Agradecemos ao pessoal da AniCan (CRCL, Lyon) para a sua assistência técnica com cuidado animal. Este trabalho foi apoiado pelo Institut National de la Santé et de la Recherche Médicale (Programa Avenir INSERM), o Ligue Nationale Contre le Câncer, pela Associação pour la Recherche sur le Cancer, pelo Institut National du Cancer, e por bolsas de o Ligue Nationale Contre le Cancer (JG), a partir do Institut National du Cancer (JG), a partir do Ministère de l&#39;Enseignement Supérieur et de la Recherche de França (PGR e DFV) e da Associação pour la Recherche sur le Cancer ( DFV).

Todos os procedimentos foram aprovados por um comitê de ética sob regulamentação da autoridade governamental (&quot;Comité d&#39;Evaluation Commun au Centre Léon Bérard, à l&#39;Animalerie de trânsito de l&#39;ENS, au PBES et au Laboratoire P4&quot; (CECCAPP)). Camundongos foram mantidos em um biotério livre de patógenos específicos no &quot;Plateforme AniCan, Centro Léon Bérard&quot; (Lyon, França) e tratados de acordo com as diretrizes institucionais. Uma representação esquemática do procedimento é mostrado na Figura 1. 1. Dissecção pâncreas e dilaceração (Dia 0) A dissecção muito rápida é crítico para um rendimento óptimo de extracção e para garantir uma boa viabilidade das células em cultura. A fim de reduzir o tempo necessário para o isolamento de pâncreas, de todos os instrumentos e equipamentos devem estar pronto antes da eutanásia do rato. <ol><li><html> Eutanásia rato por asfixia CO 2 ou luxação cervical. </li></ol> A partir deste passo, todos os procedimentos devem ser realizados em um ambiente estéril (gabinete de segurança microbiológica, o nível II) com equipamentos de dissecção estéril. <ol start="2"><li> Corrigir o mouse e pulverizar o abdômen do rato com etanol 70%. Com qualquer uma tesoura de dissecção e uma pinça, faça uma incisão em forma de V na área genital e continuá-la até o diafragma para abrir completamente a cavidade abdominal. </li><li> Posicionar os lóbulos do fígado contra o diafragma, que devem permanecer ali, se a cavidade do corpo é aberta suficientemente longe. Puxe o intestino eo cólon fora da cavidade abdominal, à sua esquerda, e encontrar o reto. Com um par de fórceps curvos e dissecando tesoura, garra e seção do reto. </li><li> Com o mesmo par de pinças, cuidadosamente desenrole totalmente o intestino do reto para o estômago, puxando o intestino à sua esquerda. </li></ol>ent &quot;&gt; Neste passo, o pâncreas pode ser distinguido como uma pequena faixa entre o estômago e no início do intestino. suas ligaduras com o baço permanecem intactos. <ol start="5"><li> Usando Noyes tesoura e um par de fórceps, cuidadosamente cortadas ao longo do pâncreas e do intestino libertá-la com o baço a partir do resto do tracto digestivo. </li><li> Pega o baço e o pâncreas secção ligado a ele (Figura 2). </li></ol> Nesta etapa, certifique-se de que nenhuma gordura mesentérica e / ou outro tecido adjacente (baço, intestino, etc) podem ser coletadas com o pâncreas, para evitar a contaminação celular. Para o resto do processo, todos os tampões devem ser preparada sem ião de cálcio Ca 2 + agentes quelantes para evitar a dissociação completa do tecido pancreático exócrino em células acinares individuais. <ol start="7"><li> Enxaguar o pâncreas duas vezes em solução salina equilibrada de Hank (HBSS) 1x. </li></ol> Neste passo, como tecido adiposo irá flutuar ao contrário do pâncreas que se afundará, é facilmente possível visualizar e remover rapidamente o tecido adiposo branco contaminante ainda ligado ao pâncreas. Se o pâncreas precisa ser transportado para a instalação de cultura de células, deve ser mantido em gelo em HBSS 1x. <ol start="8"><li> Transferir o pâncreas em uma placa de Petri estéril contendo 5 ml de HBSS 1x. Usando Noyes tesouras e um bisturi, cortar o pâncreas em pequenos pedaços de 1-3 mm 3 (Figura 3A). </li></ol> 2. Dissociations enzimática e mecânica de pâncreas (Dia 0) <ol><li> Transferi-los para um tubo de polipropileno ml estéril 50. </li><li> Centrifugar durante 2 minutos a 450 xg e 4 ° C. Aspirar e desprezar o sobrenadante para remover fragmentos de células e células do sangue. </li><li> Adicionar 10 ml de solução de colagenase IA (1x HBSS contendo 10 mM de HEPES, 200 U / ml de collagenaSE IA, e de 0,25 mg / ml de inibidor de tripsina) de secções de pâncreas. Usando uma pipeta serológica de 25 ml, transferi-los