Este trabalho foi apoiado pela UE (STREP Projeto 241577 &quot;grande coração,&quot; 7 º Programa-Quadro Europeu, CP), Menarini International Operations Luxembourg (AM), Telethon GGP07133 (PB) e Gilead Sciences (AM).

Os protocolos experimentais sobre o tecido humano foram aprovados pelo comitê de ética do Careggi Hospital Universitário (2006/0024713; renovada Maio de 2009). Cada paciente deu consentimento informado por escrito. 1. Soluções e Equipamentos Preparação As soluções são descritas na Tabela 1. Um fluxograma simplificado do processo de isolamento de células encontra-se na Figura 1. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td colspan="2"> Solução </td><td> CP </td><td> DB </td><td> KB </td><td> Tuberculose </td><td> PS </td><td> EB1 </td><td> EB2 </td></tr><tr><td rowspan="23"> Reagente (mM) </td><td> KH 2 PO 4 </td><td> 50 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> MgSO4 </td><td> 8 </td><td> 1.2 </td><td> 5 </td><td></td><td></td><td> 1.2 </td><td> 1.2 </td></tr><tr><td> HEPES </td><td> 10 </td><td></td><td></td><td> 10 </td><td> 10 </td><td></td><td></td></tr><tr><td> adenosina </td><td> 5 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> glicose </td><td> 140 </td><td> 10 </td><td> 20 </td><td> 10 </td><td></td><td> 10 </td><td> 10 </td></tr><tr><td> manitol </td><td> 100 </td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> taurina </td><td> 10 </td><td> 20 </td><td> 5 </td><td></td><td></td><td> 20 </td><td> 20 </td></tr><tr><td> NaCl </td><td></td><td> 113 </td><td></td><td> 136 </td><td></td><td> 113 </td><td> 113 </td></tr><tr><td> KCl </td><td></td><td> 4.7 </td><td> 85 </td><td> 5.4 </td><td> 25 </td><td> 4.7 </td><td> 4.7 </td></tr><tr><td> MgCl2 </td><td></td><td> &lt;/ Td&gt; <td></td><td> 1.2 </td><td> 5 </td><td></td><td></td></tr><tr><td> KH 2 PO 4 </td><td></td><td> 0,6 </td><td> 30 </td><td></td><td></td><td> 0,6 </td><td> 0,6 </td></tr><tr><td> Na 2 HPO 4 </td><td></td><td> 0,6 </td><td></td><td></td><td></td><td> 0,6 </td><td> 0,6 </td></tr><tr><td> NaHCO3 </td><td></td><td> 12 </td><td></td><td></td><td></td><td> 12 </td><td> 12 </td></tr><tr><td> KHCO3 </td><td></td><td> 10 </td><td></td><td></td><td></td><td> 10 </td><td> 10 </td></tr><tr><td> Na-piruvato </td><td></td><td> 4 </td><td></td><td></td><td></td><td> 4 </td><td> 4 </td></tr><tr><td> BDM </td><td></td><td> 10 </td><td></td><td></td><td></td><td> 10 </td><td> 10 </td></tr><tr><td> BHBA </td><td></td><td></td><td> 5 &lt;/ Td&gt; <td></td><td></td><td></td><td></td></tr><tr><td> ácido succínico </td><td></td><td></td><td> 5 </td><td></td><td></td><td></td><td></td></tr><tr><td> EGTA </td><td></td><td></td><td> 0,5 </td><td></td><td></td><td></td><td></td></tr><tr><td> K 2-ATP </td><td></td><td></td><td> 2 </td><td></td><td></td><td></td><td></td></tr><tr><td> ácido pirúvico </td><td></td><td></td><td> 5 </td><td></td><td></td><td></td><td></td></tr><tr><td> creatina </td><td></td><td></td><td> 5 </td><td></td><td></td><td></td><td></td></tr><tr><td> KMES </td><td></td><td></td><td></td><td></td><td> 115 </td><td></td><td></td></tr><tr><td rowspan="2"> Enzimas (U / ml) </td><td> Colagenase tipo V </td><td></td><td></td><td></td><td></td><td></td><td> 250 &lt;/ Td&gt; <td> 250 </td></tr><tr><td> Protease Tipo XXIV </td><td></td><td></td><td></td><td></td><td></td><td> 4 </td><td></td></tr><tr><td> pH </td><td></td><td> 7.4 KOH </td><td> NaOH 7,3 </td><td> 7.1 KOH </td><td> NaOH 7,35 </td><td> 7.2 KOH </td><td> NaOH 7,3 </td><td> NaOH 7,3 </td></tr></tbody></table> . Tabela 1 Soluções utilizadas para a coleta de amostras, isolamento de células e caracterização funcional de miócitos CP = solução cardioplégica.; DB = tampão de dissociação; KB = solução Kraft-Brühe; TB = tampão Tyrode; PS = solução da pipeta; EB1 = tampão de enzima 1; EB2 = tampão de enzima 2. <ol><li> Solução cardioplégica (CP) Preparar. CP solução pode ser armazenada a 4 ° C durante até 1 