Name: selective harvesting of marginating-hepatic leukocytes
Uploaded: 2016-07-21T13:00:00.000+00:00
Duration: 6 min 53 s
Description: Watch this Scientific Journal Video about Selective Harvesting of Marginating-hepatic Leukocytes at JoVE.com

The authors and this work were supported by NIH/NCI grant # R01CA172138 (to SBE).

Declaração de Ética: Procedimentos envolvendo indivíduos animais foram aprovados pelo Comitê Institucional de Animal Care and Use (IACUC) da Universidade de Tel-Aviv. Protocolo 1. Ratos <ol><li> preparativos <ol><li> Preparar a solução de PBS heparinizado (30 unidades / ml) para ser utilizado à temperatura ambiente (RT) por adição de 30 unidades de heparina livre de conservante por ml de tampão fosfato salino (PBS) 1x solução. Calcular 35 ml por animal. </li><li> Passe heparinizado PBS através das linhas de bomba peristáltica e as agulhas borboleta para eliminar bolhas de ar. </li><li> Esterilizar instrumentos cirúrgicos - 2 pares de tesouras, fórceps embotados-2 gumes, hemostáticos, e uma pinça de dente-de tecido (autoclave a 121 ° C durante pelo menos 30 min no ambiente gravidade (seco)). </li></ol></li><li> Perfusão do Fígado e Recolha de MH-leucócitos <ol><li> Eutanásia do animal por uma overdose de 8% de isoflurano. Como medida de precaução, coloque um tubo de 50 ml contendo um isofluranopad -soaked em torno da cabeça do rato até que a abertura de sua caixa torácica. </li><li> Abrir as cavidades peritoneais e tórax para expor o fígado e o complexo cardiopulmonar imediatamente após a cessação da respiração. Evitar o sangramento através deste processo. <ol><li> Especificamente, iniciar a incisão no ponto da linha média abdominal inferior, sem órgãos internos prejudiciais, e progresso para ambos os lados na diagonal em direção as costelas, cortando-os a níveis peito-cavidade superior. </li></ol></li><li> Dentro de aproximadamente um minuto, recolher o máximo de sangue possível (~ 6 ml de um 250 g de animais) do ventrículo direito para uma seringa. </li><li> Prender a veia cava caudal com uma pinça hemostática tão próximo quanto possível do coração, para permitir a recolha do perfundido. </li><li> Retire os intestinos e lugar fora do animal para a direita do experimentador, para expor a veia portal. </li><li> Inserir um cateter IV 25 L, ligado a uma bomba peristáltica, na veia porta, como caudal quanto possível,mas rostral à veia do baço. </li><li> Inserir uma agulha G 25 borboleta, ligado a uma seringa de 5 ml, para a veia cava inferior, caudal para a pinça hemostática. </li><li> Ligar a bomba peristáltica a uma velocidade de aproximadamente 3 ml / min e recolhem suavemente os primeiros ml de perfusato que está contaminada com sangue para a seringa. Continue até que a cor do perfusato é ficar vermelho pálido (aproximadamente 3-5 ml). Note-se que a cor do fígado está mudando no sentido castanho claro. </li><li> Sem parar a bomba peristáltica, substituir a seringa de 5 mL com uma seringa de colheita de 20 ml de colheita e recolha de pelo menos 20 ml de perfusato fígado empregando uma velocidade mais elevada de perfusão (até 4 ml / min). monitorar continuamente o fluxo de perfusato para a seringa de coleta, e evitar vácuo que é muito forte (que podem entupir a agulha através colapso das paredes da veia cava). </li><li> Terminar a perfusão, quando a cor do fígado vira-se para castanho claro. </li></ol></li><li> leucócitos Extração <ol&gt; <li> Centrifuga-se o perfusato durante 10 minutos a 400 xg, 24 ° C. </li><li> Aspirar o sobrenadante </li><li> Adicionar 10 ml de PBS, centrifugação a 400 xg durante 10 min, e o sobrenadante aspirado. Com base em metas de estudo, utilize preparação como está, ou realizar purificações adicionais (por exemplo, separação, em gradiente de