células primarias del epitelio pigmentado de la retina del ratón

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...