semana. </li><li> Prepare Ca 2 tampão de dissociação livre de + (DB). Esta solução deve ser usado durante o dia. </li><li> Preparar a solução Kraft-Brühe (KB). KB solução pode ser armazenada a 4 ° C durante até uma pequeninak. </li><li> Prepare Ca tampão Tyrode livre de + 2 (TB). Esta solução deve ser usado durante o dia. </li><li> Filtrar todas as soluções usando filtros de seringa, antes da utilização. </li><li> Preparar o aparelho de digestão (Figura 2), um recipiente de raspagem feita de duas escovas de frente de elastómero de silicone, um dos quais rodados por um motor eléctrico. O dispositivo digestão é feito sob encomenda. Detalhes sobre o dispositivo de digestão são na Figura 8; imagens do dispositivo estão nas Figuras 2 C e 2D. Lava-se a câmara de tecido com 70% de etanol e água. </li></ol> 2. Coleta e Processamento de Amostras do miocárdio <ol><li> Verter 40 ml da solução cardiplegic (CP) num tubo de 50 ml e armazena-o em gelo durante o transporte da amostra a partir do quarto dispositivo para o isolamento de células de laboratório. </li><li> Colete a amostra do miocárdio ventricular da sala operatória imediatamente após a excisão, lave-o com gelo frioSolução CP e armazená-lo no tubo. Use espécimes endocárdio excisadas do septo ventricular, entre superior durante a cirurgia de coração aberto, ponderação&gt; 100 mg. </li><li> Rapidamente transferir a amostra para a área do laboratório; iniciar o processamento da amostra dentro de 10 minutos a partir de amostra de excisão. </li><li> Enquanto mantém a amostra em tampão gelado CP, retire cuidadosamente a camada fibrosa endocárdica usando tesoura fina sob estereomicroscópio; depois, cortar o tecido do miocárdio para pequenas peças (2-3 mm de comprimento). Dependendo do tamanho da amostra de tecido, cortar uma quantidade total de miocárdio ventricular entre 100 mg e 1 g para cada isolamento. </li><li> Após a conclusão da picagem tecido, transfira os pedaços do miocárdio no dispositivo digestão, com gelo limpo solução CP frio. Evitar preenchendo todo o volume entre as duas escovas de silício (3-4 ml) com os pedaços do miocárdio, utilizando-se não mais do que 1 g de tecido total. </li></ol> 3. Lavar e digestão de miocárdio Pedaços <ol><li> À réer os pedaços são transferidos para a câmara de raspagem do dispositivo digestão, mudar o tampão CP na câmara de frio Ca 2 + tampão de dissociação-free (DB). </li><li> Colocar o dispositivo de digestão num banho termostático, de modo para a câmara de estar em contacto com a água aquecida no banho (Figura 1). Definir o banho a 37,5 ° C e ligá-lo, a fim de aumentar lentamente a temperatura da câmara de tecido. Ligar o motor do dispositivo de digestão, a definição da velocidade de rotação de 1 volta / segundo. </li><li> Realizar 3 ciclos de lavagem com DB, mudando a solução na câmara de limpeza com DB a cada 8 minutos. A DB é aquecido (37 ° C) e oxigênio saturado antes de entrar em contato com os pedaços do miocárdio. </li><li> Preparar tampão de enzima 1 (EB1) por adição de 250 U / ml de colagenase de tipo V e 4 U / mL de protease do tipo XXIV solução DB. Prepare tampão da enzima 2 (EB2), adicionando 250 U / ml de colagenase tipo V a solução DB. Aquecimento (37 ° C) e oxygenate EB1 e EB2. </li><li> Realizar dois ciclos de 12 min de digestão no dispositivo de digestão rotativo com 100% EB1 oxigenado (a 37 ° C). Em cada ciclo, utilizar ~ 3 ml de EB1. Remover a solução com uma pipeta de aspiração e descartá-lo após cada ciclo. </li><li> Prepare a 6 tubos de 15 ml para recolha de células, e ~ 80 mL de água fria (4 ° C) solução KB para a eluição dos tampões. </li><li> Realizar um primeiro ciclo de digestão de 15 minutos com 3 ml de 100% EB2 oxigenada a 37 ° C. Após o ciclo de digestão, recolher a solução contendo os primeiros miócitos dissociadas num tubo de 15 ml e dilui-se a suspensão de células com 12 ml de solução fria KB. Guarde o apartamento tubo à temperatura ambiente. </li><li> Diluir a solução EB2 restante