densidade). <ol><li> Especificamente, a camada de 4 ml de perfusato mais de 4 ml de massa separação em gradiente de densidade num tubo de centrifugação de polipropileno de 50 cc. Gire os tubos a 754 xg, RT, durante 30 minutos com a quebra de fora. </li><li> Obter as camadas mononucleares na interface de separação por gradiente de densidade de sobrenadante por pipetagem manual de, lavar em PBS, e inserir um tubo de 5 ml. </li></ol></li></ol></li></ol> 2. Rato Protocolo <ol><li> preparativos <ol><li> Preparar a solução de PBS heparinizado (30 unidades / ml) para ser utilizado à temperatura ambiente (RT) por adição de 30 unidades de heparina livre de conservante por ml de fosfato salino (PBS) tamponada 1x solution. Calcular 20 ml por animal. </li><li> Passe heparinizado PBS através das linhas de bomba peristáltica e as agulhas borboleta para eliminar bolhas de ar. </li><li> Esterilizar instrumentos cirúrgicos - 2 pares de tesouras, fórceps embotados-2 gumes, hemostáticos, e uma pinça de dente-de tecido (autoclave a 121 ° C durante pelo menos 30 min no ambiente gravidade (seco)). </li></ol></li><li> Perfusão do Fígado e Recolha de MH-Leucócitos <ol><li> Eutanásia do animal por uma overdose de 8% de isoflurano. Como medida de precaução, coloque um tubo de 15 ml contendo uma almofada encharcada de isoflurano em torno da cabeça do rato até a abertura de sua caixa torácica. </li><li> Corrigir os membros do mouse para uma cama. </li><li> Abrir as cavidades peritoneais e tórax para expor o fígado e o complexo cardiopulmonar imediatamente após a cessação da respiração, mantendo o aspecto inferior do diafragma intacto (para criar uma cavidade torácica piscina drenagem). Evitar o sangramento através deste processo. <ol><li> Especificamente, iniciar a incisão na parte inferiorponto abdominal na linha média, sem órgãos internos prejudiciais, e progresso para ambos os lados na diagonal em direção as costelas, cortando-os a níveis peito-cavidade superior. </li></ol></li><li> Arrancamento dos intestinos e lugar fora do animal para a direita do experimentador, para expor a veia portal. </li><li> Inserir uma agulha 30 g, ligada a uma bomba peristáltica para a veia porta rostral à veia esplénica. </li><li> Cortar a veia cava inferior acima do diafragma para permitir a drenagem e recolha do perfundido da cavidade torácica. </li><li> Ligar a bomba peristáltica a uma velocidade de aproximadamente 3 ml / min e suavemente recolher os primeiros 3 ml de perfusato que está contaminada com sangue a partir da piscina cavidade torácica. </li><li> Descarte este perfusato. Repita essa lavar novamente, se necessário, usando solução salina, e só então começar a recolher perfusato fígado. </li><li> Re-iniciar a perfusão a uma velocidade de até 4 ml / min, e recolher 10 ml de perfusato da cavidade torácica usandouma borboleta ligado a uma seringa. </li><li> Terminar a perfusão, quando a cor do fígado vira-se para castanho claro. </li></ol></li><li> leucócitos Extração <ol><li> Centrifugar o perfusado durante 10 min a 400 x g. </li><li> Aspirar o sobrenadante. </li><li> Adicionar 10 ml PBS, centrifugação a 400 xg durante 10 min, e aspirar o sobrenadante com base em metas de estudo, utilize preparação como é, ou realizar purificações adicionais (por exemplo, separação, em gradiente de densidade). <ol><li> Especificamente, a cada camada de 4 ml de perfusato mais de 4 ml de massa separação em gradiente de densidade num tubo de centrifugação de polipropileno de 50 cc. Gire os tubos a 754 xg, RT, durante 30 minutos com a quebra de fora. </li><li> Obter as camadas mononucleares na interface de separação por gradiente de densidade de sobrenadante por pipetagem manual de, lavar em PBS, e inserir um tubo de 5 ml. </li></ol></li></ol></li></ol>

