Los autores agradecen a la Patología Molecular and Imaging Core del NIDDK Centro de la Universidad de Pensilvania de Estudios Moleculares de las Enfermedades Digestivas y Hepáticas (DK50306 P30) para la asistencia con la imagen. Este trabajo fue apoyado por becas de los Institutos Nacionales de Salud (R01 DK-092111) y de la Fred y Suzanne Biesecker Pediatric Liver Center (a RGW) y por una beca de la Red de Investigación de Enfermedades del hígado infantil y Educación (SK).

Todo el procedimiento se lleva a cabo a temperatura ambiente a menos que se especifique lo contrario. Todo animal de trabajo debe llevarse a cabo en condiciones humanas bajo un protocolo aprobado por el Comité de Cuidado de Animales institucional y el uso local (IACUC). 1. Preparación de Equipos y Soluciones <ol><li> Configuración de la mesa de operaciones del ratón en estrecha proximidad a la campana de cultivo de tejido y incubadora (Figura 1A). Nota: El equipo requerido incluye un microscopio de disección, una fuente de luz, 12,5 cm de largo tijeras iris recta, 6 pulgadas pinzas curvas dentadas no con puntas finas y 4 pulgadas pinzas dentadas con puntas curvas (fig. 1B). </li><li> Coloque los instrumentos esterilizados en un vaso de vidrio de 70% de alcohol en la mesa quirúrgica. </li><li> Llenar tres 60 mm placas de Petri estériles con medios de aislamiento estéril (Tabla 1); colocar dos en la mesa quirúrgica y una en la capilla, todo en hielo. </li><li> Coloque colágeno de cola de rata,estéril 10x tampón fosfato salino (PBS), agua estéril, estéril 1 N de NaOH y células epiteliales biliares (BEC) medios de comunicación (Tabla 1) en la campana de cultivo de tejido. Nota: El colágeno debe estar en hielo. </li></ol> 2. Aislamiento y cultivo del conducto biliar <ol><li> La eutanasia a los ratones neonatales entre 0-3 días de vida, por la exposición a dióxido de carbono hasta que inconsciente seguido por dislocación cervical, según las directrices del IACUC (Figura 1C). Nota: Los ratones más viejos pueden ser utilizados en función de las necesidades experimentales específicas. </li><li> Coloque animal en decúbito supino y hacer una pequeña incisión horizontal que cruza la línea media en la región de la pelvis. Extienda esta incisión lateralmente y hacia arriba a ambos lados del abdomen, formando una aleta en forma de U. Gire la solapa hacia arriba para destapar los órganos abdominales. Una vez que el abdomen está abierto, mover cuidadosamente los intestinos de la cavidad abdominal hacia el lado derecho del animal para una mejor visualización del conducto biliar común y del hígado. Nota: puede ser necesario extender las incisiones laterales en la caja torácica para obtener una mejor identificación del hígado. </li><li> Usando el microscopio de disección, identificar el conducto biliar común, el hígado y los intestinos. Identificar el conducto biliar común primero (Figura 1 D), utilizando cautela, ya que las estructuras son muy delicados. Con las pinzas dentadas de sierra y no gubernamentales, meticulosamente limpia y acaba con el tejido conectivo que incluye la grasa que rodea a la bilis. Utilice uno fórceps para sujetar el conducto y el otro para limpiar. </li><li> Utilice una técnica similar para limpiar la vesícula biliar. </li><li> Coloque los conductos limpios y la vesícula hasta el primer plato de medios de aislamiento en el hielo. Masajea suavemente las estructuras con los forceps no dentados, mientras que en los medios de comunicación para eliminar más tejido conectivo y eliminar la bilis desde la vesícula biliar. </li><li> Traslado conductos y vesícula biliar con el segundo plato de medios de aislamiento. </li><li> Después del aislamiento de los conductos y vesículas biliares de 5 animales, transferir elpiezas a la tercera placa de Petri, en el capó. Nota: Si las células han de ser aislado a partir de más de 5 animales, se puede hacer en lotes, utilizando soluciones frescas para cada grupo de 5 animales. </li><li> Preparar geles de colágeno gruesas en la campana de cultivo de tejidos: Nota: cualquier plato tamaño puede utilizarse. Una placa de cultivo de 60 mm con un volumen final de 3 ml de colágeno para cada grupo de 5 neonatos funciona bien. Esto debe ser preparado fresco en el momento de la incorporación de conductos recién aisladas. <ol><li> Coloque colágeno de cola de rata, estéril 10x tampón fosfato salino (PBS 10x), dH 2 O estéril, y estéril de NaOH 1 N en hielo. </li><li> Cálculo de los volúmenes de dH 2 0, 10x de PBS, NaOH, y el colágeno necesario para geles con una concentración final de 2 colágeno mg / ml