com uma quantidade igual de DB, a fim de reduzir pela metade a concentração de colagenase V para os seguintes ciclos de digestão. </li><li> Realizar outras cinco ciclos de 12 min de digestão com 3 ml EB2 a 37 ° C; depois de cada um deles recolher de miócitos contendo tampão, num tubo cónico de 15 ml ediluir com 12 ml de solução KB. Armazenar as 6 células contendo os tubos à temperatura ambiente durante 30 min. </li></ol> 4. Resuspensão Celular e Ca 2 + Readaptação <ol><li> Adicionar 1 mg / ml de albumina de soro bovino (BSA) a 20 ml de tampão de Ca 2 +-Tyrode livre (TB). Filtrar a solução. </li><li> Centrifugar os seis miócitos contendo tubos cônicos a 100 xg por 5 min para forçar miócitos para resolver. Remover o sobrenadante e ressuspender as células em cada um dos tubos com uma quantidade variável (1-3 ml, dependendo do rendimento) de BSA contendo TB, à TA. </li><li> Aumentar gradualmente a concentração de Ca2 + na célula contendo tampão através da adição de pequenas alíquotas de 100 mmol / L de solução de CaCl 2. Na primeira e na segunda etapas concentração de Ca2 + é elevado até 50 pmol / L e 100 pmol / L, respectivamente. Os seguintes passos de Ca 2 + de adição são realizadas a cada 5 min e a concentração é aumentada em 100 mmol / L em cada passo toa concentração final de 0,9 mmol / L. </li><li> Avaliar o rendimento do processo de isolamento. Transferir 0,5 ml de solução contendo miócitos para a câmara de fundo de vidro de microscópio. Avaliar 15 campos do microscópio em 10x objetivo e calcular a percentagem de miócitos saudáveis ​​(por exemplo, em forma de bastonetes com estrias claras e sem inclusões significativas, Figura 2). O rendimento previsto é de cerca de 20%. </li></ol> 5. Avaliação Funcional de isolado cardiomiócitos. O protocolo a seguir é um exemplo de avaliação funcional cardiomiócitos humano, incluindo gravações simultâneas de potenciais de ação e Ca 2 + intracelular fluxos. <ol><li> Preparar a solução de pipeta (PS) para experimentos de patch clamp na configuração de patch perfurado. A solução pode ser armazenada a -20 ° C durante até 3 meses. </li><li> Adicionar 1,8 mmol / L de CaCl 2 a 2 Ca tampão Tyrode + livre (TB). Use esta solução para superfusão de cardiomiócitos durante as experiências braçadeira patch / fluorescência. </li><li> Transferir 1 ml de suspensão de células para um tubo de 1,5 ml e adicionam-se 10 mmol / L e 10 ul Fluoforte Concentrado carga de potência. Incubar durante 30 minutos à temperatura ambiente. Em seguida, defina o tubo na posição vertical e deixar a célula de se contentar com 5 min; ressuspender as células em Ca 2 + contendo TB. </li><li> Transferir 0,25 ml de suspensão de células para um pequeno (0,5 ml), temperatura controlada microscópio montado câmara de registo, superfundidas por gravidade com um sistema microperfusor aquecido a uma taxa de fluxo de 0,3 ml / min (temperatura: 37 ± 0,5 ° C). </li><li> Usando um puxador de micropipeta, preparar pipetas de fixação de membranas com um diâmetro de ponta de 3-5 um e uma resistência de 3 a 4,5 M, quando cheio com o PS. </li><li> Adicionar anfotericina B para um lote de PS (250 ug / ml) e usá-lo para preencher os eléctrodos. </li><li> Selecione uma célula em forma de haste com estrias claras, desprovido de inclusões, form o selo giga e esperar de 5 a 10 min, até que a resistência de acesso for inferior a 20 mohms. </li><li> Provocar potenciais de ação no modo de fixação de corrente usando pulsos de curta duração (&lt;3 ms) em diferentes frequências de estimulação (0,2 Hz, 0,5 Hz e 1 Hz, 1 min em cada freqüência). Durante a fase de gravação, ligar a iluminação de campo claro a 492 ± 3 nm e detectar Fluoforte fluorescência em 505-520 nm. Adquirir fluorescência e potencial de membrana usando sinais Digidata 1440A e 10,0 software pClamp. Repita a seqüência de gravação várias vezes, se necessário; no entanto, manter o tempo total de gravação inferior a 15 min, para cada célula. </li></ol>