<ol>
	<li>Melamed, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+marginating-pulmonary+immune+compartment+in+rats:+characteristics+of+continuous+inflammation+and+activated+NK+cells.">The marginating-pulmonary immune compartment in rats: characteristics of continuous inflammation and activated NK cells.</a> J Immunother. 33, 16-29 (2010).</li><li>Benish, M., Melamed, R., Rosenne, E., Neeman, E., Sorski, L., Levi, B., Shaashua, L., Matzner, M., Ben-Eliyahu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+marginating-pulmonary+immune+compartment+in+mice+exhibits+increased+NK+cytotoxicity+and+unique+cellular+characteristics.">The marginating-pulmonary immune compartment in mice exhibits increased NK cytotoxicity and unique cellular characteristics.</a> Immunologic Research. 58, 28-39 (2014).</li><li>Wisse, E., van't Noordende, J. M., van der Meulen, J., Daems, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pit+cell:+description+of+a+new+type+of+cell+occurring+in+rat+liver+sinusoids+and+peripheral+blood.">The pit cell: description of a new type of cell occurring in rat liver sinusoids and peripheral blood.</a> Cell Tissue Res. 173, 423-435 (1976).</li><li>Vermijlen, D., 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=Hepatic+natural+killer+cells+exclusively+kill+splenic/blood+natural+killer-resistant+tumor+cells+by+the+perforin/granzyme+pathway.">Hepatic natural killer cells exclusively kill splenic/blood natural killer-resistant tumor cells by the perforin/granzyme pathway.</a> J Leukoc Biol. 72, 668-676 (2002).</li><li>Gao, B., Radaeva, S., Park, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+natural+killer+and+natural+killer+T+cells:+immunobiology+and+emerging+roles+in+liver+diseases.">Liver natural killer and natural killer T cells: immunobiology and emerging roles in liver diseases.</a> J Leukoc Biol. 86, 513-528 (2009).</li><li>Kaneda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver-associated+large+granular+lymphocytes:+morphological+and+functional+aspects.">Liver-associated large granular lymphocytes: morphological and functional aspects.</a> Arch Histol Cytol. 52, 447-459 (1989).</li><li>Podack, E. R., Hengartner, H., Lichtenheld, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+central+role+of+perforin+in+cytolysis?.">A central role of perforin in cytolysis?.</a> Annu Rev Immunol. 9, 129-157 (1991).</li><li>Kamada, M. M., 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=Identification+of+carboxypeptidase+and+tryptic+esterase+activities+that+are+complexed+to+proteoglycans+in+the+secretory+granules+of+human+cloned+natural+killer+cells.">Identification of carboxypeptidase and tryptic esterase activities that are complexed to proteoglycans in the secretory granules of human cloned natural killer cells.</a> J Immunol. 142, 609-615 (1989).</li><li>Wisse, E., 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=On+the+function+of+pit+cells,+the+liver-specific+natural+killer+cells.">On the function of pit cells, the liver-specific natural killer cells.</a> Semin Liver Dis. 17, 265-286 (1997).</li><li>Bouwens, L., Remels, L., Baekeland, M., Van Bossuyt, H., Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+granular+lymphocytes+or+&amp;#34;pit+cells&amp;#34;+from+rat+liver:+isolation,+ultrastructural+characterization+and+natural+killer+activity.">Large granular lymphocytes or &amp;#34;pit cells&amp;#34; from rat liver: isolation, ultrastructural characterization and natural killer activity.</a> Eur J Immunol. 17, 37-42 (1987).</li><li>Vanderkerken, K., 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=Origin+and+differentiation+of+hepatic+natural+killer+cells+(pit+cells).">Origin and differentiation of hepatic natural killer cells (pit cells).