Os autores agradecem à Patologia Molecular e Imagem Núcleo do Centro de NIDDK UPenn para estudos moleculares em Doenças Digestivas e do Fígado (P30 DK50306) para assistência com imagens. Este trabalho foi financiado por concessões do National Institutes of Health (R01 DK-092111) e do Fred e Suzanne Biesecker Pediatric Liver Center (para RGW) e por uma bolsa da Liver Disease Research e Rede de Educação Infantil (a SK).

Todo o procedimento é realizado à temperatura ambiente, a menos que especificado de outra forma. Todo o trabalho animal deve ser realizado em condições humanas no âmbito de um protocolo aprovado pelo Comitê Institucional locais Animal Care e Use (IACUC). 1. Preparação de equipamentos e soluções <ol><li> Configure mesa cirúrgica o mouse em estreita proximidade com a capa de cultura de tecidos e incubadora (Figura 1A). Nota: Equipamento necessário inclui um microscópio de dissecação, uma fonte de luz, 12,5 cm de comprimento tesoura de íris reta, pinça curva de 6 polegadas não serrilhadas com pontas finas e 4 de polegada de fórceps serrilhados, com ponta curva (Figura 1B). </li><li> Coloque instrumentos esterilizados em um copo de vidro de 70% de álcool na mesa cirúrgica. </li><li> Preencha três estéreis 60 milímetros placas de Petri com meios de isolamento estéril (Tabela 1); colocar dois na mesa cirúrgica e um no capô, tudo em gelo. </li><li> Coloque rato colágeno cauda,estéril 10x tampão fosfato salino (PBS), água estéril, estéril NaOH 1 N e biliar de células epiteliais (BEC) mídia (Tabela 1) na capa de cultura de tecidos. Nota: O colagénio deve ser em gelo. </li></ol> 2. Colagoga Isolamento e Cultura <ol><li> Eutanásia ratos neonatal entre 0-3 dias de vida, por exposição ao dióxido de carbono até inconsciente, seguido por deslocamento cervical, por diretrizes IACUC (Figura 1C). Nota: ratos mais velhos podem ser usados, dependendo das necessidades específicas experimental. </li><li> Coloque o animal em decúbito dorsal e fazer uma pequena incisão horizontal cruzando a linha média na região da pelve. Estender esta incisão lateral e os dois lados do abdômen, formando uma ponta em forma de U. Vire aba para cima para expor os órgãos abdominais. Uma vez que o abdômen é aberto, mover cuidadosamente os intestinos para fora da cavidade abdominal para o lado direito do animal para melhor visualização do ducto hepático e biliar comum. Nota: pode ser necessário prolongar os cortes laterais para a caixa torácica para obter uma melhor identificação do fígado. </li><li> Utilizando o microscópio de dissecação, identificar o ducto biliar comum, fígado e intestinos. Identificar o ducto biliar comum em primeiro lugar (Figura 1D), utilizando-se cautela, pois as estruturas são muito delicadas. Com a pinça serrilhadas dentadas e não, meticulosamente limpo e apanhar o tecido conjuntivo, incluindo a gordura em torno do bile. Use uma pinça para segurar a conduta e o outro para limpar. </li><li> Use uma técnica similar para limpar a vesícula biliar. </li><li> Coloque os dutos limpos e vesícula biliar para o primeiro prato de meios de isolamento no gelo. Massageie suavemente as estruturas com a pinça não serrilhadas enquanto nos meios de comunicação para remover mais tecido conjuntivo e remover bile da vesícula biliar. </li><li> Transferência de dutos e da vesícula biliar para o segundo prato de meios de isolamento. </li><li> Após o isolamento de dutos e vesículas biliares de 5 animais, transferir opeças para a terceira placa de Petri, no bairro. Nota: Se as células são para ser isolado a partir de mais de 5 animais, fazer em lotes, usando soluções frescas para cada grupo de 5 animais. </li><li> Prepare géis de colágeno grossas na capa de cultura de tecidos: Nota: todos os tamanhos do prato podem ser utilizados. Uma placa de cultura de 60 milímetros, com um volume final de 3 mL de colagénio para cada grupo de 5 recém-nascidos funciona bem. Isto deve ser preparado fresco, no momento de incorporação de condutas isoladas de fresco. <ol><li> Coloque colagénio de cauda de rato, estéril de 10x tampão fosfato salino (PBS 10x), dH 2 O estéril e estéril de NaOH 1 N em gelo. </li><li> Calcular as quantidades de dH 2 0, 10x PBS, NaOH, e colagénio necessária para géis com uma concentração final de colagénio de 2 mg / mL em 1x PBS, utilizando um volume de NaOH a 1 N igual a 0,023 vezes o volume de