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M., Rudy, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+IKr+and+IKs+heterogeneity+on+action+potential+duration+and+its+rate+dependence:+a+simulation+study.">Effects of IKr and IKs heterogeneity on action potential duration and its rate dependence: a simulation study.</a> Circulation. 99, 2466-2474 (1999).</li><li>Volders, P. G., 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=Probing+the+contribution+of+IKs+to+canine+ventricular+repolarization:+key+role+for+beta-adrenergic+receptor+stimulation.">Probing the contribution of IKs to canine ventricular repolarization: key role for beta-adrenergic receptor stimulation.</a> Circulation. 107, 2753-2760 (2003).</li><li>Sanguinetti, M. C., Jurkiewicz, N. K., Scott, A., Siegl, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoproterenol+antagonizes+prolongation+of+refractory+period+by+the+class+III+antiarrhythmic+agent+E-4031+in+guinea+pig+myocytes.+Mechanism+of+action.">Isoproterenol antagonizes prolongation of refractory period by the class III antiarrhythmic agent E-4031 in guinea pig myocytes. Mechanism of action.</a> Circulation Research. 68, 77-84 (1991).</li><li>Coppini, 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=A+translational+approach+to+treatment+of+hypertrophic+cardiomyopathy:+pre-clinical+rationale+and+design+of+a+prospective+randomized+pilot+trial+with+ranolazine.">A translational approach to treatment of hypertrophic cardiomyopathy: pre-clinical rationale and design of a prospective randomized pilot trial with ranolazine.</a> Circulation. 125, 1 (2012).</li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