</a> Hepatology. 18, 919-925 (1993).</li><li>Luo, D., 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=The+role+of+adhesion+molecules+in+the+recruitment+of+hepatic+natural+killer+cells+(pit+cells)+in+rat+liver.">The role of adhesion molecules in the recruitment of hepatic natural killer cells (pit cells) in rat liver.</a> Hepatology. 24, 1475-1480 (1996).</li><li>Shresta, S., MacIvor, D. M., Heusel, J. W., Russell, J. H., Ley, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+killer+and+lymphokine-activated+killer+cells+require+granzyme+B+for+the+rapid+induction+of+apoptosis+in+susceptible+target+cells.">Natural killer and lymphokine-activated killer cells require granzyme B for the rapid induction of apoptosis in susceptible target cells.</a> Proc Natl Acad Sci U S A. 92, 5679-5683 (1995).</li><li>Luo, D., 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=Involvement+of+LFA-1+in+hepatic+NK+cell+(pit+cell)-mediated+cytolysis+and+apoptosis+of+colon+carcinoma+cells.">Involvement of LFA-1 in hepatic NK cell (pit cell)-mediated cytolysis and apoptosis of colon carcinoma cells.</a> J Hepatol. 31, 110-116 (1999).</li><li>Wiltrout, R. H., 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=Augmentation+of+mouse+liver-associated+natural+killer+activity+by+biologic+response+modifiers+occurs+largely+via+rapid+recruitment+of+large+granular+lymphocytes+from+the+bone+marrow.">Augmentation of mouse liver-associated natural killer activity by biologic response modifiers occurs largely via rapid recruitment of large granular lymphocytes from the bone marrow.</a> J Immunol. 143, 372-378 (1989).</li><li>Schluter, K., 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=Organ-specific+metastatic+tumor+cell+adhesion+and+extravasation+of+colon+carcinoma+cells+with+different+metastatic+potential.">Organ-specific metastatic tumor cell adhesion and extravasation of colon carcinoma cells with different metastatic potential.</a> Am J Pathol. 169, 1064-1073 (2006).</li><li>Wiltrout, R. H., 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=Augmentation+of+organ-associated+natural+killer+activity+by+biological+response+modifiers.+Isolation+and+characterization+of+large+granular+lymphocytes+from+the+liver.">Augmentation of organ-associated natural killer activity by biological response modifiers. Isolation and characterization of large granular lymphocytes from the liver.</a> J Exp Med. 160, 1431-1449 (1984).</li><li>Winnock, M., 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=Functional+characterization+of+liver-associated+lymphocytes+in+patients+with+liver+metastasis.">Functional characterization of liver-associated lymphocytes in patients with liver metastasis.</a> Gastroenterology. 105, 1152-1158 (1993).</li><li>Hata, K., 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,+phenotyping,+and+functional+analysis+of+lymphocytes+from+human+liver.">Isolation, phenotyping, and functional analysis of lymphocytes from human liver.</a> Clin Immunol Immunopathol. 56, 401-419 (1990).</li><li>Vanderkerken, K., Bouwens, L., Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+phenotypically+and+functionally+distinct+subset+of+large+granular+lymphocytes+(pit+cells)+in+rat+liver+sinusoids.">Characterization of a phenotypically and functionally distinct subset of large granular lymphocytes (pit cells) in rat liver sinusoids.</a> Hepatology. 12, 70-75 (1990).</li><li>Sorski, L., Melamed, R., Lavon, H., Matzner, P., Rosenne, E., Ben-Eliyahu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (2015).</li></ol>

