Este trabajo fue financiado por el NIH R01 DK05823 (de pasarela residencial), F32 DK083213 (para JWW), F30 DK081265 (a ALO), y por una beca de la Fred y Suzanne Biesecker Centro de hepatología pediátrica (de pasarela residencial).

Todo el procedimiento se lleva a cabo a temperatura ambiente a menos que se especifique lo contrario. 1. Preparación de las soluciones enzimáticas <ol><li> Preparar soluciones de enzima de acuerdo con la Tabla 1 y el filtro estéril utilizando un filtro de 0,22 micras. Mantener la solución 1 y 2 solución a temperatura ambiente y la solución 3 a 4 ° C hasta el momento de uso. Todas las soluciones y equipos que entran en contacto con células aisladas debe ser estéril. </li></ol> 2. Preparación del equipo de perfusión <ol><li> Cebe el sistema de perfusión con HBSS menos de Ca 2 + / Mg 2 +, que ha sido calentado a 37 ° C. Nota: Para asegurarse de que la perfusión es de 37 ° C cuando se llega al hígado, HBSS se calienta en un baño de agua a 37 ° C. Después pasa a través de la bomba de perfusión, y sale a través de un condensador de vidrio encamisado que está conectado a un conjunto de recirculación de agua a 40 ° C (Fig. 1). </li><li> Esterilizar quirúrgico paraoles en un vaso de precipitados con etanol al 70%. </li></ol> 3. La perfusión del hígado <ol><li> Anestesie un adulto macho Sprague-Dawley, ratas por inyección intraperitoneal de Nembutal (50-100 mg / kg de peso corporal como sea necesario para obtener una anestesia adecuada). </li><li> Colocar el animal en decúbito supino sobre una bandeja de plástico y fijar las extremidades de la bandeja con cinta adhesiva. </li><li> Limpie el abdomen con un 70% de EtOH. </li><li> Haga una pequeña incisión en la piel del abdomen en la línea media. Disecar la piel lejos de la fascia haciendo un gran corte en forma de U en el abdomen. Cortar el músculo abdominal con tijeras después de la misma forma para exponer la cavidad peritoneal. </li><li> Exponer la vena porta mediante el uso de 2 hisopos de algodón para mover suavemente las vísceras abdominales hacia el lado derecho. </li><li> Pase dos suturas estériles de seda 2.0 por debajo de la vena porta y corbata flojo. </li><li> Canular la vena porta con un 16-18 de calibre del catéter IV y asegurar el catéter en su lugar apretando ligeramente la TIed suturas de la etapa anterior (fig. 2). </li><li> Inyectar heparina diluida a través del catéter IV (1 ml de heparina 5000 unidades USP / ml se diluyó con 4 ml de HBSS menos 2 + Ca / Mg 2 +). El hígado debe blanquear. </li><li> Conectar el catéter IV del sistema de perfusión. Perfundir con HBSS menos 2 + Ca / Mg 2 + a una velocidad de 20 ml / min durante 10 minutos. Transecto la VCI inferior al hígado para permitir el drenaje de la sangre y perfundido. Aspirar sangre acumulada de la cavidad abdominal. </li><li> Cambiar el líquido de perfusión a una solución de colagenasa 0,3% (150 mg de colagenasa tipo 2 en 500 ml de HBSS más Ca 2 + / Mg 2 +) y perfundir durante 25 minutos. Mantenga la humedad del hígado cubriéndolo con la solapa previamente disecados abdominal. Ponga una cubierta placa de Petri sobre la solapa abdominal y la posición de una lámpara de calor sobre el área para mantener la temperatura intra-abdominal a aproximadamente 37 ° C. </li></ol> 4. Preparación de ªe árbol biliar <ol><li> Retire el hígado por sacarla de la cavidad abdominal con una pinza