Este trabalho foi financiado por um UNCF-Merck de Pós-Graduação Pesquisa em Ciências Dissertation Fellowship (JLM), National Heart, Lung, and Blood Institute concede RO1-HL-62228 (JSJ) e RO1-HL-073990 (JSJ).

1. Preparação de sal de Hanks Balance Solution (HBSS) <ol><li> Autoclave um copo de vidro L, 1000 mL proveta graduada, e mexa bar. </li><li> Reconstituir a solução HBSS pó no copo de vidro autoclavados 1 L para a marca de 900 mL com água destilada duas vezes. </li><li> Misturar a solução HBSS com 3,4 g de sal Hepes Na + e ajustar o pH para um valor entre 7,35-7,41 usando HCL concentrado ou NaOH. </li><li> Uma vez que o pH desejado foi atingido e permanece estável, adicionar 5 g de soro albumina bovina para a solução à temperatura ambiente até que a albumina é completamente dissolvido. </li><li> Adicionar 10 mL da água antibiótico, antimyotic, para a solução e duplamente destilada até o volume total é de 1000 marca mL. </li><li> Agitar a solução completamente. </li><li> Filtrar a solução HBSS e separá-lo em 50 mL tubos Falcon para uso posterior. Tubos adicionais podem ser congelados e utilizados em uma data posterior. </li></ol> 2. PREPARAÇÃO animaisn - Inserção de tubo traqueal <ol><li> Prepare uma bandeja com instrumentos necessários e material para inserção de um tubo traqueal (fórceps ou seja, tesoura, tubo traqueal, 3-0 sutura trançada preta, ventilador de pequenos animais, etc.) </li><li> Quando o rato é completamente anestesiado com um cocktail de cetamina / xilazina na proporção de 2 para 1 quetamina xilazina, raspar o abdômen, tórax e garganta do rato. Para garantir que um estado anestésico profundo foi alcançado, pinch dedo do rato e verificar se há retirada pedal do membro. Certifique-se que o rato está profundamente anestesiados antes de fazer todas as incisões. </li><li> Coloque o rato com a cabeça levantada em uma placa de corte que é inclinada em um ângulo de 30 ° </li><li> Faça uma incisão na pele a partir de meados do abdômen para abaixo do maxilar inferior. Dissecar longe os músculos do triângulo anterior do pescoço para expor a traquéia. </li><li> Coloque um 3-0 sutura trançada preta sob a traquéia. </li><li> Faça uma incisão na traquéia entre os anéis cartilaginosos umd inserir o tubo traqueal. Amarre o fio firmemente para proteger o tubo traqueal até a traquéia. </li><li> Conecte o tubo traqueal a um ventilador de pequenos animais. </li></ol> 3. Preparação instrumento para isolamento de células imunes <ol><li> Prepare uma bandeja com os instrumentos para o processo imunológico cela de isolamento (fórceps ou seja, tesouras, HBSS, aparados macio Teflon calibre 24 Baxter intravascular sobre a agulha quick-cath) </li><li> Prepare duas seringas 10cc, encha uma seringa com HBSS para a introdução do tampão dentro do saco pericárdio e reservar a outra para aspirar as células contendo HBSS. 3.2.1. Reduzir a ponta de teflon do calibre 24 Baxter intravascular sobre a agulha quick-cath (1,6 cm) por 1 / 2 a 3 / 4 de sua extensão original para o procedimento de isolamento. </li><li> Coloque todas as seringas, buffers, e um vazio 15 mL do tubo Falcon (para conter o fluido extraído uma vez o procedimento esteja concluído) no gelo. </li></ol> &lt;class = p &quot;jove_title&quot;&gt; 4. Isolamento de células imunes <ol><li> Faça uma incisão mediana na parede abdominal (ao longo da linha alba) até chegar ao apêndice xifóide. </li><li> Começando um pouco lateralmente ao processo xifóide, fazer incisões bilaterais através da caixa torácica contínua em um ângulo em direção à axila. Tenha cuidado para não perfurar o pulmão, coração ou pericárdio. </li><li> Uma vez que as incisões em ambos os lados do esterno são completos, dissecar afastado do diafragma das costelas inferiores, o corte lateral para medial. Nota: Esteja sempre ciente de onde o pericárdio se conecta ao diafragma e esterno para evitar danos. Use o ligamento falciforme como um marco para observar onde o pericárdio é anexado no lado oposto do diafragma. Além disso, se o pericárdio é cortado, a capacidade de isolar as células é perdido. </li><li> Levantar delicadamente um dos lados da caixa torácica para expor a cavidade torácica e do coração (ainda encapsulados pelo pericárdio).