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O método de perfusão do fígado aqui apresentada permite uma colheita selectiva e estudo da população única de marginating leucócitos hepáticas. Células hepáticas NK, também chamados de células pit 3, constituem uma população distinta de células NK que residem nos sinusóides hepáticos. Eles são encontrados em ratos, ratos 17 e em humanos 18,19. Em comparação com células NK periféricas isoladas, células pit demonstraram maior citotoxicidade contra YAC-1 e CC531s linh...

Os sinusóides do fígado contêm numerosos subtipos de leucócitos de várias actividades imunes críticos para o organismo. Por exemplo, marginating hepática células natural killer (MH) (NK), também conhecidas como células pit, são caracterizados morfologicamente como grandes linfócitos granulares (LGLs) e funcionalmente como leucócitos com capacidade citotóxica espontânea, permitindo hepática resistência contra o estabelecimento de análises ao sangue metástases tumorais Borne. O objectivo do método aqui apresentado é o de permitir a colheita selectiva de leucócitos MH, a fim de estudar esta população de células importante e original (e compartimento imune), e para elucidar o impacto de várias manipulações (por exemplo, activação imune) sobre essas células específicas. Apesar do fracasso da imunidade para eliminar o desenvolvimento de um tumor primário, provas em pacientes com cancro e em modelos animais indicam que o sistema imune pode controlar células tumorais, micrometástases e de doença residual que circula atraimunidade mediada por células GH (CMI). No entanto, existe uma aparente inconsistência entre estes em capacidades in vivo e in vitro em seres humanos e em animais, que demonstram que a maioria das células tumorais autólogas são resistentes a citotoxicidade pela circulação de leucócitos ou esplénicas 1,2. Esta inconsistência pode ser atribuída, pelo menos em parte, à existência in vivo de uma subpopulação de leucócitos distintas, a saber, os sinusóides (leucócitos) marginating-hepático e a sua sub-população de células NK activadas, nomeadamente células pit 3. Com efeito, dados não publicados do nosso laboratório indicaram que em ratos F344 células tumorais singénicas (MADB106), as quais foram consideradas resistentes à circulação e esplénicos leucócitos, foram submetidas a lise por células NK MH-4. Assim, as células tumorais que são &quot;resistente NK-&quot; alegadamente para leucócitos circulantes podem ser controlados por células NK-MH. Notável, em ratos o aumento da atividade de MH-NK é evidente apenas seguir in vivo imunológicoestimulação (por exemplo, através da utilização de poli I: C ou CpG-C) 5. células NK específico do fígado (MH-NK) estão situados dentro do lúmen sinusoidais, aderir às células endoteliais e células de Kupffer. Células MH-NK são caracterizados exclusivamente por grânulos densos esféricas e vesículas com núcleo de haste 6, que contêm fosfatase ácida como enzimas lisossomais e perforina e granzimas como substâncias bioativas 7,8. Em comparação com células NK circulantes, células-NK MH exibem um maior número e tamanho dos grânulos e vesículas 9-11. Sob condições inflamatórias, células-NK foram MH mostrado exibir maior expressão de LFA-1 12, em comparação com células NK circulantes. Esta expressão aumentada pode constituir um mecanismo pelo qual as células NK-MH é mais citotóxico contra certas células tumorais do que as células NK circulantes 13,14. nterestingly, após incubação in vitro com a interleucina (IL) -2, células-NK MH tornou alargada, eo seu número e tamanho de grânulos de aumentar, os quais são consistentes com uma pro fi le de assassinas activadas por linfocina (LAK) células 15. leucócitos MH activas não podem ser obtidos exclusivamente através dos métodos de colheita de leucócitos no fígado padrão, os quais são baseados na moedura e degradação biológica do tecido. A nossa abordagem perfusão aqui descrito possui duas grandes vantagens em comparação com os métodos normais. Em primeiro lugar, a abordagem de perfusão colhe seletivamente leucócitos MH, evitando a contaminação por outros leucócitos de outros compartimentos do fígado. Em segundo lugar, a técnica de perfusão melhor preserva a integridade, a actividade, e o meio fisiológico de leucócitos MH, ao contrário do processamento de tecidos abordagens que danificam as células ou alteram a sua morfologia, e devido ao dano do tecido, induzir a libertação de vários factores que marcadamente modular imune atividade. O fígado é um dos principais alvos de metástase de câncer dend para várias infecções 16. Como as células MH apresentam características únicas que é importante estudar essa população específica sob várias condições no que diz respeito a estas patologias. Por exemplo, é digno de nota que a activação imunitária sistémica por vários BRMs (por exemplo, poli I: C ou CpG-C) têm mostrado activar MH-leucócitos mais do que 5 leucócitos circulantes.

<table><tbody><tr><td>Autoclud Peristaltic pump</td><td></td><td></td><td></td></tr><tr><td>Butterfly needle</td><td>OMG</td><td></td><td>26 G</td></tr><tr><td>Butterfly needle</td><td>OMG</td><td></td><td>21 G*3/4&quot;</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>1 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>2.5 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>5 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>10 ml</td></tr><tr><td>Syringe</td><td>Pic solution</td><td></td><td>25 ml</td></tr><tr><td>Blunted-edged forceps</td><td></td><td></td><td></td></tr><tr><td>Scissors</td><td></td><td></td><td></td></tr><tr><td>hemostat</td><td></td><td></td><td></td></tr><tr><td>Tissue forceps</td><td></td><td></td><td></td></tr><tr><td>22 W Fluorescent Daylight Magnifier Lamp</td><td></td><td></td><td></td></tr></tbody></table>