y colocarla en un plato de cultivo de tejidos estéril llena de L-15 de Leibovitz fría medios de comunicación. </li><li> Trabajo en la campana de cultivo de tejidos, la cáscara de la cápsula hepática y suavemente burlarse del parénquima hepático, aparte con pinzas. Mueva el hígado a través de varios platos llenos de L-15 Liebovitz los medios de comunicación sin dejar de burlarse de distancia del parénquima, hasta que finalmente se fue con el árbol biliar aislada. El parénquima hepático es de color crema, mientras que el árbol biliar restante es blanco. </li><li> Coloque el árbol biliar aislado en un tubo Falcon de 50 ml lleno con 10 ml de penicilina / estreptomicina (10.000 UI / ml de penicilina y 10.000 mg / ml de estreptomicina); dejar en hielo durante 15 minutos. </li></ol> 5. Aislamiento de la fracción de fibroblastos portal <ol><li> Coloque el árbol biliar en un tubo estéril de 50 ml Falcon y picar con unas tijeras estériles. </li><li> Añadir una pequeña cantidad de enzima Sollución # 1 en el tubo (aproximadamente 5 ml) y continuar para picar el árbol biliar hasta que alcanza la consistencia de una suspensión. </li><li> Verter la mezcla en una botella de vidrio estéril. Lavar tubo Falcon con el resto de la solución n º 1, a continuación, se vierte en la botella de vidrio. Incubar a 37 ° C con agitación a 100 rpm durante 30 minutos. </li><li> Se filtra la suspensión a través de un vaso de precipitados cubierto con una sola capa de malla de nylon (30-micras de tamaño de poro). </li><li> Transferir cualquier tejido restante en la parte superior de la malla a una botella de vidrio estéril por lavado de la malla con una solución de enzima # 2. Empezar vertiendo medio (25 ml) de la solución en un estéril de 15 cm placa de Petri. Retirar con cuidado la malla del vaso de precipitados manteniendo los bordes de la malla sin contaminar el área central, a continuación, invirtiendo la malla y sumergiéndola en la solución en la placa de Petri para eliminar cualquier resto de tejido sobre la malla. Deseche la malla. Transferir la solución en la placa de Petri en una botella de vidrio estéril. Enjuague la placa de Petri con la remanando 25 ml de solución # 2 y transferirlo a la misma botella de vidrio. Incubar a 37 ° C con agitación a 100 rpm durante 30 minutos. </li><li> Mientras tanto, tome el filtrado en el vaso y se vierte en un tubo Falcon de 50 ml. Centrifugar a 1.600 rpm (460 xg) durante 5 minutos a temperatura ambiente. </li><li> Aspirar medios y resuspender el sedimento en 25 ml de solución # 3. </li><li> Tomar la solución de la etapa 5,5 y filtrar en un segundo vaso de precipitados estéril cubierta con 30-micras de malla. </li><li> Deseche la malla. Centrifugar el filtrado a 1600 rpm durante 5 minutos a temperatura ambiente. Aspirar y resuspender el precipitado con 25 ml de solución # 3 como se hizo en el paso 5,7. </li><li> Se combinan las fracciones de 2 pasos de 5.7 y 5.9 y centrifugar de nuevo a 1600 rpm durante 5 minutos a temperatura ambiente. </li><li> Aspirar los medios de comunicación y resuspender el precipitado en 10 ml de portales de medios de cultivo de fibroblastos (DMEM/F12 suplementado con FBS al 10%, 1% penicilina / estreptomicina, gentamicina 0,3% y 0,2% fungizona). </li><li> Contar las células para la viabilidad y la placa en placas de cultivo de tejidos. Dependiendo de la aplicación deseada, de 0,5 