Este trabalho foi financiado pelo NIH R01 DK05823 (para RGW), F32 DK083213 (para JWW), F30 DK081265 (para ALO), e por uma concessão do Fred e Biesecker Suzanne Liver Center Pediátrica (para RGW).

Todo o procedimento é realizado à temperatura ambiente, a menos que especificado em contrário. 1. Preparação de soluções enzimáticas <ol><li> Preparam-se soluções de enzimas de acordo com a Tabela 1 e estéreis, utilizando um filtro de 0,22 um filtro. Manter a solução 1 e solução 2, à temperatura ambiente e uma solução de 3 a 4 ° C até ao momento de utilização. Todas as soluções e equipamentos que entram em contacto com as células isoladas devem ser estéreis. </li></ol> 2. Preparação da máquina de perfusão <ol><li> Primeiro o sistema de perfusão com HBSS menos Ca 2 + / Mg 2 +, que foi aquecido a 37 ° C. Nota: Para assegurar que o perfusato é de 37 ° C, quando chega ao fígado, HBSS é aquecida num banho de água ajustado para 37 ° C. Depois ela passa através da bomba de perfusão, ele passa por um condensador de vidro encamisado que está ligado a um conjunto circulador de água a 40 ° C (Fig. 1). </li><li> Esterilizar cirúrgico paraóis em um copo com etanol a 70%. </li></ol> 3. Perfusão do Fígado <ol><li> Anestesiar um adulto de rato Sprague-Dawley machos por injecção intraperitoneal de Nembutal (50-100 peso corporal mg / kg, conforme necessário para se obter anestesia adequada). </li><li> Coloque o animal em decúbito dorsal sobre uma bandeja de plástico e corrigir os membros para a bandeja com fita adesiva. </li><li> Limpe o abdômen com EtOH 70%. </li><li> Fazer uma pequena incisão na pele sobre o abdómen na linha média. Dissecar a pele para longe do painel frontal por meio de corte em forma de U grande no abdómen. Cortar o músculo abdominal com uma tesoura na sequência da mesma forma para expor a cavidade peritoneal. </li><li> Expor a veia porta usando 2 cotonetes para mover suavemente as vísceras abdominais para o lado direito. </li><li> Passe duas suturas de seda estéril 2.0 debaixo da veia porta e gravata frouxa. </li><li> Canular a veia porta com um cateter calibre 16-18 IV e fixe o cateter no lugar apertando a ti vagamentesuturas ed a partir do passo anterior (Fig. 2). </li><li> Injectar heparina diluída através do cateter IV (1 ml de unidades de heparina 5000 USP / ml diluído com 4 ml de HBSS menos Ca 2 + / Mg 2 +). O fígado deve branquear. </li><li> Ligue o cateter IV para o sistema de perfusão. Perfundir com HBSS menos Ca 2 + / Mg + 2 a uma taxa de 20 ml / min durante 10 minutos. O transecto IVC inferior ao fígado para permitir a drenagem do sangue e do perfusato. Aspirar sangue reunidas a partir da cavidade abdominal. </li><li> Alterar o fluido de perfusão a uma solução de colagenase 0,3% (150 mg de colagenase tipo 2 em 500 ml HBSS mais Ca 2 + / Mg 2 +) e perfundir durante 25 minutos. Manter a umidade do fígado, cobrindo-o com o retalho previamente dissecados abdominal. Colocar uma tampa placa de Petri sobre a aba abdominal ea posição de uma lâmpada de calor sobre a área a manter a temperatura intra-abdominal, aproximadamente 37 ° C. </li></ol> 4. Preparação do diae árvore biliar <ol><li> Remover o fígado, levantando-o