</li><li> Pegue na seringa preenchida e HBSS encontrar um ponto no saco pericárdico, aproximadamente a meio caminho entre o esterno eo coração (de preferência mais rostral do caudal) e insira a ponta de teflon da encurtado quick-cath através das duas camadas do pericárdio. </li><li> Uma vez que o cateter está em vigor, proceder gentilmente preencher o espaço pericárdico com 2 a 3 ml de tampão HBBS. Você saberá imediatamente se o cateter é colocado corretamente quando você percebe o espaço pericárdico em expansão. Se não houver preenchimento é testemunhado, retirar e reposicionar o quick-cath. </li><li> Quando o saco pericárdico é preenchido, tirar a seringa outros 10 cc e colocar a ponta de volta no pericárdio e começar a aspiração do buffer. O buraco mesma entrada pode ser usado ou de outra pode ser criada. Certifique-se que a ponta não vem muito perto das paredes do saco pericárdico, proporcionando sucção, especialmente na parte de baixo do coração. Isto poderia levar a punção do saco pericárdico e perda de células contendo célulass. </li><li> Enquanto aspiração do buffer, a ponta do cateter pode ser movido dentro do saco pericárdico, a fim de coletar tampão, tanto quanto possível. </li></ol> Nota: Se ao fazê-lo um pouco rasgar o pericárdio e fazer o furo na parte superior do coração maior que o buraco original, não entre em pânico. Enquanto o pericárdio ainda cerca o suficiente do coração, você ainda pode realizar outra lavagem sobre o coração, embora com uma menor quantidade de buffer. Para a coleta de uma quantidade ideal de células, 3-4 lavagens adicionais são necessários. <ol start="9"><li> Coloque o tampão extraído em tubos de Falcon que foram colocados no gelo. </li><li> Remover coração, pulmões, e / ou qualquer outro tecido, se necessário para estudos posteriores. Após a coleta de células do sistema imunológico e outros tecidos, o rato deve ser rápida e humanitariamente sacrificados. </li><li> Centrifugar a coletaram amostras de células imunes por 10 minutos a 200g a 4 ° C. Mantenha sempre os tubos no geloquando não em uso. </li><li> Remover o sobrenadante e reconstituir o sedimento em 1 mL de tampão ou Hyclone HBSS. </li></ol> Nota: É importante contar o número de células imunes obtidos em cada coração após o procedimento imunológico cela de isolamento. 5. Resultados representativos: Um exemplo isolado mastócitos cardíacos obtidos com esta técnica e corados com azul de toluidina e safranina O / alcian blue são apresentados nas figuras 2A e 2B, respectivamente. Como pode ser visto na Figura 3, O mastro cardíaca, mantiveram a sua capacidade de liberação de histamina após o tratamento com ativadores conhecidos, como composto 48/80 e substância P, conforme descrito no Morgan et al. 9 parcelas Representante dispersão de citometria de fluxo de células CD4 + e CD8 + são apresentados nas figuras 4 e 5, respectivamente. Os linfócitos T são ainda identificadas e fenotipadas como uma população de células a partir dessa técnica. Figura Fluxograma 1. Procedimento de isolamento das células imunes cardíaca. <img alt="Figura 2" src="/files/ftp_upload/3020/3020fig2.jpg" /> . Figura 2 imagens representativas histológica de mastócitos cardíacos estão manchadas como se segue: (A) com azul de toluidina e (B) são safranina O / alcian blue. <img alt="Figura 3" src="/files/ftp_upload/3020/3020fig3.jpg" /> Figura 3. Estudo funcional de mastócitos cardíacos isolados da região epicárdica do miocárdio e estimulados com composto 48/80 (10μg/mL) e substância P (10μM). <img alt="Figura 4" src="/files/ftp_upload/3020/3020fig4.jpg" /> Figura 4. Representante fluxo parcelas citometria de dispersão de CD4 + isoladas células marcadas com Substância P (1:100 diluindoion). <img alt="Figura 5" src="/files/ftp_upload/3020/3020fig5.jpg" /> Figura 5. Citometria de fluxo Representante do isolado CD8 + células marcadas com Substância P (1:100).