a 5 x 10 6 células por 10-cm placa de cultivo de tejidos son apropiados. Desde un adulto rata Sprague-Dawley, que típicamente obtener 2 a 10 millones de células viables. </li><li> Vuelva a colocar los medios de cultivo al día siguiente para retirar los escombros no adherente. Posteriormente, reemplazar los medios de cultivo cada 2 días. </li></ol> 6. Los resultados representativos Fibroblastos primarios portal aisladas a partir de hígado de rata adulta se muestran en 1, 3 y 7 días después del aislamiento (Fig. 3). Nótese que las células son alargadas, con una morfología típica de los fibroblastos. Las células pueden ser teñidas con anti-elastina de anticuerpos (CL55041AP, los laboratorios de Cedarlane, Burlington, Carolina del Norte) para confirmar la pureza. Fibroblastos Portal someterse a la diferenciación miofibroblástico en la cultura. Esto se puede demostrar por inmunotinción para actina de músculo liso-α (α-SMA; A2547, Sigma, St. Louis, MO). <tableborder = &quot;1&quot;&gt; <tbody><tr><td rowspan="2"> 0,3% de colagenasa solución </td><td> Tipo 2 colagenasa </td><td> 150 mg </td></tr><tr><td> HBSS con Ca 2 + / Mg 2 + </td><td> 500 ml </td></tr><tr><td rowspan="7"> Solución n º 1 (Pronasa) </td><td> Albúmina de suero bovino </td><td> 50 mg </td></tr><tr><td> La colagenasa tipo 2 </td><td> 25 mg </td></tr><tr><td> Pronasa </td><td> 18 mg </td></tr><tr><td> DNasa </td><td> 3 mg </td></tr><tr><td> DMEM/F12 </td><td> 47,5 ml </td></tr><tr><td> Pen / Strep </td><td> 1 ml </td></tr><tr><td> Suero bovino fetal </td><td> 1,5 ml </td></tr><tr><td> Solución n º 2 </td><td> Albúmina de suero bovino </td><td> 50 mg </td></tr><tr><td rowspan="6"> (Hialuronidasa) </td><td> La colagenasa tipo 2 </td><td> 25 mg </td></tr><tr><td> Hialuronidasa </td><td> 22 mg </td></tr><tr><td> DNasa </td><td> 3 mg </td></tr><tr><td> DMEM/F12 </td><td> 47,5 ml </td></tr><tr><td> Pen / Strep </td><td> 1 ml </td></tr><tr><td> Suero bovino fetal </td><td> 1,5 ml </td></tr><tr><td rowspan="2"> Solución n º 3 </td><td> DNasa </td><td> 3 mg </td></tr><tr><td> Medio RPMI 1640 </td><td> 50 ml </td></tr></tbody></table> Tabla 1. Soluciones enzimáticas. <img alt="Figura 1" src="/files/ftp_upload/3669/3669fig1.jpg" /> Figura 1. La perfusión de configurar. A. El agua de circulación para la columna de calentamiento. Columna B. que contiene los medios de comunicación de perfusión. C. llave de paso para controlar flujo de salida de la bomba. D. Bomba de entrada. E. de velocidad variable de la bomba peristáltica. F. frasco vacío con Bio-Clean detergente. G. lámpara de calor. H. El agua de baño para los medios de comunicación de perfusión calentamiento. <img alt="Figura 2" src="/files/ftp_upload/3669/3669fig2.jpg" /> FiguRe 2. En perfusión in situ. A. Catéter IV insertada en la vena porta, se fija con sutura y conectado a la tubería de perfusión a través de llave de paso. B. pipeta de vidrio conectado a frasco de vacío y succión de pared para extraer el líquido de perfusión se escape hacia afuera de la vena cava inferior seccionado. I. intestinos. L. hígado. <img alt="Figura 3" src="/files/ftp_upload/3669/3669fig3.jpg" /> Figura 3. El contraste de fases y de inmunofluorescencia tinción de fibroblastos portales de rata en cultivo. Portal fibroblastos cultivados durante 1, 3 y 7 días después de que el aislamiento se fijan y se tiñen de la elastina, α-SMA y DAPI.