en PBS 1x, utilizando un volumen de 1 N de NaOH igual a 0,023 veces el volumen de colágeno añadido. Nota: Si se utiliza una fuente alternativa de colágeno, siga las instrucciones del fabricante para la neutralización y la formación de gel. </li><li> Mezcle dH 2 0, 10x PBS, y 1 N de NaOH, a continuación, añadir colágeno y así pipeta para garantizar una mezcla uniforme. </li></ol></li><li> Una vez solución de colágeno se ha espesado a un gel de consistencia similar a temperatura ambiente, inmediatamente incrustar los conductos en el colágeno. </li><li> Coloque en la incubadora a 37 ° C, 5% de CO 2 durante 30 min para permitir que el colágeno se solidifique. </li><li> Una vez gel se ha solidificado (color cambia de transparente a blanco), overlay con 3,5 ml de medio BEC (Tabla 1) y se incuba a 37 ° C, 5% de CO 2. </li><li> Retire el medio de BEC 3 veces por semana y reemplazarla con 3,5 ml de medio fresco. </li></ol> 3. Aislamiento cholangiocytes primarias de grueso colágeno geles Nota: en el plazo de 3 semanas, hojas de cholangiocytes (células con núcleo grande que se extiende directamente desde conductos integrados) serán visibles en el colágeno de espesor. Si hay un gran número de fibroblastos en el plato, debe desecharse. Si un par de regiones del plato tienende crecimiento de fibroblastos, éstos se puede cortar con un bisturí y se retira antes de la división de los colangiocitos. <ol><li> Preparar geles de colágeno delgadas: <ol><li> Diluir colágeno a una concentración de 1 mg / ml con PBS estéril. Nota: Utilice 1 ml por placa de 100 mm; ajustar en consecuencia para otros platos de tamaño. </li><li> Corre la solución de colágeno de manera uniforme con un esparcidor de células (grandes platos) o puntas de pipeta (pequeños platos), a continuación, dejar a temperatura ambiente durante 10 min. </li><li> Cubrir la placa con DMEM/F12 y se incuba a 37 ° C, 5% de CO 2 durante 10 min. </li><li> Retirar la placa de la incubadora y lavar con PBS 1x. Inmediatamente aspirar el PBS 1x. </li><li> Escudo con una segunda capa delgada de colágeno, la difusión mediante la rotación de la placa o la colocación de la placa en un agitador. Incubar a 37 ° C, 5% de CO 2 durante 10 min. </li><li> Después de colágeno se ha solidificado, la placa de cubierta con DMEM/F12 e incubar a 37 ° C, 5% de CO2 durante 30 min. </li></ol></li><li> Dividir celdas de thgeles de colágeno ick: <ol><li> Con espátula de cultivo celular o rascador de células, raspar gel de colágeno suavemente la placa de modo que está flotando. </li><li> Preparar la solución de colagenasa diluyendo Tipo XI colagenasa (vea la lista de materiales) a 10 mg / ml con DMEM/F12. Mezclar bien y filtrar de forma estéril. </li><li> Añadir 1 ml de solución de colagenasa (arriba) por 60 mm de placa de células en colágeno de espesor. No extraiga el soporte BEC (que debe ser 3 ml). Incubar plato durante 30 min a 37 ° C, 5% de CO 2. </li><li> Retire la solución que contiene células, y el lugar en 15 ml tubo cónico. </li><li> Centrifugar a 2200 xg durante 5 min. Descartar el sobrenadante y volver a suspender el pellet en volumen similar de DMEM/F12. </li><li> Girar solución de nuevo en 2200 xg durante 5 min. Aspirar el sobrenadante. </li><li> Resuspender el sedimento en 3 ml de 0,5% de tripsina, y se incuba a 37 ° C, 5% de CO 2 durante 5 min. Después de la incubación, la solución de la pipeta hacia arriba y abajo para romper las hojas de cholangiocytes. Añadir 5 ml de los medios de comunicación un BECsolución giro ª a 1500 × g durante 5 min. </li><li> Extraer el sobrenadante, y resuspender el sedimento en DMEM/F12, a continuación, girar solución a 1500 xg durante 5 min. </li><li> Eliminar el sobrenadante y volver a suspender el pellet en medios BEC (Tabla 1). Las células de la placa en geles de colágeno delgadas hechas previamente. </li></ol></li></ol> 4. Pasaje y almacenamiento de células Nota: las células en geles de colágeno delgadas se pueden dividir según sea necesario. <ol><li> Lavar las placas con PBS estéril antes de la incubación en tripsina al 0,25% a 37 ° C durante 10-15 min. </li><li> Neutralizar con un volumen igual de medio de BEC, y sedimentar a 1500 xg durante 5 min. Enjuague de pellets con DMEM/F12, entonces sedimentar a 1500 xg durante 5 min. </li><li> Resuspender las células en los medios de comunicación de BEC para la distribución de colágeno recubierto placas de cultivo celular. Nota: Alternativamente, las células pueden ser resuspendieron en medios de almacenamiento y se almacenan en un congelador de -80 ° C o nitrógeno líquido (Tabla 1). </li></ol>