<ol>
	<li>Brower, G. L., Chancey, A. L., Thanigaraj, S., Matsubara, B. B., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cause+and+effect+relationship+between+myocardial+mast+cell+number+and+matrix+metalloproteinase+activity.">Cause and effect relationship between myocardial mast cell number and matrix metalloproteinase activity.</a> Am. J. Physiol. Heart. Circ. Physiol. 283, H518-H525 (2002).</li><li>Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacologic+inhibition+of+mast+cell+degranulation+prevents+left+ventricular+remodeling+induced+by+chronic+volume+overload+in+rats.">Pharmacologic inhibition of mast cell degranulation prevents left ventricular remodeling induced by chronic volume overload in rats.</a> Journal of Cardiac Failure. 11, 548-556 (2005).</li><li>Chancey, A. L., Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+mast+cell-mediated+activation+of+gelatinase+and+alteration+of+ventricular+diastolic+function.">Cardiac mast cell-mediated activation of gelatinase and alteration of ventricular diastolic function.</a> Am. J. Physiol. Heart. Circ. Physiol. 282, H2152-H2158 (2002).</li><li>Levick, S. P., Gardner, J. D., Holland, M., Hauer-Jensen, M., Janicki, J. S., Brower, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+from+adverse+myocardial+remodeling+secondary+to+chronic+volume+overload+in+mast+cell+deficient+rats.">Protection from adverse myocardial remodeling secondary to chronic volume overload in mast cell deficient rats.</a> Journal of Molecular and Cellular Cardiology. 43, 56-61 (2008).</li><li>Levick, S. P., McLarty, J. L., Murray, D. B., Freeman, R. M., Carver, W. E., Brower, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+Mast+Cells+Mediate+Left+Ventricular+Fibrosis+in+the+Hypertensive+Rat.">Cardiac Mast Cells Mediate Left Ventricular Fibrosis in the Hypertensive Rat.</a> Hypertension. 53, 1041-1047 (2009).</li><li>Batista, M. L., Santos, R. V., Cunha, L. M., Mattos, K., Oliveira, E. M., Seelander, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+pro-inflammatory+cytokine+production+and+peritoneal+macrophage+function+in+rats+with+chronic+heart+failure.">Changes in the pro-inflammatory cytokine production and peritoneal macrophage function in rats with chronic heart failure.</a> Cytokine. 34, 284-290 (2006).</li><li>Galli, S. J., Tsai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+Cells:+Versatile+regulator+of+inflammation,+tissue+remodeling,+host+defense+and+homeostasis.">Mast Cells: Versatile regulator of inflammation, tissue remodeling, host defense and homeostasis.</a> Journal of Dermatological Science. 49, 7-19 (2008).