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	<li>Parola, M., Marra, F., Pinzani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofibroblast+-+like+cells+and+liver+fibrogenesis:+Emerging+concepts+in+a+rapidly+moving+scenario.">Myofibroblast - like cells and liver fibrogenesis: Emerging concepts in a rapidly moving scenario.</a> Mol. Aspects Med. 29, 58-66 (2008).</li><li>Dranoff, J. A., Wells, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Portal+fibroblasts:+Underappreciated+mediators+of+biliary+fibrosis.">Portal fibroblasts: Underappreciated mediators of biliary fibrosis.</a> Hepatology. 51, 1438-1444 (2010).</li><li>Tuchweber, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation+and+phenotypic+modulation+of+portal+fibroblasts+in+the+early+stages+of+cholestatic+fibrosis+in+the+rat.">Proliferation and phenotypic modulation of portal fibroblasts in the early stages of cholestatic fibrosis in the rat.</a> Lab Invest. 74, 265-278 (1996).</li><li>Beaussier, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prominent+contribution+of+portal+mesenchymal+cells+to+liver+fibrosis+in+ischemic+and+obstructive+cholestatic+injuries.">Prominent contribution of portal mesenchymal cells to liver fibrosis in ischemic and obstructive cholestatic injuries.</a> Lab Invest. 87, 292-303 (2007).</li><li>Kinnman, N., Housset, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peribiliary+myofibroblasts+in+biliary+type+liver+fibrosis.">Peribiliary myofibroblasts in biliary type liver fibrosis.</a> Front Biosci. 7, d496-d503 (2002).</li><li>Kinnman, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myofibroblastic+conversion+of+peribiliary+fibrogenic+cells+distinct+from+hepatic+stellate+cells+is+stimulated+by+platelet-derived+growth+factor+during+liver+fibrogenesis.">The myofibroblastic conversion of peribiliary fibrogenic cells distinct from hepatic stellate cells is stimulated by platelet-derived growth factor during liver fibrogenesis.</a> Lab Invest. 83, 163-173 (2003).</li><li>Uchio, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+retinol-binding+protein-1+expression+and+modulation+during+in+vivo+and+in+vitro+myofibroblastic+differentiation+of+rat+hepatic+stellate+cells+and+portal+fibroblasts.">Cellular retinol-binding protein-1 expression and modulation during in vivo and in vitro myofibroblastic differentiation of rat hepatic stellate cells and portal fibroblasts.</a> Lab Invest. 82, 619-628 (2002).</li><li>Kruglov, E. A., Jain, D., Dranoff, 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=Isolation+of+primary+rat+liver+fibroblasts.">Isolation of primary rat liver fibroblasts.</a> J. Investig. Med. 50, 179-184 (2002).</li><li>Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor-beta+and+substrate+stiffness+regulate+portal+fibroblast+activation+in+culture.">Transforming growth factor-beta and substrate stiffness regulate portal fibroblast activation in culture.</a> Hepatology. 46, 1246-1256 (2007).</li><li>Bosselut, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+proteomic+features+of+two+fibrogenic+liver+cell+populations:+hepatic+stellate+cells+and+portal+myofibroblasts.">Distinct proteomic features of two fibrogenic liver cell populations: hepatic stellate cells and portal myofibroblasts.</a> Proteomics. 10, 1017-1028 (2010).</li><li>Dranoff, 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=The+ecto-nucleoside+triphosphate+diphosphohydrolase+NTPDase2/CD39L1+is+expressed+in+a+novel+functional+compartment+within+the+liver.">The ecto-nucleoside triphosphate diphosphohydrolase NTPDase2/CD39L1 is expressed in a novel functional compartment within the liver.</a> Hepatology. 36, 1135-1144 (2002).</li></ol>