Nós descrevemos e validado um método para isolar os miócitos viáveis ​​a partir de amostras cirúrgicas do miocárdio ventricular humano. A partir de protocolos descritos anteriormente que tinham sido usados ​​com sucesso para células isoladas a partir de amostras cirúrgicas atriais, a técnica para permitir a separação de miócitos viáveis ​​individuais de miocárdio ventricular doente foi desenvolvido e afinado. Os primeiros relatos mostraram que o isolamento dos cardiomiócitos individuais de ped...

Dissecção das propriedades eletrofisiológicas do miocárdio progrediu acentuadamente após o desenvolvimento de técnicas para o isolamento individual dos miócitos cardíacos. Os recentes avanços na compreensão da excitação cardíaca Contração Coupling (EC-acoplamento) também têm sido possível graças à capacidade de isolar cardiomiócitos únicos viáveis ​​que conservam todas as propriedades fisiológicas do tecido intacto. Métodos de patch clamp são rotineiramente utilizados para estudar a função e modulação farmacológica de correntes iônicas sarcolemais cardíaca. Gravações da dinâmica de cálcio intracelular com Ca 2 + corantes sensíveis também são realizados regularmente em miócitos cardíacos individuais a partir de uma variedade de modelos saudáveis ​​e doentes, fornecendo dados vitais sobre a fisiologia da EC-acoplamento, bem como sobre as alterações patológicas de Ca 2 + intracelular homeostase levando a prejuízo mecânica e aumento da carga arritmogênica em doenças cardíacas. Informação a partir desses estudos é fundamental para a compreensão dos efeitos eletrofisiológicos e mecânicas de drogas no ambiente clínico. No entanto, existem diferenças específicas de espécies nas correntes transmembrana e nas proteínas EC-de acoplamento que são responsáveis ​​por características específicas do potencial de ação cardíaco e mecânica cardíaca. Assim, enquanto os estudos de miócitos isolados de mamíferos não humanos elucidaram as propriedades biofísicas e as funções fisiológicas dos canais iônicos transmembrana específicos e proteínas EC-acoplamento, eles não fornecem necessariamente modelos relevantes de miócitos cardíacos humanos. Isolamento de miócitos viáveis ​​do miocárdio humano é, portanto, essencial para compreender plenamente a fisiopatologia das doenças cardíacas e validar novas abordagens terapêuticas. Tecido atrial humano está prontamente disponível como apêndices atriais estão muitas vezes descartados durante os procedimentos cirúrgicos. Estudos quantitativos iniciais do potencial de ação cardíaco humanos adultos e cu iônicarrents empregada células atriais enzimaticamente isolados 1-4. Gravações de potenciais de ação ou correntes a partir de células humanas adultas ventriculares isoladas foram posteriormente relatado 3,5-10. A maioria destes estudos utilizaram células obtidas a partir de corações de explante e utilizados ou de perfusão com colagenase de um segmento da artéria coronária ou a exposição de quantidades relativamente grandes de tecido excisado de colagenase para obter células isoladas. Esses estudos permitiram uma caracterização detalhada de uma série de correntes de íons transmembrana de cardiomiócitos do ventrículo humanos de corações saudáveis ​​e de pacientes com insuficiência cardíaca terminal. As gravações de tipo-L de Ca 2 + de corrente (I Ca-L) 5-7, a corrente de potássio para fora transitórias (I) 8, para dentro de potássio rectificador de corrente (I κ1) 8, os diferentes componentes da corrente de potássio rectificador retardado (I κ ) 9 foram relatados. Avanços e refino deo procedimento de isolamento 10, permitiu uma caracterização clara da base iônica do aumento do potencial arritmogênico na insuficiência cardíaca terminal, compreendendo acção potencial prolongamento 11, adiada após despolarizações 12 e aumentou engraçado atual 13 levando a despolarização diastólica e batimentos prematuros. Miócitos cardíacos adultos são normalmente isolados a partir de pequenos animais por perfusão retrógrada do coração com toda a várias misturas de enzimas, uma técnica que produz rendimentos elevados de Ca 2 + as células tolerantes a 14. Isolamento de miócitos cardíacos de fragmentos de tecido é inerentemente menos bem sucedida, provavelmente devido ao acesso limitado de enzimas de miócitos individuais em comparação com a obtida por perfusão das artérias coronárias. Por causa da disponibilidade muito limitada de corações doados não utilizados, a única maneira prática de obter células ventriculares humanos normais em uma base regular é de digestio enzimátican dos fragmentos de tecido, muitas vezes muito pequenas excisadas durante procedimentos cirúrgicos eletivos. O único modelo de doença humana, que foi completamente caracterizada a nível celular é a insuficiência cardíaca terminal, devido à acessibilidade aos corações transplantados. No entanto, insuficiência cardíaca terminal ocorre numa minoria de pacientes e muitas vezes envolve uma via comum de remodelação grave de células do miocárdio, que é relativamente independente da causa subjacente 15. A capacidade de avaliar a função dos cardiomiócitos individuais de pacientes em um estágio não falha precoce da doença é fundamental para entender a fisiopatologia específica de diferentes condições hereditárias ou adquiridas. A cardiomiopatia hipertrófica (CMH) é um bom exemplo. HCM é uma comum (1/500 indivíduos) condição cardíaca hereditária caracterizada por hipertrofia cardíaca, aumento do risco e alterações contráteis arritmogénicos devido à obstrução da via de saída e disfunção diastólica 16. Cardiomiócitos de HCM corações undergo um processo de remodelação complexa, envolvendo mudanças na estrutura celular (hipertrofia, desarranjo miofibrilar) e EC-Coupling 17. No entanto, a maioria das informações de disfunção dos miócitos na CMH vem de modelos animais transgênicos. Uma vez que apenas uma minoria dos pacientes com CMH evolui para insuficiência cardíaca terminal e requer transplante cardíaco, HCM corações estão muito raramente disponíveis para o isolamento de células com métodos padrão. No entanto, pelo menos 30% dos portadores de CMH desenvolver sintomas obstrutivos, devido ao enorme fluxo de sangue do trato hipertrofia septal alterando fluxo durante a sístole (HCM) 18. A opção terapêutica disponível mais eficaz para o alívio da obstrução na CMH é a miectomia septal cirúrgica: durante o procedimento cirúrgico, uma parcela variável tamanho do septo superior é removido por abordagem da aorta trans. Esta porção do septo hipertrofiado é, por conseguinte, disponível para o isolamento de células a partir de tecido fresco. Um método para o isolamento de ventricula humanor miócitos de, pequenas amostras de biópsia endomiocárdica transvenous único previamente desenvolvido e publicado em 19. Implementamos um método para isolar miócitos septo individuais a partir de amostras do miocárdio ventricular de pacientes submetidos à cirurgia cardíaca, incluindo pacientes com CMH submetidos a miectomia septal e pacientes submetidos a procedimentos de substituição de válvulas. Para além de uma descrição detalhada do protocolo de isolamento, electrofisiológico representativa e Ca 2 + de fluorescência as medições são apresentados, demonstrando a viabilidade dos isolados miócitos ventriculares humanos e a viabilidade de patch-clamp e intracelulares de Ca 2 + estudos.