<ol>
	<li>Paradis, K., Sharp, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+duct-like+structure+formation+after+isolation+of+bile+ductular+cells+from+a+murine+model.">In vitro duct-like structure formation after isolation of bile ductular cells from a murine model.</a> J. Lab. Clin. Med. 113 (6), 689-694 (1989).</li><li>Vroman, B., LaRusso, N. F. <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+characterization+of+polarized+primary+cultures+of+rat+intrahepatic+bile+duct+epithelial+cells.">Development and characterization of polarized primary cultures of rat intrahepatic bile duct epithelial cells.</a> Lab. Invest. 74 (1), 303-313 (1996).</li><li>Kumar, U., Jordan, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+biliary+epithelial+cells+from+the+biliary+tract+fraction+of+normal+rats.">Isolation and culture of biliary epithelial cells from the biliary tract fraction of normal rats.</a> Liver. 6 (6), 369-378 (1986).</li><li>Ishii, M., Vromen, B., LaRusso, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+morphologic+characterization+of+bile+duct+epithelial+cells+from+normal+rat+liver.">Isolation and morphologic characterization of bile duct epithelial cells from normal rat liver.</a> Gastroenterology. 97 (5), 1236-1247 (1989).</li><li>Strazzabosco, M., Fabris, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+bile+ducts:+Essentials+for+the+clinical+hepatologist.">Development of the bile ducts: Essentials for the clinical hepatologist.</a> J. Hepatol. 56 (5), 1159-1170 (2012).</li><li>Carpentier, R., 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=Embryonic+ductal+plate+cells+give+rise+to+cholangiocytes,+periportal+hepatocytes,+and+adult+liver+progenitor+cells.">Embryonic ductal plate cells give rise to cholangiocytes, periportal hepatocytes, and adult liver progenitor cells.</a> Gastroenterology. 141 (4), 1432-1438 (2011).</li><li>Cho, W. K., Mennon, A., Boyer, J. L. <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+functional+polarized+bile+duct+units+from+mouse+liver.">Isolation of functional polarized bile duct units from mouse liver.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 280 (2), (2001).</li><li>Karjoo, S., Hand, N. J., Loarca, L., Russo, P. A., Friedman, J. R., 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=Extra-hepatic+cholangiocyte+cilia+are+abnormal+in+biliary+atresia.">Extra-hepatic cholangiocyte cilia are abnormal in biliary atresia.</a> J. Pediatr. Gastroenterol. Nutr. 57 (1), 96-101 (2013).</li><li>Sutton, M. E., op den Dries, S., Koster, M. H., Lisman, T., Gouw, A. S., Porte, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+human+extrahepatic+biliary+epithelium:+the+peribiliary+glands+as+progenitor+cell+compartment.">Regeneration of human extrahepatic biliary epithelium: the peribiliary glands as progenitor cell compartment.</a> Liver Int. 32 (4), 554-559 (2012).</li><li>Cardinale, V., 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=Multipotent+stem/progenitor+cells+in+human+biliary+tree+give+rise+to+hepatocytes,+cholangiocytes,+and+pancreatic+islets.">Multipotent stem/progenitor cells in human biliary tree give rise to hepatocytes, cholangiocytes, and pancreatic islets.</a> Hepatology. 54 (6), 2159-2172 (2011).</li></ol>