</li><li>Yndestad, A., Holm, A. M., Muller, F., Simonsen, S., Froland, S. S., Gullested, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+expression+of+inflammatory+cytokines+and+activation+markers+in+T-cells+from+patients+with+chronic+heart+failure.">Enhanced expression of inflammatory cytokines and activation markers in T-cells from patients with chronic heart failure.</a> Cardiovasc. Res. 60, 141-146 (2003).</li><li>Bozkurt, B., Kribbs, S. B., Clubb, F. J., Michael, L. H., Didenko, V. V., Hornsby, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiologically+Relevant+Concentrations+of+Tumor+Necrosis+Factor-&#x0251;+Promote+Progressive+Left+Ventricular+Dysfunction+and+Remodeling+in+Rats.">Pathophysiologically Relevant Concentrations of Tumor Necrosis Factor-&#x0251; Promote Progressive Left Ventricular Dysfunction and Remodeling in Rats.</a> Circulation. 97, 1382-1391 (1998).</li><li>Frangogiannis, N. G., Lindsey, M. L., Michael, L. H., Youker, K. A., Bressler, R. B., Mendoza, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resident+Cardiac+Mast+Cells+Degranulate+and+Release+Preformed+TNF-&#x0251;,+Initiating+the+Cytokine+Cascade+in+Experimental+Canine+Myocardial+Ischemia/Reperfusion.">Resident Cardiac Mast Cells Degranulate and Release Preformed TNF-&#x0251;, Initiating the Cytokine Cascade in Experimental Canine Myocardial Ischemia/Reperfusion.</a> Circulation. 98, 699-710 (1998).</li><li>Jobe, L. J., Melendez, G. C., Levick, S. P., Du, Y., Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-&#945;+inhibition+attenuates+adverse+myocardial+remodeling+in+a+rat+model+of+volume+overload.">TNF-&#945; inhibition attenuates adverse myocardial remodeling in a rat model of volume overload.</a> Am. J. Physiol. Heart. Circ. Physiol. 297, H1462-H1468 (2009).</li><li>Forman, M. F., Brower, G. L., Janicki, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+histamine+secretion+during+isolation+of+rat+cardiac+mast+cells.">Spontaneous histamine secretion during isolation of rat cardiac mast cells.</a> Inflammation Research. 53, 453-457 (2004).</li><li>Levick, S. P., Murray, D. B., Janicki, J. S., Brower, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sympathetic+nervous+system+modulation+of+inflammation+and+remodeling+in+the+hypertensive+heart.">Sympathetic nervous system modulation of inflammation and remodeling in the hypertensive heart.</a> Hypertension. 55, 270-276 (2010).</li><li>Morgan, L., Levick, S., Voloshenyuk, T., Murray, D., Forman, M., Brower, 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+novel+technique+for+isolating+functional+mast+cells+from+the+heart.">A novel technique for isolating functional mast cells from the heart.</a> Inflammation Research. 57, 241-246 (2008).</li></ol>