para um balão de 25 cm2. Incubar para 20-30 min a 37 ° C. Durante este tempo (5 minutos cada), executar uma dissociação mecânica por energeticamente movimento de vai-e-vem os fragmentos pâncreas cerca de dez vezes, em pipetas estéreis de tamanho decrescente (25, 10, e 5 ml pipetas serológicas). </li></ol> Neste passo, é frequentemente essencial para controlar a extensão da dissociação enzimática de secções pancreáticas. <ol start="4"><li> Quando o tecido pancreático parece ser bem dissociado (de acordo com o desaparecimento de fragmentos pancreáticas e ao aumento da turbidez da solução) (Figura 3B), parar a reacção enzimática por adição de 10 ml de solução de lavagem tamponada fria (HBSS 1x contendo 5 % Soro Fetal Bovino (FBS) e 10 mM de HEPES). </li><li> Transferi-lo para um tubo de 50 ml de polipropileno estéril e centrifuge durante 2 min a 450 xg e 4 ° C. Aspirar cuidadosamente e descartar o sobrenadante para remover a solução de colagenase IA. </li><li> Ressuspender o sedimento e lava-se com 10 ml de solução de lavagem tamponada. Centrifugar durante 3 min a 450 xg e 4 ° C. Aspirar cuidadosamente e descartar o sobrenadante. Repita este passo mais duas vezes. </li></ol> 3. Filtração e semeadura de Dispersos Acini (Dia 0) <ol><li> Ressuspender o sedimento celular em 7 ml de meio de Waymouth contendo 2,5% de FBS, 1% de Penicilina-Estreptomicina mistura (PS), 0,25 mg / ml de inibidor de tripsina, e 25 ng / mL de Factor de Crescimento Epidérmico recombinante humana (EGF). </li><li> Filtra-se a mistura de células, permitindo-lhe passar através de um filtro de 100 um para reter os fragmentos não digeridos (dutos, vasos sanguíneos, e as ilhotas de Langerhans). Estruturas acinares pancreáticas (acinus de 10-15 células) passar. </li><li> Lavar o filtro com 6 ml de meio contendo FBS de Waymouth, PS, tripsina inhibitor e EGF. </li></ol> Após este passo, as células têm de ser tratadas com muito cuidado, para evitar qualquer ácinos dissociação. <ol start="4"><li> Semente ácinos isolado em uma placa de cultura de 6 poços (2 ml por poço) (Figura 3C). Cultura los a 37 ° C sob 5% (v / v) de atmosfera de CO 2. </li></ol> Após este passo, as células acinares são cultivadas em suspensão. 4. Acinar Cultura de Células (dia 1 a 10) <ol><li> Vinte e quatro horas depois, transferir os ácinos (em suspensão) para uma nova placa de cultura de 6 poços, para eliminar as células contaminantes e restos celulares que tenham aderido durante a noite (Figura 4). </li></ol> Se a cultura de células tem de ser alargado durante vários dias, ou se as condições experimentais requerem células cultivadas em monocamada, recomenda-se a transferência e sementes em ácinos matriz andaimes. <ol start="2"><li> Um dia antes de verding em apoio matricial (dia 0), casaco de uma placa de cultura de 6 poços com colágeno tipo I (5 mg / cm 2). Adicionar 1 ml de solução de colagénio do tipo I (50 ug / ml em ácido acético 0,02 M, 0,2 um por filtração) a cada poço e permitir que adsorva passivamente em plástico, durante 1 hora a 37 ° C (ou durante a noite a 4 ° C, ). </li><li> Aspirar a solução de colágeno tipo I e lavar a poço revestido duas vezes com tampão fosfato 1x. </li><li> Permitir que o poço revestido para secar (em um gabinete de segurança microbiológica) pelo menos 12 horas antes do uso. </li><li> Transferir a ácinos primário isolado (obtido no Passo 4.1) para o prato de colagénio tipo I revestido de 6 poços de cultura e da cultura deles em condições idênticas às descritas anteriormente (a 37 ° C sob 5% (v / v) de atmosfera de CO2) . As células aderem ao substrato de colagénio de tipo I durante 2 dias. </li><li> No Dia 3, mudar o meio de cultura para eliminar as células não viáveis, que não aderiram. Com o tempo na cultura, as células vão progressivamente spler com o apoio contendo colágeno. Alterar o meio de cultura a cada 3 dias (Figura 5). </li></ol> As células acinares isolados obtidos podem ser contadas, depois de uma completa dissociação mecânica, usando uma câmara de contagem Thoma célula. Note-se que as células acinares isoladas não podem ser mantidas em cultura em seguida. A qualidade da cultura acinar obtido pode ser controlado por controlo da expressão de marcadores específicos tais como Tripsinogênio acinares, Pâncreas transcription factor 1 subunidade alfa, ou A1 carboxipeptidase (por imunocitoquímica ou experiências de imunofluorescência).