colagénio adicional. Nota: Se é usada fonte alternativa de colágeno, siga as instruções do fabricante para a neutralização e formação de gel. </li><li> Misture dH 2 0, 10x PBS, e NaOH 1 N, em seguida, adicionar o colagénio e pipeta bem para assegurar uma mistura uniforme. </li></ol></li><li> Depois solução de colagénio engrossou para um gel de consistência semelhante à temperatura ambiente, imediatamente inserir as condutas do colagénio. </li><li> Colocar na incubadora a 37 ° C, 5% de CO 2 durante 30 minutos para permitir que o colagénio para solidificar. </li><li> Depois de gel ter solidificado (a cor muda de transparente para branco), sobreposição com 3,5 ml de meio de BEC (Tabela 1) e incubar a 37 ° C, 5% de CO 2. </li><li> Remova a mídia BEC 3 vezes por semana e substituir com 3,5 ml de mídia fresco. </li></ol> 3. Isolando primárias cholangiocytes de Thick Colágeno Gel Nota: dentro de 3 semanas, folhas de cholangiocytes (células com núcleos grandes estender diretamente de dutos embutidos) será visível no colágeno de espessura. Se houver um grande número de fibroblastos no prato, ele deve ser descartado. Se algumas regiões do prato temde crescimento de fibroblastos, estes podem ser cortados com um bisturi e removido antes da divisão das cholangiocytes. <ol><li> Prepare géis finas de colágeno: <ol><li> Dilui-se o colagénio a uma concentração de 1 mg / ml com PBS estéril. Nota: utilizar 1 ml para placa de 100 mm; ajustar em conformidade para outros pratos de tamanho. </li><li> Espalhe a solução de colágeno de forma uniforme com um espalhador de celular (pratos grandes) ou ponta de pipeta (pequenos pratos), deixe em temperatura ambiente por 10 min. </li><li> Espelho com DMEM/F12 e incubar a 37 ° C, 5% de CO 2 durante 10 min. </li><li> Retire a placa da incubadora e lave com 1x PBS. Imediatamente a aspirar 1x PBS. </li><li> Revestimento com uma segunda camada de colagénio finas, espalhando por rotação da placa de colocação ou de placa num agitador. Incubar a 37 ° C, 5% de CO 2 durante 10 min. </li><li> Depois de colagénio tenha solidificado, a placa de cobertura com DMEM/F12 e incubar a 37 ° C, 5% de CO 2 durante 30 min. </li></ol></li><li> Dividir células a partir de diagéis de colágeno ick: <ol><li> Com uma espátula de cultura de células ou um raspador de células, raspar gel de colagénio suavemente a placa a fim de que ele está a flutuar. </li><li> Preparar a solução de colagenase diluindo XI Tipo de colagenase (ver lista de materiais) para 10 mg / ml com meio DMEM/F12. Misturar bem e filtro estéril. </li><li> Adicionar 1 ml de solução de colagenase (acima) por 60 milímetros prato de células no colagénio de espessura. Não remova a mídia BEC (que deve ser de 3 ml). Incubar prato durante 30 minutos a 37 ° C, 5% de CO 2. </li><li> Remover solução contendo as células, e coloque no tubo de 15 ml. </li><li> Gire para baixo a 2.200 xg por 5 min. Desprezar o sobrenadante e re suspender pellet em volume similar de DMEM/F12. </li><li> Rotação solução novamente a 2.200 xg durante 5 min. Remover o sobrenadante. </li><li> Ressuspender o sedimento em 3 ml de 0,5% de tripsina e incubação a 37 ° C, 5% de CO 2 durante 5 min. Após a incubação, pipetar a solução para cima e para baixo para quebrar as folhas de cholangiocytes. Adicionar 5 ml de BEC mídia umasolução de spin ª a 1.500 xg por 5 min. </li><li> Remover o sobrenadante e ressuspender o pellet em DMEM/F12, em seguida girar a solução 1.500 xg durante 5 min. </li><li> Remover o sobrenadante e re suspender pellet em mídia BEC (Tabela 1). Células placa na feitas anteriormente géis de colágeno finas. </li></ol></li></ol> 4. Passage e Armazenamento de Células Nota: as células em géis de colagénio finas pode ser dividido conforme necessário. <ol><li> Lavar as placas com PBS estéril, antes da incubação em 0,25% de tripsina, a 37 ° C durante 10-15 min. </li><li> Neutraliza-se com um volume igual de meio de BEC, e sedimentar a 1500 x g durante 5 min. Lavar pelete com DMEM/F12, então sedimentar a 1500 x g durante 5 min. </li><li> Ressuspender as células em meios BEC para a distribuição de colágeno revestido placas de cultura de células. Observação: Como alternativa, as células podem ser novamente suspensas em meios de armazenamento e armazenado num congelador a -80 ° C ou azoto líquido (Quadro 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>