selective harvesting of marginating-hepatic leukocytes

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Specifically, evidence suggests a distinct pro- and anti-inflammatory profile of the MH-leukocyte population and higher cytotoxicity of liver-specific NK cells (namely, pit cells) compared to circulating or splenic immunocytes in both mice and rats. The method presented herein enables selective harvesting of MH leukocytes by forced perfusion of the liver in mice and rats. In contrast to other methods used to extract liver-leukocytes, including tissue grinding and biological degradation, this method exclusively yields leukocytes from the liver sinusoids, uncontaminated by cells from other liver compartments. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MH leukocytes, sparing known physiological responses to tissue processing. As many circulating malignant cells and infected cells are detained while passing through the liver sinusoids, physically interacting with endothelial cells and resident leukocytes, the unique MH leukocyte population is strategically located to interact, identify, and react towards aberrant circulating cells. Thus, selective harvesting of MH-leukocytes and their study under various conditions may advance our understanding of the biological and clinical significance of MH leukocytes, specifically with respect to circulating aberrant cells and liver-related diseases and cancer metastases.

Em ratos F344 comparou-se a citotoxicidade de células NK-MH (recolhida das sinusóides do fígado por perfusão do fígado forçada) para citotoxicidade de toda a população de células de fígado após trituração mecânica do tecido do fígado, e para a citotoxicidade de leucócitos circulantes. Todas as preparações celulares foram lavadas, pelo menos, 3 vezes, como rotina em ensaios imunológicos, e como linhas de células alvo foi utilizado o alogénico de YAC-1 ou as linhas de ...

Watch this Scientific Journal Video about Selective Harvesting of Marginating-hepatic Leukocytes at JoVE.com

Selective Harvesting of Marginating-hepatic Leukocytes

Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Colheita seletiva de Marginating-hepática Leucócitos

Marginating-hepatic (MH) leukocytes (leukocytes adhering to the sinusoids of the liver), were shown to exhibit unique composition and characteristics ...

selective-harvesting-of-marginating-hepatic-leukocytes

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

5.7K visualizações.  Tel-Aviv University. Marginating-hepatic leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe a method for selective harvesting of this specific hepatic cell population, through forced perfusion of the liver of rats or mice. Marginating-hepatic leukocytes seem critical in determining susceptibility to hepatic-related diseases and metastases.

Vídeo: Selective Harvesting of Marginating-hepatic Leukocytes

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Um método simples e eficiente para isolar Macrófagos de Mixed culturas primárias de células do fígado Adulto

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Imunologia e Infecção

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

Isolamento e Enriquecimento de fígado progenitoras Subconjuntos identificado por um marcador combinação de superfícies Novel

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

Biologia do Desenvolvimento

Selective Harvesting of Marginating-pulmonary Leukocytes

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit unique composition and characteristics compared to leukocytes of other immune compartments. Evidence suggests higher cytotoxicity of natural killer cells, and a distinct pro- and anti-inflammatory profile of the MP-leukocyte population compared to circulating or splenic immunocytes. The method presented herein enables selective harvesting of MP-leukocytes by forced perfusion of the lungs in either mice or rats. In contrast to other methods used to extract lung-leukocytes, such as tissue grinding and biological degradation, this method exclusively yields leukocytes from the lung capillaries, uncontaminated with parenchymal, interstitial, and broncho-alveolar cells. In addition, the perfusion technique better preserves the integrity and the physiological milieu of MP-leukocytes, without inducing physiological responses due to tissue processing. This unique MP leukocyte population is strategically located to identify and react towards abnormal circulating cells, as all circulating malignant cells and infected cells are detained while passing through the lung capillaries, physically interacting with endothelial cells and resident leukocytes,. Thus, selective harvesting of MP-leukocytes and their study under various conditions may advance our understanding of their biological and clinical significance, specifically with respect to controlling circulating aberrant cells and lung-related diseases.

Marginating-pulmonary leukocytes exhibit unique characteristics and distinct immunological functions compared to other leukocyte populations. Here we describe selective harvesting of this subpopulation of pulmonary leukocytes, by forced perfusion of the lungs in rats and mice. Marginating-pulmonary leukocytes seem critical in determining susceptibility to blood-borne and lung-related diseases.