<ol>
	<li>Lardon, J., Bouwens, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metaplasia+in+the+pancreas.">Metaplasia in the pancreas.</a> Differentiation. 73, 278-286 (2005).</li><li>Sphyris, N., Logsdon, C. D., Harrison, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+retention+of+zymogen+granules+in+cultured+murine+pancreatic+acinar+cells+and+induction+of+acinar-ductal+transdifferentiation+in+vitro.">Improved retention of zymogen granules in cultured murine pancreatic acinar cells and induction of acinar-ductal transdifferentiation in vitro.</a> Pancreas. 30, 148-157 (2005).</li><li>Amsterdam, A., Jamieson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+functional+characterization+of+isolated+pancreatic+exocrine+cells.">Structural and functional characterization of isolated pancreatic exocrine cells.</a> Proc. Natl. Acad. Sci. U.S.A. 69, 3028-3032 (1972).</li><li>Amsterdam, A., Jamieson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+dispersed+pancreatic+exocrine+cells.+I.+Dissociation+technique+and+morphologic+characteristics+of+separated+cells.">Studies on dispersed pancreatic exocrine cells. I. Dissociation technique and morphologic characteristics of separated cells.</a> J. Cell. Biol. 63, 1037-1056 (1974).</li><li>Amsterdam, A., Jamieson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+dispersed+pancreatic+exocrine+cells.+II.+Functional+characteristics+of+separated+cells.">Studies on dispersed pancreatic exocrine cells. II. Functional characteristics of separated cells.</a> J. Cell. Biol. 63, 1057-1073 (1974).</li><li>Schultz, G. S., 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=Guinea+pig+pancreatic+acini+prepared+with+purified+collagenase.">Guinea pig pancreatic acini prepared with purified collagenase.</a> Exp. Cell Res. 130, 49-62 (1980).</li><li>Williams, J. A., Korc, M., Dormer, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Action+of+secretagogues+on+a+new+preparation+of+functionally+intact,+isolated+pancreatic+acini.">Action of secretagogues on a new preparation of functionally intact, isolated pancreatic acini.</a> Am. J. Physiol. 235, 517-524 (1978).</li><li>Logsdon, C. D., Williams, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+binding+and+biologic+effects+on+mouse+pancreatic+acini.">Epidermal growth factor binding and biologic effects on mouse pancreatic acini.</a> Gastroenterology. 85, 339-345 (1983).</li><li>Han, B., Logsdon, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholecystokinin+induction+of+mob-1+chemokine+expression+in+pancreatic+acinar+cells+requires+NF-kappaB+activation.">Cholecystokinin induction of mob-1 chemokine expression in pancreatic acinar cells requires NF-kappaB activation.</a> Am. J. Physiol. 277, 74-82 (1999).</li><li>Ji, B., Kopin, A. S., Logsdon, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species+differences+between+rat+and+mouse+CCKA+receptors+determine+the+divergent+acinar+cell+response+to+the+cholecystokinin+analog+JMV-180.">Species differences between rat and mouse CCKA receptors determine the divergent acinar cell response to the cholecystokinin analog JMV-180.