Os autores declaram que não têm interesses conflitantes.

Descrito aqui é uma técnica para isolar cholangiocytes puros das vias biliares extra-hepáticas de ratos de ratos de todas as idades, incluindo recém-nascidos. A técnica oferece a vantagem de que cholangiocytes extra-hepáticas podem ser estudadas separadamente cholangiocytes intra-hepática, e pode facilitar a estudos para identificar as principais diferenças entre estas populações de células. Publicamos recentemente um estudo demonstrando diminuição cílios em cholangiocytes extra-hepáticos isolados por est...

A origem eo desenvolvimento da intra e extra-hepáticos cholangiocytes são marcadamente diferentes. O fígado se desenvolve a partir de um divertículo do intestino anterior endoderme ventral 5. A região caudal do divertículo forma a árvore biliar extra-hepática, enquanto que a região craniana gera a árvore biliar intra-hepática 5. Cholangiocytes duto intra-hepática são derivadas de células progenitoras em torno da placa ductal nas regiões peri portal 6. Estas células têm a capacidade de se diferenciar em qualquer hepatócitos ou cholangiocytes 6. Isto tem implicações clínicas significativas, dado que cholangiopathies podem objetivar especificamente uma categoria de cholangiocytes. Por exemplo, atresia biliar afeta inicialmente os ductos extra-hepáticos do recém-nascido, enquanto a Síndrome de Alagille afeta ductos intra-hepáticos. Há várias descrições do isolamento de cholangiocytes intra e unidades biliares de camundongos e ratos. Emvestigators isolaram células individuais pelo isolamento do duto e conseqüência subseqüente, ou por digestão do fígado e do anticorpo puxar para baixo de cholangiocytes expressam marcadores de superfície celular específicos 1-4. Unidades biliares funcionais e polarizadas foram isoladas por digestão do fígado e tamanho filtração 7. Unidades de ductos biliares são capazes de responder a estímulos secretora e demonstram a secreção de fluido 7, ao passo que cholangiocytes isolados têm sido mostrados para desenvolver estruturas ductulares in vitro 1,2. Limitações destes métodos incluem a necessidade de conhecimento técnico especializado e equipamento especial para perfusão de fígado com enzimas digestivas. Além disso, existe o risco de contaminação com células mesenquimais 3. Um método para isolar extra-hepáticos cholangiocytes do ducto biliar, especificamente a partir das vias biliares extra-hepáticos neonatal, não foi anteriormente descrito. Este artigo descreve uma técnica simplificada para isolar um neonatalé bem como as células dos ductos biliares extra-hepáticos adultos em altos níveis de pureza. Esta técnica irá facilitar o estudo das diferenças entre cholangiocytes e pesquisas sobre mecanismos de doenças como atresia biliar que envolvem os ductos extra-hepáticos e extra-hepáticos intra.