<table border="0" cellpadding="0" cellspacing="0" width="1059">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Potassium phosphate monobasic (KH2PO4)</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td style="width:139px;"><a href="http://www.sigmaaldrich.com/catalog/product/sigma/p9791?lang=it&amp;region=IT">P9791&#160;</a></td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium sulfate heptahydrate(MgSO4 * 7H2O)</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/m1880?lang=it&amp;region=IT">M1880&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/h3375?lang=it&amp;region=IT">H3375&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a9251?lang=it&amp;region=IT">A9251&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-(+)-Glucose</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/g8270?lang=it&amp;region=IT">G8270&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mannitol</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/m4125?lang=it&amp;region=IT">M4125&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taurine</td>
			<td style="width:172px;">Sigma-Aldrich</td>
			<td>T0625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium hydroxide (KOH)</td>
			<td>Sigma-Aldrich</td>
			<td>P5958</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s7653?lang=it&amp;region=IT">S7653</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/p9333?lang=it&amp;region=IT">P9333&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate dibasic (Na2HPO4)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s7907?lang=it&amp;region=IT">S7907&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s6297?lang=it&amp;region=IT">S6297&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium bicarbonate (KHCO3)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/237205?lang=it&amp;region=IT">237205</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/p2256?lang=it&amp;region=IT">P2256&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,3-Butanedione monoxime</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/b0753?lang=it&amp;region=IT">B0753&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium hydroxide(NaOH)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s8045?lang=it&amp;region=IT">S8045&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Glutamic acid monopotassium salt monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/fluka/49601?lang=it&amp;region=IT">49601</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pyruvic acid</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/aldrich/107360?lang=it&amp;region=IT">107360</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-Hydroxybutyric acid</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/aldrich/166898?lang=it&amp;region=IT">166898</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenosine 5&#8242;-triphosphate dipotassium salt dihydrate (K2-ATP)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a8937?lang=it&amp;region=IT">A8937</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Creatine</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/c0780?lang=it&amp;region=IT">C0780&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Succinic Acid</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/s3674?lang=it&amp;region=IT">S3674&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid (EGTA)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sial/e0396?lang=it&amp;region=IT">E0396&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin from bovine serum</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a0281?lang=it&amp;region=IT">A0281</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride (MgCl2)</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/m8266?lang=it&amp;region=IT">M8266&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase from Clostridium histolyticum, Type V</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/c9263?lang=it&amp;region=IT">C9263&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase, Bacterial, Type XXIV</td>
			<td>Sigma-Aldrich</td>
			<td>P8038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride solution, ~1 M in H2O</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/21115?lang=it&amp;region=IT">21115</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride 0.1 M solution</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/53704?lang=it&amp;region=IT">53704</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium methanesulfonate</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/aldrich/83000?lang=it&amp;region=IT">83000</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FluoForte Reagent</td>
			<td>Enzo Life Sciences</td>
			<td>ENZ-52015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Powerload concentrate, 100X</td>
			<td>Life Technologies</td>
			<td>P10020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perfusion Fast-Step System</td>
			<td>Warner Instruments</td>
			<td>VC-77SP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amphotericin B solubilized</td>
			<td>Sigma-Aldrich</td>
			<td><a href="http://www.sigmaaldrich.com/catalog/product/sigma/a9528?lang=it&amp;region=IT">A9528&#160;</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multiclamp 700B patch-clamp amplifier</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digidata 1440A</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pClamp10.0&#160;</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="108">
			<td height="108" style="height:108px;">Digestion Device</td>
			<td>CUSTOM</td>
			<td>CUSTOM</td>
			<td style="width:255px;">The device is custome made in our laboratory using plastic tubes, cast Sylgard and a motor; it is described in detail in Fig 1 C-D and in Fig.7. We can provide further details if requested</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone elastomer for the digestion device&#39;s brushes</td>
			<td>Dow Corning</td>
			<td>SYLGARD&#174; 184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Variable speed rotating motor for the digestion device</td>
			<td>Crouzet</td>
			<td>Crouzet 178-4765&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Mold for brushes casting</td>
			<td>N.A.</td>
			<td>N.A.</td>
			<td style="width:255px;">The mold is custom made from standard PTFE 2.5 cm diameter rods</td>
		</tr>
	</tbody>
</table>