</a> J. Biol. Chem. 275, 19115-19120 (2000).</li><li>Ji, K. A., Yang, M. S., Jou, I., Shong, M. H., Joe, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombin+induces+expression+of+cytokine-induced+SH2+protein+(CIS)+in+rat+brain+astrocytes:+involvement+of+phospholipase+A2,+cyclooxygenase,+and+lipoxygenase.">Thrombin induces expression of cytokine-induced SH2 protein (CIS) in rat brain astrocytes: involvement of phospholipase A2, cyclooxygenase, and lipoxygenase.</a> Glia. 48, 102-111 (2004).</li><li>Gaiser, S., 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=Intracellular+activation+of+trypsinogen+in+transgenic+mice+induces+acute+but+not+chronic+pancreatitis.">Intracellular activation of trypsinogen in transgenic mice induces acute but not chronic pancreatitis.</a> Gut. 60, 1379-1388 (2011).</li><li>Logsdon, C. D., Williams, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pancreatic+acinar+cells+in+monolayer+culture:+direct+trophic+effects+of+caerulein+in+vitro.">Pancreatic acinar cells in monolayer culture: direct trophic effects of caerulein in vitro.</a> Am. J. Physiol. 250, 440-447 (1986).</li><li>Stanger, B. Z., Dor, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+cellular+origins+of+pancreatic+cancer.">Dissecting the cellular origins of pancreatic cancer.</a> Cell Cycle. 5, 43-46 (2006).</li><li>Vincent, D. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tif1gamma+suppresses+murine+pancreatic+tumoral+transformation+by+a+smad4-independent+pathway.">Tif1gamma suppresses murine pancreatic tumoral transformation by a smad4-independent pathway.</a> Am. J. Pathol. 180, 2214-2221 (2012).</li><li>Vincent, D. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+TIF1gamma+cooperates+with+Kras+to+induce+cystic+tumors+of+the+pancreas.">Inactivation of TIF1gamma cooperates with Kras to induce cystic tumors of the pancreas.</a> PLoS Genet. 5, e1000575 (2009).</li><li>Dorrell, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mouse+pancreatic+alpha,+beta,+duct+and+acinar+populations+with+cell+surface+markers.">Isolation of mouse pancreatic alpha, beta, duct and acinar populations with cell surface markers.</a> Mol. Cell Endocrinol. 339, 144-150 (2011).</li><li>Case, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+intracellular+transport+and+discharge+of+exportable+proteins+in+the+pancreatic+acinar+cell+and+other+cells.">Synthesis, intracellular transport and discharge of exportable proteins in the pancreatic acinar cell and other cells.</a> Biol. Rev. Camb. Philos. Soc. 53, 211-354 (1978).</li><li>Blauer, M., Nordback, I., Sand, J., Laukkarinen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+explant+outgrowth+culture+model+for+mouse+pancreatic+acinar+cells+with+long-term+maintenance+of+secretory+phenotype.">A novel explant outgrowth culture model for mouse pancreatic acinar cells with long-term maintenance of secretory phenotype.</a> Eur. J. Cell. Biol. 90, 1052-1060 (2011).</li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