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Este método de isolamento de células do sistema imunológico cardíaca permite a análise de sua função, o fenótipo, e maturidade, sem a ativação indesejados que normalmente ocorre durante a técnica de isolamento enzimático de digestão tradicional. O número médio total de células obtidas com esta técnica é de cerca de 400.000 por coração de rato, e coloração diferencial e análise de citometria de fluxo destas células indica população mista de linfócitos T (~ 70%), macrófagos (~ 12%) e mastócito...

<table border="1"><tbody><tr><td> Nome do reagente e Equipamentos </td><td> Companhia </td><td> Número Catálogo </td><td> Comentários </td></tr><tr><td> Salina balanceada de Hanks </td><td> Sigma </td><td> H4891 </td><td> Uma garrafa faz 1 L de Salt Hanks Balance Solução. Sal reconstituir Hanks Balance Solução, como parte do procedimento. </td></tr><tr><td> Antibióticos Antimyotic </td><td> Gibco </td><td> 15240-062 </td><td></td></tr><tr><td> HEPES </td><td> Sigma </td><td> H3784 </td><td></td></tr><tr><td> Albumina de soro bovino </td><td> Sigma </td><td> A3912 </td><td></td></tr><tr><td> 250 mL Nalogene Bottle Filtro </td><td> Thermoscientific </td><td> 157-0020 </td><td></td></tr><tr><td> 12 N HCL </td><td> Fisher Scientific </td><td> CAS 7647-01-0 </td><td> Por 6 N, adicionar volumes iguais de HCL para deionizadada água. </td></tr><tr><td> 10 N NaOH </td><td> Fisher Scientific </td><td> SC267 </td><td> Por 5 N, adicionar volumes iguais de NaOH à água deionizada. </td></tr><tr><td> Água Destilada dupla </td><td></td><td></td><td></td></tr></tbody></table> Tabela de equipamentos específicos: <table border="1"><tbody><tr><td> Nome do reagente e Equipamentos </td><td> Companhia </td><td> Número Catálogo </td><td> Comentários </td></tr><tr><td> 3-O fio de seda trançada </td><td> Ethicon Produtos </td><td> SA64H </td><td></td></tr><tr><td> Ventilador de Pequenos Animais </td><td> CWE Incorporated </td><td> SAR-830 </td><td> Defina a taxa de respiração para 40,5 respirações por min. </td></tr><tr><td> Bisel da agulha de calibre 18 </td><td> BD Biosciences </td><td> 305199 </td><td> Guarnição da agulha 1 / 2 a 3 / 4 de seu comprimento original para ser usado como um tubo traqueal. </td></tr><tr><td> Dica Teflon (com agulha removida) de calibre 24 Baxter cateter quick-cath </td><td> Baxter </td><td> 2N111 </td><td> Reduzir o comprimento Dica Teflon por 1 / 2 a 04/03. </td></tr><tr><td> Seringa de 10 cc </td><td> Fisher Scientific </td><td> 14-823-16B </td><td></td></tr><tr><td> 15 mL Falcon Tubes </td><td> BD Biosciences </td><td> 352097 </td><td></td></tr><tr><td> Centrífuga Refrigerada Sorvall RT6000B </td><td> Fisher Scientific </td><td> 75-004-377 </td><td> Lembre-se de pré-cool e manter a 4 ° C. </td></tr><tr><td> Hyclone </td><td> Thermoscientific </td><td> S300030.02 </td><td></td></tr><tr><td> Hemocitômetro </td><td> Hausser Scientific </td><td> HS-LB-1483 </td><td></td></tr></tbody></table>

isolation of functional cardiac immune cells

Cardíacas células do sistema imunológico estão ganhando interesse para os papéis que desempenham no remodelamento patológico em muitas doenças cardíacas 05/01 Estas células do sistema imunológico, que incluem os mastócitos, células T e macrófagos;. Armazenar e liberar uma variedade de mediadores biologicamente ativos, incluindo citocinas e proteases, como triptase. 6-8 Esses mediadores têm se mostrado os jogadores-chave no metabolismo da matriz extracelular pela ativação de metaloproteinases de matriz ou causando acúmulo de colágeno por função modular os fibroblastos cardíacos. 9-11 No entanto, as técnicas disponíveis para isolar células do sistema imunológico têm cardíaca sido problemática, porque eles usam a colagenase bacteriana para digerir o tecido do miocárdio. Esta técnica faz com que a ativação das células imunes e, portanto, uma perda de função. Por exemplo, cardíaco mastócitos tornam-se significativamente menos sensíveis aos compostos que causam degranulação. 12 Por isso, desenvolvemos uma técnica que permite a tele isolamento de células do sistema imunológico funcional cardíaca que levaria a uma melhor compreensão do papel destas células na doença cardíaca. 13, 14 Este método requer uma familiaridade com a localização anatômica do rato processo xifóide axila, e ligamento falciforme, e pericárdio do coração. Estes marcos são importantes para aumentar o sucesso do procedimento e garantir uma maior produção de células do sistema imunológico cardíaca. Estes isolados cardíaca células do sistema imunológico podem então ser utilizados para a caracterização do fenótipo, funcionalidade, maturidade e co-cultura experiências com outras células cardíacas para obter uma melhor compreensão de suas interações.