Colheita seletiva de Marginating-pulmonar Leucócitos

Marginating-pulmonary (MP) leukocytes are leukocytes that adhere to the inner endothelium of the lung capillaries. MP-leukocytes were shown to exhibit ...

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

Digestão do fígado murino para um fluxo Cytometric análise de células endoteliais linfáticas

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

Protocolo para o isolamento de hepatócitos humanos primários e Correspondente principais populações de células do fígado não-parenquimatosas

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

Biologia

Estabelecimento de culturas LLEC in vitro usando seleção de células magnéticas

Harvest and Primary Culture of Leptomeningeal Lymphatic Endothelial Cells

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct from peripheral lymphatic endothelial cells. Their cellular function and clinical implications remain largely unknown. Consequently, the availability of a supply of LLECs is essential for conducting functional research in vitro. However, there is currently no existing protocol for harvesting and culturing LLECs in vitro.
This study successfully harvested LLECs using a multi-step protocol, which included coating the flask with fibronectin, dissecting the leptomeninges with the assistance of a microscope, enzymatically digesting the leptomeninges to prepare a single-cell suspension, inducing the expansion of LLECs with vascular endothelial growth factor-C (VEGF-C), and selecting lymphatic vessel hyaluronic receptor-1 (LYVE-1) positive cells through magnetic-activated cell sorting (MACS). This process ultimately led to the establishment of a primary culture. The purity of the LLECs was confirmed through immunofluorescence staining and flow cytometric analysis, with a purity level exceeding 95%. This multi-step protocol has demonstrated reproducibility and feasibility, which will greatly facilitate the exploration of the cellular function and clinical implications of LLECs.

Autor em destaque: Revelando funções celulares e potenciais implicações clínicas das células endoteliais linfáticas leptomeníngeas 

Leptomeningeal lymphatic endothelial cells (LLECs), a recently identified intracranial cell type, have poorly understood functions. This study presents a reproducible protocol for harvesting LLECs from mice and establishing in vitro primary cultures. This protocol is designed to enable researchers to delve into the cellular functions and potential clinical implications of LLECs.

Colheita e Cultura Primária de Células Endoteliais Linfáticas Leptomeníngeas

Leptomeningeal lymphatic endothelial cells (LLECs) are a recently discovered intracranial cellular population with a unique distribution clearly distinct ...

Neurociência

Isolation of Regenerating Hepatocytes after Partial Hepatectomy in Mice

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream single-cell applications is challenging. A marked accumulation of lipids within regenerating hepatocytes is observed during the first 2 days of normal liver regeneration in mice. This so-called transient regeneration-associated steatosis (TRAS) is temporary but partially overlaps the major proliferative phase. Density-gradient purification is the backbone of most existing protocols for the isolation of primary hepatocytes. As gradient purification relies on the density and size of cells, it separates non-steatotic from steatotic hepatocyte populations. Therefore, fatty hepatocytes often are lost, yielding non-representative hepatocyte fractions. 
The presented protocol describes an easy and reliable method for the in vivo isolation of regenerating hepatocytes regardless of their lipid content. Hepatocytes from male C57BL/6 mice are isolated 24-48 h after hepatectomy by a classic two-step collagenase perfusion approach. A standard peristaltic pump drives the warmed solutions via the catheterized inferior vena cava into the remnant, using a retrograde perfusion technique with outflow through the portal vein. Hepatocytes are dissociated by collagenase for their release from the Glisson's capsule. After washing and careful centrifugation, the hepatocytes can be used for any downstream analyses. In conclusion, this paper describes a straightforward and reproducible technique for the isolation of a representative population of regenerating hepatocytes after partial hepatectomy in mice. The method may also aid the study of fatty liver disease.

Lipid-laden hepatocytes are inherent to liver regeneration but are usually lost upon density-gradient centrifugation. Here, we present an optimized cell isolation protocol that retains steatotic hepatocytes, yielding representative populations of regenerating hepatocytes after partial hepatectomy in mice.

Isolamento de Hepatócitos Regeneradores após Hepatectomia Parcial em Camundongos

Partial hepatectomy has been widely used to investigate liver regeneration in mice, but the isolation of high yields of viable hepatocytes for downstream ...

Medicina

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. ...

Colheita seletiva de Marginating-hepática Leucócitos

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