Los autores declaran que no tienen intereses en conflicto.

Aquí se describe es una técnica para aislar cholangiocytes puros a partir de las vías biliares extrahepáticas de los ratones de los ratones de todas las edades, incluidos los recién nacidos. La técnica ofrece la ventaja de que cholangiocytes extrahepáticos se pueden estudiar por separado de cholangiocytes intrahepáticas, y puede facilitar los estudios para identificar las diferencias clave entre estas poblaciones de células. Hemos publicado recientemente un estudio que demuestra disminuyó cilios en cholangiocy...

El origen y desarrollo de cholangiocytes intra y extrahepáticas son marcadamente diferentes. El hígado se desarrolla a partir de un divertículo del intestino anterior endodermo ventral 5. La región caudal del divertículo forma el árbol biliar extrahepático, mientras que la región craneal genera el árbol biliar intrahepático 5. Cholangiocytes conductos intrahepáticos se derivan de células progenitoras alrededor de la placa ductal en las regiones portal peri 6. Estas células tienen la capacidad de diferenciarse en hepatocitos o colangiocitos 6. Esto tiene importantes implicaciones clínicas dado que cholangiopathies pueden dirigirse específicamente a una categoría de cholangiocytes. Por ejemplo, atresia biliar afecta inicialmente los conductos extrahepáticos del recién nacido, mientras que el síndrome de Alagille afecta conductos intrahepáticos. Hay múltiples descripciones de la aislamiento de cholangiocytes intrahepáticos y unidades de conducto biliar de ratones y ratas. Entigadores han aislado células individuales mediante el aislamiento de conductos y la posterior derivación o por digestión del hígado y el anticuerpo desplegable de cholangiocytes que expresan marcadores de superficie celular específicos 1-4. Unidades funcionales de las vías biliares y polarizados se han aislado por digestión del hígado y la filtración de tamaño 7. Unidades de conductos biliares son capaces de responder a los estímulos secretora y demostrar la secreción de líquido 7, mientras cholangiocytes aislados se ha demostrado que el desarrollo de las estructuras ductular in vitro 1,2. Las limitaciones de estos métodos incluyen la necesidad de conocimientos técnicos especializados y equipos especiales para la perfusión del hígado con las enzimas digestivas. Además, hay un riesgo de contaminación con células mesenquimales 3. Un método para aislar extrahepáticos cholangiocytes del conducto biliar, específicamente de los conductos biliares extrahepáticos neonatales, no se ha descrito previamente. En este trabajo se describe una técnica simplificada para aislar un neonatals así como las células de las vías biliares extrahepáticas adultos en los altos niveles de pureza. Esta técnica facilitará el estudio de las diferencias entre cholangiocytes y la investigación sobre los mecanismos de enfermedades como la atresia biliar que implican los conductos intra y extrahepáticas extrahepáticas.