isolation and functional characterization of human ventricular cardiomyocytes from fresh surgical samples

Cardiomiócitos de corações doentes são submetidos a processos de remodelação complexas que envolvem mudanças na estrutura celular, acoplamento excitação contração e correntes de íons de membrana. Essas mudanças tendem a ser responsáveis ​​pelo aumento do risco arritmogênica e as alterações contráteis levando à disfunção sistólica e diastólica em pacientes cardíacos. No entanto, a maior parte da informação sobre as alterações da função dos miócitos em doenças cardíacas veio a partir de modelos animais. Aqui vamos descrever e validar um protocolo para isolar miócitos viáveis ​​a partir de pequenas amostras cirúrgicas do miocárdio ventricular de pacientes submetidos a operações de cirurgia cardíaca. O protocolo está descrito em detalhe. Medições de cálcio e eletrofisiológicos intracelulares são relatados para demonstrar a viabilidade de uma série de medições de células individuais em cardiomiócitos ventriculares humanos obtidos com este método. O protocolo relatado elere pode ser útil para futuras investigações de base celular e molecular das alterações funcionais do coração humano, na presença de doenças cardíacas diferentes. Além disso, este método pode ser utilizado para identificar novos alvos terapêuticos a nível celular e para testar a eficácia de novos compostos em cardiomiócitos humanos, com valor de translação directa.

O método descrito acima foi utilizado para caracterizar as anormalidades funcionais de cardiomiócitos isolados do septo interventricular de pacientes com cardiomiopatia hipertrófica (CMH) que se submeteram à operação miectomia, em comparação com pacientes não falhando não hipertróficas cirúrgicos 21. Os resultados contidos nesta secção são derivados de trabalho 21 e que são mostrados aqui como um exemplo de como esta técnica pode ser utilizada para caracterizar as alterações da f...

Watch this Scientific Journal Video about Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples at JoVE.com

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

O conhecimento atual sobre a base celular de doenças cardíacas se baseia principalmente em estudos em modelos animais. Aqui nós descrevemos e validar um novo método para obter simples cardiomiócitos viáveis ​​a partir de pequenas amostras cirúrgicas do miocárdio ventricular humano. Miócitos ventriculares humanos podem ser usados ​​para estudos eletrofisiológicos e testes de drogas.

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 ...

isolation-functional-characterization-human-ventricular

Research

JoVE Journal

Medicine

17.1K visualizações.  University of Florence.  O conhecimento atual sobre a base celular de doenças cardíacas se baseia principalmente em estudos em modelos animais. Aqui nós descrevemos e validar um novo método para obter simples cardiomiócitos viáveis ​​a partir de pequenas amostras cirúrgicas do miocárdio ventricular humano. Miócitos ventriculares humanos podem ser usados ​​para estudos eletrofisiológicos e testes de drogas. 