Neste protocolo, descrevemos um procedimento para isolar células acinares pancreáticas. Este método torna possível isolar mais do que 20 x 10 6 células acinares por animal, em menos de 1 hora. Graças à sua implementação rápida e simples (até 10 pâncreas podem ser processados ​​de forma independente por cada experiência em paralelo), este protocolo surge como um bom compromisso entre métodos de isolamento existentes 3-5,9-12,17. Etapas críticas / r...

Um problema frequentemente encontrado para laboratórios de investigação úteis em tecido pancreático exócrino é a dificuldade de se cultivar células acinares in vitro durante um período de tempo suficientemente longo para permitir uma experiência de longa duração. Um factor impedindo o desenvolvimento de tais sistemas de cultura representa a sensibilidade intrínseca do tecido pancreático para manipulação experimental devido ao elevado teor em glicolíticas, proteolítica, lipolítica e de enzimas, que literalmente digerir o tecido pancreático, quando eles são libertados durante o isolamento das células pancreáticas. Um segundo factor é a notável na plasticidade in vitro de células acinares, que tendem a perder as suas características de secreção e transdiferenciam a outras células maduras, tais como células do ducto pancreático hepatócitos ou células do tipo 1. In vitro, este plasticidade das células varia de acordo com as condições experimentais (tais como composição do meio de cultura) 2 </sup&gt; e apresenta um grau de complexidade no design das condições de cultivo apropriadas para células pancreáticas exócrinas 1. Vários métodos têm sido desenvolvidos para o isolamento e cultura de células acinares, primeiro a partir do pâncreas de porco da guiné 3-5. Inicialmente, esses protocolos envolvidos digestão do tecido pancreático com colagenase, quimotripsina, e um cocktail de protease, com isolamento final por dissociação mecânica vigorosa. As células pancreáticas isoladas desta forma exibido características estruturais e funcionais anormais, nomeadamente uma perda de estruturas apicais e danos significativos aos seus receptores de membrana. Células isoladas permaneceu viável por apenas 1 ou 2 dias. Preparação de ácinos dispersos mantém a sua arquitectura intra e intercelular, preservando as membranas celulares, limitando os danos para os receptores da superfície e, assim, melhorar a secreção exócrina em resposta a secreção de 6-8. Como result, este método oferece a grande vantagem de prolongar a viabilidade das células acinares de 7-10 dias in vitro. Além disso, este método é actualmente preferido para isolamento de células acinares 9-12, porque a manutenção de contactos intercelulares, incluindo o acoplamento por junções de hiato célula, é um determinante essencial da pancreática exócrina acinar fenótipo celular 13. À medida que a desdiferenciação de células acinares e do seu transdiferenciação de células ductais é um dos mecanismos propostos para a génese de cancros agressivos pancreáticas exócrinas 14, o modelo ácinos dispersos é também um sistema adequado para estudar a plasticidade do pâncreas e dos seus mecanismos moleculares subsequentes. Além disso, em combinação com a utilização de animais geneticamente modificados 15,16 e o desenvolvimento de técnicas de transferência de genes (adenovirais 2 ou transdução lentiviral, da utilização de nanopartículas, etc), neste modelo in vitro de células acinares primário podeser muito útil na determinação de como diversas disfunções genéticas afectam a regulação da diferenciação de células acinares, ou de desdiferenciação, e deverá proporcionar uma melhor compreensão dos eventos moleculares responsáveis ​​pelo início da pancreatite, lesões pré-cancerosas, e as alterações na plasticidade celular. Isolamento de ácinos dispersos é a abordagem usamos no nosso laboratório para cultura de células acinares pancreáticas. Nós aqui descrever e discutir o método utilizado. Ele envolve a dissociação enzimática de tecido pancreático (com uma colagenase bacteriana), acoplado ao rompimento mecânico, sem dissociação de células acinares. Enquanto a maioria dos protocolos envolvem a cultura do ácinos, em suspensão ou em substratos de plástico com tratamento especial, nós cultivá-las em suspensão apenas brevemente (por 24 horas), semeando-los depois para andaimes matriz se cultura de células prolongada é necessária. Este protocolo permite o isolamento rápido (em menos de 1 hora) de corrente alternada dispersos de pâncreasini, sustentável durante mais de uma semana de cultura. Ele permite o isolamento de mais de 20 x 10 6 por células acinares do pâncreas do rato. A sua simplicidade permite processar independentemente tantos como 10 pâncreas em paralelo. Ao manter a arquitectura intra e intercelular de ácinos e assim o fenótipo das células acinares primárias isoladas, este modelo constitui um sistema de escolha para o estudo de mecanismos de transdiferenciação, como todos os outros modelos pancreáticas exócrinas actualmente disponíveis são derivadas de tumores pancreáticos exibindo muitos genética alterações que levam a transformação celular.