<table border="0" cellpadding="0" cellspacing="0" style="width:943px;" width="942">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Name of Material/Equipment</td>
			<td style="width:323px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">DMEM/F12 (1:1)</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">11320-033</td>
			<td>500ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">FBS</td>
			<td style="width:323px;">Atlanta Biologicals</td>
			<td style="width:119px;">S11150</td>
			<td>25 ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">MEM with non-essential amino acids</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">11140-019</td>
			<td>5 ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Insulin-transferrin-selenium</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">51300-044</td>
			<td>5 ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Na Pyruvate</td>
			<td style="width:323px;">Cellgro</td>
			<td style="width:119px;">25-000-CL</td>
			<td>5ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Chemically-defined lipid concentrate</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">11905-031</td>
			<td>5ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Penicillin-Streptomycin</td>
			<td style="width:323px;">Cellgro</td>
			<td style="width:119px;">30-002-CI</td>
			<td>5ml, used in BEC media and 500 ul in the isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Gentamicin</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">15750-060</td>
			<td>0.2ml, used in BEC media and 500 ul in the isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Ethanolamine</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">E9508-100ml</td>
			<td>0.13ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">MEM vitamin solution</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">11120-052</td>
			<td>5ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Soybean trypsin inhibitor</td>
			<td style="width:323px;">Biowhittaker</td>
			<td style="width:119px;">17-605E</td>
			<td>5ml, used in BEC media. Solvent is PBS, mix to 5mg/ml stock concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">L-glutamine</td>
			<td style="width:323px;">Cellgro</td>
			<td style="width:119px;">25-005-CL</td>
			<td>5ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Bovine pituitary extract</td>
			<td style="width:323px;">Gemini</td>
			<td style="width:119px;">500-102</td>
			<td>1.1ml, used in BEC media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Dexamethasone</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">D4902</td>
			<td>0.5ml, used in BEC media, Stock conc 393ug/ml dilute with ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">3 3&#39;,5-triiodo-L-thyronine</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">T6397</td>
			<td>0.5ml, used in BEC media, Stock conc 3.4 mg/ml dilute with ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Epidermal growth factor</td>
			<td style="width:323px;">millipore</td>
			<td style="width:119px;">01-101</td>
			<td>0.5ml, used in BEC media, 25ug/ml dilute with DMEM F12+1%BSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Forskolin</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">F6886</td>
			<td>5ml, used in BEC media, use at stock concentration of 0.411mg/ml and dilute with DMSO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Fungizone</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">15290-018</td>
			<td>1ml, used in BEC media and 500 ul in the isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Rat-tail collagen</td>
			<td style="width:323px;">BD Biosciences</td>
			<td align="right" style="width:119px;">354236</td>
			<td>variable depending on concentration of collagen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">PBS 10X</td>
			<td style="width:323px;">USB Corporation</td>
			<td align="right" style="width:119px;">75889</td>
			<td>use at 10x, sterlie, used to make collagen, amount used depends on collagen concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">dH2O</td>
			<td style="width:323px;">N/A</td>
			<td style="width:119px;">N/A</td>
			<td>sterile, used to make collagen, amount used depends on collagen concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">NaOH 10N</td>
			<td style="width:323px;">Fischer Scientific</td>
			<td style="width:119px;">ss255-1</td>
			<td>Dilute to 1N, sterile, used to make collagen, amount used depends on collagen concentration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">collagenase type XI from Clostridium histolyticum</td>
			<td style="width:323px;">Sigma Aldrich</td>
			<td style="width:119px;">C7657</td>
			<td>dilute in DMEM and sterile filter before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">trypsin-EDTA (1x) 0.25%</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">25200-056</td>
			<td>3 ml, incubate max 10 minutes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">trypsin-EDTA (10x) 0.5%</td>
			<td style="width:323px;">Gibco/ Life technologies</td>
			<td style="width:119px;">15400-054</td>
			<td>3 ml, incubate max 10 minutes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;"></td>
			<td style="width:323px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">Dissecting microscope</td>
			<td style="width:323px;">Nikon</td>
			<td style="width:119px;">SMZ645</td>
			<td>Other models acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:335px;">Light source (fiberoptic illuminator)</td>
			<td style="width:323px;">Schott-Fostec</td>
			<td style="width:119px;">Ace EKE LR 92240</td>
			<td>Other models acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">12.5 cm straight iris scissors</td>
			<td style="width:323px;">Kent Scientific</td>
			<td style="width:119px;"></td>
			<td>Other models acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">6&#34; non-serrated curved forceps with fine tips</td>
			<td style="width:323px;">Electron Microscopy Sciences</td>
			<td style="width:119px;"></td>
			<td>Other models acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:335px;">4&#34; serrated stainless forceps with fine tips</td>
			<td style="width:323px;">Electron Microscopy Sciences</td>
			<td style="width:119px;"></td>
			<td>Other models acceptable</td>
		</tr>
	</tbody>
</table>