Watch this Scientific Journal Video about Isolation of Functional Cardiac Immune Cells at JoVE.com

Isolation of Functional Cardiac Immune Cells

Este método para isolar células do sistema imunológico funcional do coração fornece uma alternativa aos métodos convencionais de digestão da colagenase, que causa a ativação de células imunes indesejadas, resultando em uma capacidade de resposta diminuiu dessas células. Nosso método de isolamento de rendimentos funcional cardíaca células do sistema imunológico, evitando os problemas associados com a digestão enzimática.

Isolamento de células do sistema imunológico funcional cardíaca

Cardiac immune cells are gaining interest for the roles they play in the pathological remodeling in many cardiac diseases.1-5  These immune cells, which ...

isolation-of-functional-cardiac-immune-cells

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

15.0K visualizações.  University of South Carolina- School of Medicine. Este método para isolar células do sistema imunológico funcional do coração fornece uma alternativa aos métodos convencionais de digestão da colagenase, que causa a ativação de células imunes indesejadas, resultando em uma capacidade de resposta diminuiu dessas células. Nosso método de isolamento de rendimentos funcional cardíaca células do sistema imunológico, evitando os problemas associados com a digestão enzimática.

Vídeo: Isolation of Functional Cardiac Immune Cells

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

Isolamento de perivascular Multipotentes populações de células precursoras a partir de tecido cardíaco humano

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Biologia do Desenvolvimento

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Isolamento e caracterização funcional de Ventricular Humano cardiomiócitos a partir de amostras cirúrgicas frescos

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Medicina

Isolation and Identification of Extravascular Immune Cells of the Heart

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with functional compartmentalization both during steady state and during different forms of inflammation. Until recently, the study of immune cells in the heart required the use of microscopy or poorly developed digestion protocols, which provided enough sensitivity during severe inflammation but were unable to confidently identify small &#8212; but key &#8212; populations of cells during steady state. Here, we discuss a simple method combining enzymatic (collagenase, hyaluronidase and DNAse) and mechanical digestion of murine hearts preceded by intravascular administration of fluorescently-labelled antibodies to differentiate small but unavoidable intravascular cell contaminants. This method generates a suspension of isolated viable cells that can be analyzed by flow cytometry for identification, phenotyping and quantification, or further purified with fluorescence-activated cell sorting or magnetic bead separation for transcriptional analysis or in vitro studies. We include an example of a step-by-step flow cytometric analysis to differentiate the key macrophage and dendritic cell populations of the heart. For a medium sized experiment (10 hearts) the completion of the procedure requires 2&#8211;3 h.

This protocol presents a simple and efficient method to isolate, identify and quantify immune cells residing in the myocardium of mice during steady state or inflammation. The protocol combines enzymatic and mechanical digestion for the generation of a single cell suspension that can be further analyzed by flow cytometry.

Isolamento e identificação de células do sistema imunológico Extravascular do coração

The immune system is an essential component of a healthy heart. The myocardium is home to a rich population of different immune cell subsets with ...

Imunologia e Infecção

Isolation of Macrophage Subsets and Stromal Cells from Human and Mouse Myocardial Specimens

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative responses after cardiac injury. How various subsets of macrophages orchestrate the immune responses after cardiac injury is an active area of research. Presented here is a simple protocol that our lab performs routinely, for the extraction of macrophages from mouse and human myocardium specimens obtained from healthy and diseased individuals. Briefly, this protocol involves enzymatic digestion of cardiac tissue to generate a single cell suspension, followed by antibody staining, and flow cytometry. This technique is suitable for functional assays performed on sorted cells as well as bulk and single cell RNA sequencing. A major advantage of this protocol is its simplicity, minimal day to day variation and wide applicability allowing investigation of macrophage heterogeneity across various mouse models and human disease entities.