para fora da cavidade abdominal com uma pinça e colocá-lo num prato de cultura de tecido estéril preenchido com L-15 de Leibovitz frio meios de comunicação. </li><li> Trabalhando na capa cultura de tecidos, descascar a cápsula hepática e gentilmente burlar o parênquima hepático além com uma pinça. Mova o fígado através de vários pratos cheios de L-15 Liebovitz de mídia, enquanto continua a provocar longe do parênquima, até que finalmente saiu com a árvore biliar isolado. O parênquima hepático é castanhos claros, enquanto o restante da árvore biliar é branco. </li><li> Coloque a árvore biliar isolado num tubo Falcon 50 ml cheia com 10 ml de penicilina / estreptomicina (10.000 UI / ml de penicilina e 10.000 ug / ml de estreptomicina); deixar em gelo durante 15 minutos. </li></ol> 5. Isolamento da fracção de Fibroblasto Portal <ol><li> Coloque a árvore biliar em um tubo de 50 ml estéril falcão e triture com tesoura estéril. </li><li> Adicionar uma pequena quantidade de enzima solution # 1 para dentro do tubo (cerca de 5 ml) e continuar a picar a árvore biliar até atingir a consistência de uma massa pastosa. </li><li> Verter a suspensão em um frasco de vidro estéril. Lavar a tubo de falcão com a restante solução # 1, depois despejar o frasco de vidro. Incubar a 37 ° C com agitação a 100 rpm durante 30 minutos. </li><li> Filtra-se a suspensão através de um copo coberto com uma única camada de malha de nylon (30-micron de tamanho de poro). </li><li> Transferir todo o tecido restante no topo da malha para uma garrafa de vidro estéril por lavagem da malha com solução de enzima # 2. Iniciar vertendo meia (25 ml) da solução para um prato 15 centímetros estéril Petri. Cuidadosamente remover a malha a partir do copo, segurando as bordas da malha sem contaminar a área central, em seguida, invertendo a malha e mergulhando-o na solução numa placa de Petri para remover qualquer tecido restante na malha. Descarte a malha. Transferir a solução numa placa de Petri em um frasco de vidro estéril. Lavar a placa de Petri com o remaIning 25 ml de solução # 2 e transferi-lo para a garrafa de vidro mesmo. Incubar a 37 ° C com agitação a 100 rpm durante 30 minutos. </li><li> Enquanto isso, tome-se o filtrado no copo e despeje em um tubo falcon 50 ml. Centrifugar a 1600 rpm (460 x g) durante 5 minutos à temperatura ambiente. </li><li> Aspirar meios de comunicação e ressuspender o pellet em 25 ml de solução de # 3. </li><li> Tome a solução do passo 5.5 e filtrá-lo para um copo de segunda estéril coberto com 30 mícrons de malha. </li><li> Descarte a malha. Centrifugar o filtrado a 1600 rpm durante 5 minutos à temperatura ambiente. Aspirar e ressuspender o sedimento com 25 ml de solução de # 3, como foi feito no passo 5.7. </li><li> Combinar as fracções de 2 a partir de passos 5.7 e 5.9 e centrifugar de novo a 1600 rpm durante 5 minutos à temperatura ambiente. </li><li> Aspirar meios de comunicação e ressuspender o pellet em 10 ml de meio de cultura de portal de fibroblastos (DMEM/F12 suplementado com 10% de FBS, fungizona 1% de penicilina / estreptomicina, gentamicina 0,3%, e 0,2%). </li><li> Contar as células para a viabilidade da placa em pratos