isolation of neonatal extrahepatic cholangiocytes

Los conductos biliares intra y extrahepáticas del hígado son de desarrollo distinta, y puede ser diversamente afectados por ciertas enfermedades. Sin embargo, las diferencias entre cholangiocytes intra y extrahepáticas, y entre las células neonatales y de adultos, no están bien entendidas. Los métodos para el aislamiento de cholangiocytes de conductos biliares intrahepáticos están bien establecidos 1-4. Aislamiento de células ductales extrahepáticos, especialmente desde el recién nacido, todavía no se ha descrito, aunque esto sería de gran beneficio en la comprensión de las diferencias entre las poblaciones cholangiocyte distintos y en el estudio de enfermedades tales como la atresia biliar que aparecen para apuntar a los conductos extrahepáticos. Se describe aquí es una técnica optimizada para aislar las células de las vías biliares extrahepáticas, tanto neonatal y del adulto del ratón. Esta técnica permite obtener una población de células puro con un mínimo de contaminación de las células mesenquimales como los fibroblastos. Esta metod se basa en la eliminación de los conductos y de la vesícula biliar extrahepática, seguida de la disección meticulosa y raspado para remover capas de grasa y fibroblastos. Estructuras están incrustadas en capas gruesas de colágeno y se cultivaron durante aproximadamente 3 semanas para permitir el crecimiento de cholangiocytes en monocapas, que luego pueden ser tripsinizaron y re plateado para uso experimental.

El uso de este protocolo resulta en el aislamiento de una población de ratones cholangiocytes extrahepáticas neonatales con excelente pureza, como lo demuestra la tinción de inmunofluorescencia K19 (Figuras 2A y 2B); logramos resultados similares aislamiento de células a partir de ratones adultos. Hemos observado que se tarda 3 semanas para las células de los conductos biliares recién aisladas para formar monocapas sobre geles de colágeno gruesas. Los cholangiocytes crecen de forma lineal a lo la...

Watch this Scientific Journal Video about Isolation of Neonatal Extrahepatic Cholangiocytes at JoVE.com

Isolation of Neonatal Extrahepatic Cholangiocytes

Una técnica para aislar cholangiocytes de los conductos biliares extrahepáticos de ratones recién nacidos se describe. Los conductos están meticulosamente disecados, y luego las células se aíslan por consecuencia en geles de colágeno gruesas. Este método proporciona una herramienta útil para el estudio del desarrollo de vías biliares extrahepáticas y patología.