Presented here is a protocol to isolate various subsets of macrophages and other non-immune cells from human and mouse myocardium by preparing a single cell suspension through enzymatic digestion. Gating schemes for flow cytometry based identification and characterization of isolated macrophages are also presented.

Isolamento de subconjuntos de macrófagos e células estromais de espécimes miocárdios humanos e de camundongos

Macrophages represent the most heterogeneous and abundant immune cell populations in the heart and are central in driving inflammation and reparative ...

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions.

Individual cardiomyocytes from wild type and mutant mice can be isolated from the heart in order to study their contractility and calcium transients. This allows characterization of the contribution of cellular dysfunction to heart dysfunction from any cause.

Isolamento e Fisiológica Análise de rato Cardiomyocytes

Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force ...

Biologia

Isolation and Purification of Murine Cardiac Pericytes

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier integrity. However, their tissue-specific functions in the heart are not well understood. Moreover, there is currently no protocol utilizing readily accessible materials to isolate and purify pericytes of cardiac origin. Our protocol focuses on using the widely used mammalian model, the mouse, as our source of cells. Using the enzymatic digestion and mechanical dissociation of heart tissue, we obtained a crude cell mixture that was further purified by fluorescence activating cell sorting (FACS) by a plethora of markers. Because there is no single unequivocal marker for pericytes, we gated for cells that were CD31-CD34-CD45-CD140b+NG2+CD146+. Following purification, these primary cells were cultured and passaged multiple times without any changes in morphology and marker expression. With the ability to regularly obtain primary murine cardiac pericytes using our protocol, we hope to further understand the role of pericytes in cardiovascular physiology and their therapeutic potential.

We have optimized a protocol to isolate and purify murine cardiac pericytes for basic research and investigation of their biology and therapeutic potential.

Isolamento e purificação de Pericytes cardíacos murinos

Pericytes, perivascular cells of microvessels and capillaries, are known to play a part in angiogenesis, vessel stabilization, and endothelial barrier ...

Flow Cytometry-Based Quantification and Analysis of Myocardial B-Cells

A growing body of evidence shows that B-lymphocytes play an important role in the context of myocardial physiology and myocardial adaptation to injury. However, the literature reports contrasting data on the prevalence of myocardial B-cells. B-cells have been reported to be both among the most prevalent immune cells in the rodent heart or to be present, but at a markedly lower prevalence than myeloid cells, or to be quite rare. Similarly, several groups have described that the number of myocardial B-cells increases after acute ischemic myocardial injury, but one group reported no changes in the number of B-cells of the injured myocardium. Implementation of a shared, reproducible method to assess the prevalence of myocardial B-cells is critical to harmonize observations from different research groups and thus promote the advancement of the study of B-cell myocardial interactions. Based on our experience, the seemingly contrasting observations reported in the literature likely stem from the fact that murine myocardial B-cells are mostly intravascular and connected to the microvascular endothelium. Therefore, the number of B-cells recovered from a murine heart is exquisitely sensitive to the perfusion conditions used to clean the organ and to the method of digestion used. Here we report an optimized protocol that accounts for these two critical variables in a specific way. This protocol empowers reproducible, flow cytometry-based analysis of the number of murine myocardial B-cells and allows researchers to distinguish extravascular vs. intravascular myocardial B-cells.

Here we report a protocol for the quantification and differentiation of myocardial B-lymphocytes based on their location in the intravascular or endothelial space using flow cytometry.

Quantificação e Análise Baseada em Citometria de Fluxo de Células B do Miocárdio

A growing body of evidence shows that B-lymphocytes play an important role in the context of myocardial physiology and myocardial adaptation to injury. ...