No hay conflictos de interés declarado.

Portal fibroblastos juegan un papel importante en la fibrosis biliar. Aquí se describe una modificación del protocolo original publicado por Kruglov y Dranoff 8 para el aislamiento de fibroblastos portales de hígado de rata, proporcionando una manera sencilla para estudiar esta población de células in vitro. Este método utiliza la digestión de proteasa y filtración tamaño basado. La principal ventaja del método es que una población relativamente pura de células puede obtenerse sin pasaje en cultivo, lo que permite el estudio de la expresión génica o el comportamiento funcional de células de varios días después del aislamiento. Esta técnica también ofrece la posibilidad de aislar las células de hígados sanos y enfermos. Uno de los pasos más críticos de este protocolo es la digestión enzimática del parénquima hepático. Para garantizar una digestión éxito, es importante confirmar que la sangre se vacía completamente en el hígado durante el período inicial perfusiónsión, asegurándose de que no es aún escaldado del hígado. Para facilitar esto, es posible utilizar un hisopo de algodón para masajear suavemente el hígado, mientras que la perfusión. Durante la digestión de los resultados de hígado de parénquima en un bajo rendimiento de células viables, mientras que bajo-digestión hará que sea difícil separar el parénquima hepático del árbol biliar. Como los preparativos de pronasa una variabilidad en la actividad enzimática entre los diferentes lotes, la optimización de la cantidad utilizada puede ser necesaria cuando hay un bajo rendimiento de las células viables. Este protocolo puede ser modificado para aislar fibroblastos portales de hígado de ratón o de hígado de rata joven mediante el uso de un calibre más pequeño catéter IV canular la vena porta, disminuyendo el volumen de las soluciones de perfusión en proporción al peso del animal, y la disminución de la tasa de perfusión del hígado a aproximadamente 10 ml / min. Fibroblastos Portal de la cultura someterse a la diferenciación miofibroblástico en 10-14 días, como lo demuestra por el ex α-SMAdepresión 9. Diversos marcadores han sido utilizados para distinguir fibroblastos portales de las células estrelladas hepáticas, incluyendo fibulina-2, IL-6, elastina, los nucleósidos trifosfato diphosphohydrolase-2 (NTPDase2), 1-cofilina y proteínas neuronales tales como sinaptofisina 2, 6, 10, 11 . Hemos encontrado que la elastina es un buen marcador de fibroblastos portales, incluso después de la diferenciación miofibroblástico, mientras NTPDase2 se pierde después de fibroblastos portales han sido sometidos a cultivo después de varios días 9. Por lo tanto, suelen confirmar el aislamiento de fibroblastos portales por la tinción de inmunofluorescencia para la elastina. La limitación de esta técnica es que, inmediatamente después del aislamiento de fibroblastos portal, existe una pequeña fracción de contaminación de las células, incluyendo las células de Kupffer y células biliares. Fibroblastos portales se superan estas células contaminantes dentro de 2-3 días, sin embargo, dando una población relativamente puro (&gt; 98%) 9. Sina vez que los fibroblastos portal de la cultura someterse a la diferenciación miofibroblástico en plástico de cultivo de tejidos, por lo general estudian estas células dentro de los 7 días de aislamiento. Las células se mantienen en portal medios de fibroblastos de cultivo que contienen 10% de suero fetal bovino, como se describe en la sección de métodos. Sin embargo, estas células sobreviven en medio de crecimiento que contienen 2% de suero fetal bovino durante varios días. Por lo general no utilizan los medios de comunicación de baja de suero hasta por lo menos 24 horas después del aislamiento. Una vez fibroblastos portales someterse a la diferenciación miofibroblástico, pueden pasarse varias veces y se mantuvo como material congelado de miofibroblastos.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Tipo 2 colagenasa </td><td> Bioquímica Worthington (Lakewood, NJ) </td><td> LS004177 </td></tr><tr><td> Pronasa </td><td> Roche (Indianápolis, IN) </td><td> 70290120 </td></tr><tr><td> Hialuronidasa </td><td> Sigma (St. Louis, MO) </td><td> H3506 </td></tr><tr><td> Desoxirribonucleasa </td><td> Sigma </td><td> D4527 </td></tr><tr><td> DMEM/F12 </td><td> Invitrogen (Carlsbad, CA) </td><td> 11320 </td></tr><tr><td> HBSS sin Ca 2 + / Mg 2 + </td><td> Mediatech (Manassas, VA) </td><td> 21-021-CM </td></tr><tr><td> HBSS con Ca 2 + / Mg 2 + </td><td> Mediatech </td><td> 21 a 020-CM </td></tr><tr><td> Leibovitz L-15 </td><td> Invitrogen </td><td> 11415 </td></tr><tr><td> Medio RPMI 1640 </td><td> Invitrogen </td><td> 11875 </td></tr><tr><td> Albúmina de suero bovino </td><td> Sigma </td><td> A2153 </td></tr><tr><td> Suero Bovino Fetal </td><td> Gemini Bio (West Sacramento, CA) </td><td> 900-108 </td></tr><tr><td> Nitex nylon de malla de 30 micras </td><td> Genesee Científicas (San Diego, CA) </td><td> 57-105 </td></tr></tbody></table>