Aislamiento de Neonatal extrahepática cholangiocytes

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, ...

isolation-of-neonatal-extrahepatic-cholangiocytes

Research

JoVE Journal

Biology

10.2K vistas.  The Children's Hospital of Philadelphia.  Una técnica para aislar cholangiocytes de los conductos biliares extrahepáticos de ratones recién nacidos se describe. Los conductos están meticulosamente disecados, y luego las células se aíslan por consecuencia en geles de colágeno gruesas. Este método proporciona una herramienta útil para el estudio del desarrollo de vías biliares extrahepáticas y patología. 

Video: Isolation of Neonatal Extrahepatic Cholangiocytes

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

Aislamiento de neutrófilos Protocolo

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

Investigación

Biología

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

Aislamiento de células endoteliales valvular

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Aislamiento y cultivo de células endoteliales pulmonares de ratones recién nacidos

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

El aislamiento de Drosophila melanogaster Testículos

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Isolation and Biophysical Study of Fruit Cuticles

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and abiotic stresses and also regulates water flow from the external environment.1 A biopolyester (cutin) and long-chain fatty acids (waxes) form the principal structural framework of the cuticle; the functional integrity of the cuticular layer depends on the outer 'epicuticular' layer as well as the blend consisting of the cutin biopolymer and 'intracuticular' waxes.2 Herein, we describe a comprehensive protocol to extract waxes exhaustively from commercial tomato (Solanum lycopersicum) fruit cuticles or to remove epicuticular and intracuticular waxes sequentially and selectively from the cuticle composite. The method of Jetter and Sch&auml;ffer (2001) was adapted for the stepwise extraction of epicuticular and intracuticular waxes from the fruit cuticle.3,4 To monitor the process of sequential wax removal, solid-state cross-polarization magic-angle-spinning (CPMAS) 13C NMR spectroscopy was used in parallel with atomic force microscopy (AFM), providing molecular-level structural profiles of the bulk materials complemented by information on the microscale topography and roughness of the cuticular surfaces. To evaluate the cross-linking capabilities of dewaxed cuticles from cultivated wild-type and single-gene mutant tomato fruits, MAS 13C NMR was used to compare the relative proportions of oxygenated aliphatic (CHO and CH2O) chemical moieties.
Exhaustive dewaxing by stepwise Soxhlet extraction with a panel of solvents of varying polarity provides an effective means to isolate wax moieties based on the hydrophobic characteristics of their aliphatic and aromatic constituents, while preserving the chemical structure of the cutin biopolyester. The mechanical extraction of epicuticular waxes and selective removal of intracuticular waxes, when monitored by complementary physical methodologies, provides an unprecedented means to investigate the cuticle assembly: this approach reveals the supramolecular organization and structural integration of various types of waxes, the architecture of the cutin-wax matrix, and the chemical composition of each constituent. In addition, solid-state 13C NMR reveals differences in the relative numbers of CHO and CH2O chemical moieties for wild-type and mutant red ripe tomato fruits. The NMR techniques offer exceptional tools to fingerprint the molecular structure of cuticular materials that are insoluble, amorphous, and chemically heterogeneous. As a noninvasive surface-selective imaging technique, AFM furnishes an effective and direct means to probe the structural organization of the cuticular assembly on the nm-&mu;m length scale.

Aerial plant organs are protected by the cuticle, a supramolecular biopolyester-wax assembly. We present protocols to monitor selective removal of epi- and intracuticular waxes from tomato fruit cuticles on molecular and micro scales by solid-state NMR and atomic force microscopy, respectively, and to assess the cross-linking capacity of engineered cuticular biopolyesters.

El aislamiento y estudio biofísico de las cutículas de fruta

The cuticle, a hydrophobic protective layer on the aerial parts of terrestrial plants, functions as a versatile defensive barrier to various biotic and ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

Aislamiento y cultivo de cardiomiocitos neonatales Ratón

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Aislamiento de células endoteliales embrionarias ventriculares

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

El aislamiento de la arteria pulmonar células musculares lisas de los ratones recién nacidos

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Aislamiento de las células murinas de la válvula endoteliales

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.