Isolation and Culture of Resident Cardiac Macrophages from the Murine Sinoatrial and Atrioventricular Node

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by electrical impulses generated in sinoatrial node (SAN) and then conducted to ventricles via atrioventricular node (AVN). To further study the role of resident macrophages in cardiac conduction system, a proper isolation of resident macrophages from SAN and AVN is necessary, but it remains challenging. Here, we provide a protocol for the reliable microdissection of the SAN and AVN in murine hearts followed by the isolation and culture of resident macrophages.
Both, SAN which is located at the junction of the crista terminalis with the superior vena cava, and AVN which is located at the apex of the triangle of Koch, are identified and microdissected. Correct location is confirmed by histologic analysis of the tissue performed with Masson's trichrome stain and by anti-HCN4.
Microdissected tissues are then enzymatically digested to obtain single cell suspensions followed by the incubation with a specific panel of antibodies directed against cell-type specific surface markers. This allows to identify, count, or isolate different cell populations by fluorescent activated cell sorting. To differentiate cardiac resident macrophages from other immune cells in the myocardium, especially recruited monocyte-derived macrophages, a delicate devised gating strategy is needed. First, lymphoid lineage cells are detected and excluded from further analysis. Then, myeloid cells are identified with resident macrophages being determined by high expression of both CD45 and CD11b, and low expression of Ly6C. With cell sorting, isolated cardiac macrophages can then be cultivated in vitro over several days for further investigation. We, therefore, describe a protocol to isolate cardiac resident macrophages located within the cardiac conduction system. We discuss pitfalls in microdissecting and digesting SAN and AVN, and provide a gating strategy to reliably identify, count and sort cardiac macrophages by fluorescence-activated cell sorting.

The protocol presented here provides a step-by-step approach for the isolation of cardiac resident macrophages from the sinoatrial node (SAN) and atrioventricular node (AVN) region of mouse hearts.

Isolamento e Cultura de Macrófagos Cardíacos Residentes do Nó Sinoatrial e Atrioventricular Murino

Resident cardiac macrophages have been demonstrated to facilitate the electrical conduction in the heart. The physiologic heart rhythm is initiated by ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Isolamento, caracterização e diferenciação de células-tronco cardíacas do coração de rato adulto

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation of Cardiomyocytes from Fixed Hearts for Immunocytochemistry and Ploidy Analysis

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably identify nuclei of cardiomyocytes on histological sections, many groups rely on isolating viable cardiomyocytes prior to fixation to perform immunostaining. However, these live cardiomyocyte isolation techniques require optimization to maximize the yield, viability and quality of the samples, with inherent fluctuations from sample to sample despite maximum optimization. Here, we report a reproducible protocol, involving fixation prior to enzymatic digestion of the heart, which leads to maximum yield while preserving the in vivo morphology of individual cardiomyocytes. We further developed an automated analysis platform to determine the number of nuclei and DNA content per nucleus for individual cardiomyocytes. After exposing the chest cavity, the heart was arrested in diastole by perfusion with 60 mM KCl in PBS. Next, the heart was fixed in 4% paraformaldehyde (PFA) solution, and then digested with 60 mg/mL collagenase solution. After digestions, cells were singularized by trituration, and the cardiomyocyte fraction was enriched via differential centrifugation. Isolated cardiomyocytes were stained for Troponin T and &#945;-actinin to assess purity of the obtained population. Furthermore, we developed an image analysis platform to determine cardiomyocyte nucleation and ploidy status following DAPI staining. Image based ploidy assessments led to consistent and reproducible results. Thus, with this protocol, it is possible to preserve native morphology of individual cardiomyocytes to allow immunocytochemistry and DNA content analysis while achieving maximum yield.

The goal of this work is to develop a method to reproducibly isolate cardiomyocytes from the adult heart and measure DNA content and nucleation.

Isolamento de Cardiomiócitos de Corações Fixos para Imunocitoquímica e Análise ploidy

The adult mammalian heart is composed of various cell types including cardiomyocytes, endothelial cells and fibroblasts. Since it is difficult to reliably ...