de cultura de tecidos. Dependendo da aplicação desejada, de 0,5 a 5 x 10 células por 6 10-cm placa de cultura de tecido são adequados. De um rato Sprague-Dawley adultos, que tipicamente obter 2 a 10 milhões de células viáveis. </li><li> Substituir os meios de cultura, no dia seguinte para remover os detritos não-aderente. Em seguida, substitui os meios de cultura a cada 2 dias. </li></ol> 6. Os resultados representativos Primárias fibroblastos de portal isoladas de fígado de rato adulto são mostrados em 1, 3 e 7 dias após o isolamento (Fig. 3). Note-se que as células são alongados, com morfologia típica de fibroblastos. As células podem ser coradas com anti-elastina anticorpo (CL55041AP, Cedarlane Labs, Burlington, NC) para confirmar a pureza. Fibroblastos de portal sofrem diferenciação miofibroblástico em cultura. Isto pode ser demonstrado por imunocoloração para α-actina de músculo liso (α-SMA; A2547, Sigma, St. Louis, MO). <tableborder = &quot;1&quot;&gt; <tbody><tr><td rowspan="2"> Solução de colagenase 0,3% </td><td> Colagenase tipo 2 </td><td> 150 mg </td></tr><tr><td> HBSS com Ca2 + / Mg2 + </td><td> 500 ml </td></tr><tr><td rowspan="7"> Solução # 1 (Pronase) </td><td> Albumina de soro bovino </td><td> 50 mg </td></tr><tr><td> Colagenase tipo 2 </td><td> 25 mg </td></tr><tr><td> Pronase </td><td> 18 mg </td></tr><tr><td> DNase </td><td> 3 mg </td></tr><tr><td> DMEM/F12 </td><td> 47,5 ml </td></tr><tr><td> Pen / Strep </td><td> 1 ml </td></tr><tr><td> Soro fetal de bovino </td><td> 1,5 ml </td></tr><tr><td> Solução # 2 </td><td> Albumina de soro bovino </td><td> 50 mg </td></tr><tr><td rowspan="6"> (Hialuronidase) </td><td> Colagenase tipo 2 </td><td> 25 mg </td></tr><tr><td> Hialuronidase </td><td> 22 mg </td></tr><tr><td> DNase </td><td> 3 mg </td></tr><tr><td> DMEM/F12 </td><td> 47,5 ml </td></tr><tr><td> Pen / Strep </td><td> 1 ml </td></tr><tr><td> Soro fetal de bovino </td><td> 1,5 ml </td></tr><tr><td rowspan="2"> Solução # 3 </td><td> DNase </td><td> 3 mg </td></tr><tr><td> Meio RPMI 1640 </td><td> 50 ml </td></tr></tbody></table> Tabela 1. Soluções enzimáticas. <img alt="A Figura 1" src="/files/ftp_upload/3669/3669fig1.jpg" /> Figura 1. Perfusão configurar. A. Circulação de água para a coluna de aquecimento. Coluna B. contendo mídia de perfusão. C. torneira controlar saída da bomba. D. Bomba entrada. E. bomba peristáltica de velocidade variável. F. frasco de vácuo com Bio-Clean detergente. G. lâmpada de calor. Água H. banho para a mídia de perfusão aquecimento. <img alt="A Figura 2" src="/files/ftp_upload/3669/3669fig2.jpg" /> FIGUre 2. perfusão in situ. A. Cateter IV inserido na veia portal, fixada com sutura e ligado à tubagem de perfusão através torneira. B. vidro pipeta ligado ao frasco de vácuo e de sucção de parede para remover líquido de perfusão de drenagem de seccionado VCI. Intestinos I.. L. Fígado. <img alt="A Figura 3" src="/files/ftp_upload/3669/3669fig3.jpg" /> Figura 3. De contraste de fase e imunofluorescência mancha de fibroblastos de rato de portal em cultura. Fibroblastos Portal cultivadas durante 1, 3 e 7 dias após o isolamento foram fixadas e coradas para a elastina, α-SMA e DAPI.