isolation of rat portal fibroblasts by in situ liver perfusion

La fibrosis hepática se define por la deposición excesiva de matriz extracelular por miofibroblastos activados. Hay varios precursores de miofibroblastos hepáticos, incluyendo las células estrelladas hepáticas, fibroblastos portales y la médula ósea fibroblastos derivados 1. Las células estrelladas hepáticas han sido la mejor estudiada, pero fibroblastos portales son cada vez más como importantes contribuyentes a la piscina miofibroblastos, especialmente en la fibrosis biliar 2. Fibroblastos portales experimentan una proliferación en respuesta a lesiones del epitelio biliar, lo que podría jugar un papel clave en las primeras etapas de cicatrización biliar 3-5. Un método de aislamiento de fibroblastos portales permitiría estudio in vitro de esta población de células y conducir a una mayor comprensión del papel de los fibroblastos en la fibrosis portal jugar biliar. Fibroblastos portal se han aislado mediante diversas técnicas, incluyendo derivación 6, 7 y Liperfusión ver con la digestión enzimática seguida por selección del tamaño de 8. La ventaja de la digestión y la técnica de selección de tamaño en comparación con la técnica consecuencia es que las células pueden ser estudiados sin la necesidad de pasaje en cultivo. Aquí se describe una versión modificada de la técnica original descrita por Kruglov y Dranoff 8 para el aislamiento de fibroblastos portales de hígado de rata que se traduce en una población relativamente pura de células primarias.

Watch this Scientific Journal Video about Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion at JoVE.com

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Una técnica para aislar fibroblastos portales de hígado de rata se ha descrito. Los hígados se perfundieron y digerido<em&gt; In situ</em&gt; Con colagenasa, seguido por<em&gt; Ex vivo</em&gt; Digestión de la pasta de hígado y selección del tamaño de las células. Este método proporciona una población pura de fibroblastos portales sin la necesidad de pasaje en cultivo.

El aislamiento de fibroblastos portales rata por<em&gt; In situ</em&gt; Hígado de perfusión

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

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Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

19.2K vistas.  The Children's Hospital of Philadelphia. Una técnica para aislar fibroblastos portales de hígado de rata se ha descrito. Los hígados se perfundieron y digerido In situ Con colagenasa, seguido por Ex vivo Digestión de la pasta de hígado y selección del tamaño de las células. Este método proporciona una población pura de fibroblastos portales sin la necesidad de pasaje en cultivo.

Video: Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

La endocitosis en vivo de hígado seguido por la purificación de las células hepáticas de la perfusión del hígado

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

Investigación

Biología

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

Procedimientos para la Rata In situ Propiedades contráctiles del músculo esquelético

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Aislamiento de la cromatina mediante la purificación de ARN (chirp)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

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 ...