<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

Os ductos biliares intra e extra-hepáticos do fígado são developmentally distinta, e podem ser diferencialmente afectada por certas doenças. No entanto, as diferenças entre intra e extra-hepáticos cholangiocytes, e entre as células recém-nascidos e adultos, não são bem compreendidos. Os métodos para o isolamento de cholangiocytes de ductos biliares são bem estabelecida 1-4. Isolamento de células ductais extra-hepáticos, particularmente a partir do neonato, ainda não foi descrito, embora este seria de grande benefício para a compreensão das diferenças entre as populações distintas cholangiocyte e no estudo de doenças tais como a atresia biliar que aparecem para atingir os canais extra-hepáticos. Descrito aqui é uma técnica otimizada para isolar as células dos ductos biliares extra-hepáticas, tanto neonatal e adulto do rato. Esta técnica produz uma população de células pura com contaminação mínima entre as células mesenquimais como fibroblastos. Este metodod é baseado na remoção dos ductos extra-hepáticos e da vesícula biliar, seguida por dissecação meticulosa e raspagem para remover as camadas de gordura e de fibroblastos. As estruturas são incorporados em camadas de espessura de colagénio e cultivadas durante aproximadamente 3 semanas para permitir o crescimento de cholangiocytes em monocamadas, que pode, então, ser tratadas com tripsina e re-plaqueadas para o uso experimental.

A utilização destes resultados do protocolo para o isolamento de uma população de rato cholangiocytes extra-hepáticos neonatais com uma pureza excelente, como demonstrado por coloração de imunofluorescência K19 (Figuras 2A e 2B); se conseguir resultados semelhantes isolamento de células a partir de ratinhos adultos. Temos observado que leva 3 semanas para as células dos ductos biliares recém-isoladas para formar monocamadas em géis de colágeno de espessura. Os cholangiocytes crescer de form...

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

Isolation of Neonatal Extrahepatic Cholangiocytes

Uma técnica para isolar cholangiocytes das vias biliares extra-hepáticos de ratos neonatais é descrito. As condutas são meticulosamente dissecados, e, em seguida, as células são isoladas por crescimento em géis de colagénio espessas. Este método fornece uma ferramenta útil para estudar o desenvolvimento do ducto biliar extra-hepática e patologia.

Isolamento de Neonatal extra-hepáticas 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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.2K visualizações.  The Children's Hospital of Philadelphia.  Uma técnica para isolar cholangiocytes das vias biliares extra-hepáticos de ratos neonatais é descrito. As condutas são meticulosamente dissecados, e, em seguida, as células são isoladas por crescimento em géis de colagénio espessas. Este método fornece uma ferramenta útil para estudar o desenvolvimento do ducto biliar extra-hepática e patologia. 

Vídeo: 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.

Isolamento 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 ...

Biologia

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.

Isolamento de Valvular Células Endoteliais

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.

Isolamento e cultura de células endoteliais pulmonares de camundongos Neonatal

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.

Isolamento de 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 ...

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.

Isolamento e Estudo de Biofísica de Cutículas Frutas

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.

Isolamento e Cultura de Neonatais rato Cardiomyocytes

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.

Isolamento de Células Embrionárias ventriculares endoteliais

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.

Isolamento de artéria pulmonar células musculares lisas de camundongos recém-nascidos

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.

O isolamento de células endoteliais de Murino Válvula

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.