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

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Fibroblastos de portal desempenhar um papel importante na fibrose biliar. Descrevemos aqui uma modificação do protocolo original publicada por Kruglov e Dranoff 8 para isolar fibroblastos portal a partir de fígado de rato, proporcionando uma maneira simples para estudar esta população de células in vitro. Esta abordagem utiliza a digestão da protease e baseada no tamanho de filtração. A principal vantagem do método é que uma população relativamente pura de células podem ser obtidas sem a passagem em cultura, permitindo que o estudo da expressão do gene ou o comportamento de células funcional vários dias após o isolamento. Esta técnica também oferece o potencial para isolar células de fígados saudáveis ​​e doentes. Uma das etapas mais críticas neste protocolo é a digestão enzimática do parênquima hepático. Para assegurar uma digestão sucesso, é importante para confirmar que o sangue é completamente drenado para fora do fígado durante o perfusão inicialSion, certificando-se que há ainda de branqueamento do fígado. Para facilitar isto, é possível usar um cotonete para massagem suave do fígado, enquanto perfundindo. Mais de digestão de os resultados parênquima hepático em um baixo rendimento de células viáveis, enquanto sob a digestão-irá tornar mais difícil separar o parênquima do fígado a partir da árvore biliar. Desde os preparativos de pronase tem variabilidade na atividade enzimática entre diferentes lotes, a otimização da quantidade utilizada pode ser necessária quando há baixa produção de células viáveis. Este protocolo pode ser modificada para isolar fibroblastos portal a partir de fígado de rato ou do fígado de ratos jovens usando uma menor bitola cateter IV para canular a veia portal, diminuindo o volume das soluções de perfusão em proporção ao peso do animal, e diminuindo a taxa de perfusão do fígado e cerca de 10 ml / min. Fibroblastos em cultura de portal sofrem diferenciação miofibroblástico em 10-14 dias, como evidenciado por α-SMA expressão 9. Vários marcadores têm sido utilizados para distinguir os fibroblastos de portal a partir de células estreladas hepáticas, incluindo fibulin-2, IL-6, elastina, nucleósido-trifosfato difosfohidrolase-2 (NTPDase2), cofilina-1 e proteínas neuronais, tais como sinaptofisina 2, 6, 10, 11 . Nós descobrimos que a elastina é um bom marcador para fibroblastos de portal, mesmo após a diferenciação miofibroblástico, enquanto NTPDase2 é perdido após fibroblastos de portal tenham sido submetidos a cultura, após vários dias 9. Portanto, nós tipicamente confirmar o isolamento de fibroblastos portal pela técnica de imunofluorescência para elastina. A limitação desta técnica é que, imediatamente após o isolamento de fibroblastos de portal, existe uma pequena fracção de contaminar células incluindo células de Kupffer e células biliares. Fibroblastos de portal outgrow estas células contaminantes dentro de 2-3 dias, no entanto, obtendo-se uma população relativamente puro (&gt; 98%) 9. Since fibroblastos do portal de cultura sofrem diferenciação miofibroblástico em plástico de cultura de tecido, que normalmente estudar estas células dentro de 7 dias de isolamento. As células são mantidas em meios de fibroblastos de portal de cultura contendo 10% de soro fetal bovino, como descrito na secção Métodos. No entanto, estas células irão sobreviver no meio de crescimento contendo 2% de soro fetal de bovino durante vários dias. Nós normalmente não usam meios de baixo custo de soro até pelo menos 24 horas após o isolamento. Uma vez que os fibroblastos de portal sofrem diferenciação miofibroblástico, eles podem ser passadas várias vezes e mantidos como stocks congelados de miofibroblastos.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Colagenase tipo 2 </td><td> Worthington Biochemical (Lakewood, NJ) </td><td> LS004177 </td></tr><tr><td> Pronase </td><td> Roche (Indianapolis, IN) </td><td> 70290120 </td></tr><tr><td> Hialuronidase </td><td> Sigma (St. Louis, MO) </td><td> H3506 </td></tr><tr><td> Desoxirribonuclease </td><td> Sigma </td><td> D4527 </td></tr><tr><td> DMEM/F12 </td><td> Invitrogen (Carlsbad, CA) </td><td> 11320 </td></tr><tr><td> HBSS sem Ca 2 + / Mg 2 + </td><td> Mediatech (Manassas, VA) </td><td> 21-021-CM </td></tr><tr><td> HBSS com Ca2 + / Mg2 + </td><td> Mediatech </td><td> 21-020-CM </td></tr><tr><td> Leibovitz L-15 </td><td> Invitrogen </td><td> 11415 </td></tr><tr><td> Meio RPMI 1640 </td><td> Invitrogen </td><td> 11875 </td></tr><tr><td> Albumina de Soro Bovino </td><td> Sigma </td><td> A2153 </td></tr><tr><td> Soro Fetal Bovino </td><td> Gêmeos Bio (West Sacramento, CA) </td><td> 900-108 </td></tr><tr><td> Malha de nylon Nitex de 30 uM </td><td> Genesee Científica (San Diego, CA) </td><td> 57-105 </td></tr></tbody></table>