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their prominent presence is often associated with poor prognosis1,2. CAFs are an activated subpopulation of stromal fibroblasts, many of which express the myofibroblast marker &alpha;-SMA3. CAFs originate from local tissue fibroblasts as well as from bone marrow-derived cells recruited into the developing tumor and adopt a CAF phenotype under the influence of the tumor microenvironment4. CAFs were shown to facilitate tumor initiation, growth and progression through signaling that promotes tumor cell proliferation, angiogenesis, and invasion5-8. We demonstrated that CAFs enhance tumor growth by mediating tumor-promoting inflammation, starting at the earliest pre-neoplastic stages9. Despite increasing evidence of the key role CAFs play in facilitating tumor growth, studying CAFs has been an on-going challenge due to the lack of CAF-specific markers and the vast heterogeneity of these cells, with many subtypes co-existing in the tumor microenvironment10. Moreover, studying fibroblasts in vitro is hindered by the fact that their gene expression profile is often altered in tissue culture11,12 . To address this problem and to allow unbiased gene expression profiling of fibroblasts from fresh mouse and human tissues, we developed a method based on previous protocols for Fluorescence-Activated Cell Sorting (FACS)13,14. Our approach relies on utilizing PDGFR&alpha; as a surface marker to isolate fibroblasts from fresh mouse and human tissue. PDGFR&alpha; is abundantly expressed by both normal fibroblasts and CAFs9,15 . This method allows isolation of pure populations of normal fibroblasts and CAFs, including, but not restricted to &alpha;-SMA+ activated myofibroblasts. Isolated fibroblasts can then be used for characterization and comparison of the evolution of gene expression that occurs in CAFs during tumorigenesis. Indeed, we and others reported expression profiling of fibroblasts isolated by cell sorting16. This protocol was successfully performed to isolate and profile highly enriched populations of fibroblasts from skin, mammary, pancreas and lung tissues. Moreover, our method also allows culturing of sorted cells, in order to perform functional experiments and to avoid contamination by tumor cells, which is often a big obstacle when trying to culture CAFs.

Isolation of Normal and Cancer-associated Fibroblasts from Fresh Tissues by Fluorescence Activated Cell Sorting FACS

Cancer Associated Fibroblasts (CAFs) facilitate tumor initiation, growth and progression through signaling that promotes proliferation, angiogenesis, and inflammation. Here we describe a method to isolate pure populations of normal fibroblasts and CAFs from fresh mouse and human tissues by cell sorting, using PDGFR&#x03B1; as a surface marker.

Aislamiento de fibroblastos normales y el cáncer asociado-de los tejidos frescos por Clasificación células activadas por fluorescencia (FACS)

Cancer-associated fibroblasts (CAFs) are the most prominent cell type within the tumor stroma of many cancers, in particular breast carcinoma, and their ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

El aislamiento de hepatocitos humanos por una colagenasa perfusión Procedimiento de dos pasos

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

Simultaneous Isolation of High Quality Cardiomyocytes, Endothelial Cells, and Fibroblasts from an Adult Rat Heart

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular mechanisms of cardiovascular disease progression such as cardiac hypertrophy, fibrosis, and atherosclerosis. Although several attempts with variable success have been made to develop immortalized cell lines from the cardiovascular system to understand these cellular mechanisms, primary cells offer a more natural and close to in vivo environment for such studies. Therefore, different laboratories working on a particular cell type have developed protocols to isolate individual types of rat cardiac cells of interest. A protocol that allows the isolation of more than one cell type, however, is missing. Here an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation and enables their use for cellular analyses. This permits the most efficient use of available resources, which may save time and reduce research costs.

Several protocols have been developed and described for the isolation of different cardiac cell types from a rat heart. Here, an optimized protocol is described that allows the isolation of high-quality major cardiac cell types (cardiomyocytes, endothelial cells, and fibroblasts) from a single preparation, reducing the experimental costs.

Aislamiento simultáneo de cardiomiocitos de alta calidad, células endoteliales y fibroblastos de un corazón de rata adulto

The rat is an important animal model used in cardiovascular research, and rat cardiac cells are used routinely for in vitro analysis of the molecular ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Aislamiento y caracterización de fibroblastos cardíacos adultos y miofibroblastos

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Aislamiento y caracterización de exosomas de fibroblastos del músculo esquelético

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

Aislamiento de hepatocitos primarios de rata con control de perfusión multiparamétrico

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...

Methods Article

El aislamiento de fibroblastos portales rata por