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

isolation and culture of mouse primary pancreatic acinar cells

Este protocolo permite o isolamento rápido (em menos de 1 hora) de ácinos pancreáticos de murino, tornando possível mantê-las em cultura durante mais de uma semana. Mais de 20 x 10 6 células acinares podem ser obtidas a partir de um único pâncreas murino. Este protocolo oferece a possibilidade de processar independentemente tantos como 10 pâncreas em paralelo. Porque preserva arquitectura acinar, este modelo é bem adequado para o estudo da fisiologia do pâncreas exócrino, in vitro, em contraste com as linhas de células estabelecidas a partir de tumores pancreáticos, que exibem muitas alterações genéticas, resultando em perda parcial ou total da sua diferenciação acinar.

A Figura 1 esquematiza o &quot;dispersa&quot; ácinos método para isolamento de células acinares Primária. Os passos críticos, que têm de ser estritamente respeitadas ao longo do protocolo, são descritas na discussão da peça. Para facilitar a sua remoção, o pâncreas tem que ser coletadas a partir do abdômen, juntamente com o baço em anexo (Figura 2). Ambos os órgãos precisam de ser cortados em pedaços, e os tecidos de gordura residual, que po...

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

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

Nesta publicação, é descrito um procedimento rápido e conveniente para o isolamento e cultura de células acinares pancreáticas primárias do pâncreas murino. Este método constitui uma ferramenta valiosa para o estudo da fisiologia das células primárias normais do pâncreas fresco / não transformada exócrinas.

Isolamento e Cultura do mouse primárias células pancreáticas acinares

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

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

Research

JoVE Journal

Biology

35.3K visualizações.  Centre de Recherche en Cancérologie de Lyon.  Nesta publicação, é descrito um procedimento rápido e conveniente para o isolamento e cultura de células acinares pancreáticas primárias do pâncreas murino. Este método constitui uma ferramenta valiosa para o estudo da fisiologia das células primárias normais do pâncreas fresco / não transformada exócrinas. 

Vídeo: Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

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

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

Isolamento de células primário do mouse trofoblasto e Ensaio invasão do trofoblasto

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

Biologia

Isolation and Primary Culture of Rat Hepatic Cells

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

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

Isolamento e cultura primária de células hepáticas de ratos

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

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.

We describe a reproducible method of preparing mouse pancreatic acinar cells from a mouse for the purpose of examining acinar cell calcium signals and cellular injury with physiologically and pathologically relevant stimuli. A method for adenoviral infection of these cells is also provided.

Preparação de células pancreáticas acinares para Fins de cálcio Imaging, celulares Medidas ferimento e infecção adenoviral

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

Assessing the Secretory Capacity of Pancreatic Acinar Cells

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

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

Avaliando a capacidade de secreção de pâncreas Acinar Células

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

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

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

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

Isolamento e cultura primária de células de camundongo aórtica endoteliais

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

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

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

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

Isolamento e Cultura de Queratinócitos de Rato Primário de Pele de Rato Neonatal e Adulto

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

Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs

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

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

Isolamento e cultura de células endoteliais aórticas primárias de suínos em miniatura

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

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

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

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

Dissecção eficiente e cultura de células epiteliais de pigmento de retina do rato primário

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

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

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

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

Isolamento e Cultura de Queratinócitos Orais Primários do Paladar do Rato Adulto

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

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

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

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

Isolamento e Caracterização de Células Endoteliais Hemorlicais do Fígado Primário do Rato

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