isolation of rat portal fibroblasts by in situ liver perfusion

Fibrose hepática é definida pela deposição excessiva de matriz extracelular por miofibroblastos activados. Existem múltiplas de precursores miofibroblastos hepáticas, incluindo as células estreladas hepáticas, fibroblastos de portal e medula óssea fibroblastos derivados 1. Células estreladas hepáticas ter sido melhor estudada, mas fibroblastos portal estão cada vez mais reconhecidos como importantes contribuintes para a piscina de miofibroblastos, particularmente na fibrose biliar 2. Fibroblastos de portal sofrer proliferação em resposta a uma lesão epitelial biliar, potencialmente desempenhando um papel fundamental nos estágios iniciais da cicatrização biliar 3-5. Um método de isolamento de fibroblastos de portal permitiria estudo in vitro desta população de células e conduzir a uma maior compreensão do papel fibroblastos de portal desempenhar na fibrose biliar. Fibroblastos de portal foram isolados utilizando várias técnicas, incluindo excrescência 6, 7 e liperfusão ver com a digestão enzimática seguida de seleção de tamanho 8. A vantagem da digestão e técnica de selecção de tamanho em comparação com a técnica excrescência é que as células podem ser estudados sem a necessidade de passagem em cultura. Aqui, nós descrevemos uma versão modificada da técnica original descrita por Kruglov e Dranoff 8 para o isolamento de fibroblastos portal a partir de fígado de rato, que resulta em uma população relativamente pura de células primárias.

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

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Uma técnica para o isolamento de fibroblastos portal a partir de fígado de rato é descrito. Os fígados são perfundidos e digerido<em&gt; In situ</em&gt; Com colagenase, seguido por<em&gt; Ex vivo</em&gt; Digestão da lama de fígado e de selecção do tamanho das células. Este método proporciona uma população pura de fibroblastos de portal sem a necessidade de passagem em cultura.

Isolamento de fibroblastos de rato por Portal<em&gt; In situ</em&gt; Perfusão de fígado

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

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Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

19.2K visualizações.  The Children's Hospital of Philadelphia. Uma técnica para o isolamento de fibroblastos portal a partir de fígado de rato é descrito. Os fígados são perfundidos e digerido In situ Com colagenase, seguido por Ex vivo Digestão da lama de fígado e de selecção do tamanho das células. Este método proporciona uma população pura de fibroblastos de portal sem a necessidade de passagem em cultura.

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

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

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

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

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

Endocitose em Fígado vivo seguidos de purificação de células do fígado por perfusão de fígado

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

Biologia

Procedures for Rat in situ Skeletal Muscle Contractile Properties

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

Procedures for Rat in situ Skeletal Muscle Contractile Properties

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

Procedimentos para Rat<em&gt; In situ</em&gt; Propriedades contrátil do músculo esquelético

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

Chromatin Isolation by RNA Purification (ChIRP)

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

Chromatin Isolation by RNA Purification ChIRP

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

Isolamento de cromatina por purificação de RNA (CHIRP)

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

Isolation and Primary Culture of Rat Hepatic Cells

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

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

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 ...

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

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

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

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

Isolamento de fibroblastos normais e associados ao câncer de tecidos frescos, classificando células activadas por fluorescência (FACS)

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

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

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

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

Isolamento de hepatócitos humanos por duas etapas Collagenase Perfusão Procedimento

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

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

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

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

Isolamento Simultâneo de Cardiomiócitos de Alta Qualidade, Células Endoteliais e Fibroblastos de um Coração de Rato Adulto

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

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

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

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

Isolamento e Caracterização de Fibroblastos Cardíacos Adultos e Miofibroblastos

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

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

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

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

Isolamento e Caracterização de Exossomos de Fibroblastos de Músculo Esquelético

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

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

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

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

Isolamento de Hepatócitos Primários de Ratos com Controle de Perfusão Multiparâmetro

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