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

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Cultura de Células e Isolamento paciente Derivado de CD133<sup&gt; +</sup&gt; Células-tronco de câncer putativos Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Medicina

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Isolamento de células-tronco cancerosas em amostras de câncer de próstata humano

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

Coleção, isolamento e Citometria de Fluxo de Análise de Amostras endocervix Humano

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

Chronic Thromboembolic Pulmonary Hypertension and Assessment of Right Ventricular Function in the Piglet

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. Pulmonary Hypertension (PH) was induced in 3-week-old piglets by a progressive obstruction of the pulmonary vascular bed. A ligation of the left Pulmonary Artery (PA) was performed first through a mini-thoracotomy. Second, weekly embolizations of the right lower pulmonary lobe were done under fluoroscopic guidance with n-butyl-2-cyanoacrylate during 5 weeks. Mean Pulmonary Arterial Pressure (mPAP) measured by ritght heart catheterism, increased progressively, as well as Right Atrial pressure and Pulmonary Vascular Resistances (PVR) after 5 weeks compared to sham animals. Right Ventricular (RV) structural and functional remodeling were assessed by transthoracic echocardiography (RV diameters, RV wall thickness, RV systolic function). RV elastance and RV-pulmonary coupling were assessed by Pressure-Volume Loops (PVL) analysis with conductance method. Histologic study of the lung and the right ventricle were also performed. Molecular analyses on RV fresh tissues could be performed through repeated transcutaneous endomyocardial biopsies. Pulmonary microvascular disease in obstructed and unobstructed territories was studied from lung biopsies using molecular analyses and pathology. Furthermore, the reliability and the reproducibility was associated with a range of PH severity in animals. Most aspects of the human CTEPH disease were reproduced in this model, which allows new perspectives for the understanding of the underlying mechanisms (mitochondria, inflammation) and new therapeutic approaches (targeted, cellular or gene therapies) of the overloaded right ventricle but also pulmonary microvascular disease.

Chronic Thromboembolic Pulmonary Hypertension (CTEPH) and Right Ventricular (RV) dysfunction were induced in piglets by progressive obstruction of the pulmonary arteries. Consequences were remarkably similar to those observed in CTEPH patients. This animal model would be a very useful tool for pathophysiology and therapeutic experiments on CTEPH and RV failure.

Tromboembólica crônica Hipertensão Pulmonar e Avaliação da Função Ventricular Direita no Piglet

An original piglet model of Chronic Thromboembolic Pulmonary Hypertension (CTEPH) associated with chronic Right Ventricular (RV) dysfunction is described. ...

Assessment of Sarcoplasmic Reticulum Calcium Reserve and Intracellular Diastolic Calcium Removal in Isolated Ventricular Cardiomyocytes

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum (SR) serves as a Ca2+ reservoir for contraction, which reuptakes intracellular Ca2+ during relaxation. The SR Ca2+ reserve available for beats is determinate for cardiac contractibility, and the removal of intracellular Ca2+ is critical for cardiac diastolic function. Under some pathophysiological conditions, such as diabetes and heart failure, impaired calcium clearance and SR Ca2+ store in cardiomyocytes may be involved in the progress of cardiac dysfunction. Here, we describe a protocol to evaluate SRCa2+ reserve and diastolic Ca2+ removal. Briefly, a single cardiomyocyte was enzymatically isolated, and the intracellular Ca2+ fluorescence indicated by Fura-2 was recorded by a calcium imaging system. To employ caffeine for inducing total SR Ca2+ release, we preset an automatic perfusion switch program by interlinking the stimulation system and the perfusion system. Then, the mono-exponential curve fitting was used for analyzing decay time constants of calcium transients and caffeine-induced calcium pulses. Accordingly, the contribution of the SR Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) to diastolic calcium removal was evaluated.

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Here, we describe a protocol to evaluate sarcoplasmic reticulum Ca2+ reserve and diastolic calcium removal function in cardiomyocytes by a calcium imaging system.

Avaliação da remoção de reserva e intracelular do cálcio diastólica cálcio sarcoplasmático em Cardiomyocytes Ventricular isolado

Intracellular calcium recycling plays a critical role in regulation of systolic and diastolic function in cardiomyocytes. Cardiac sarcoplasmic reticulum ...

Isolation and Cultivation of Adult Rat Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC).
The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells.
Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are &#946;-myosin heavy chain (&#946;-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

Here, we present a protocol for the isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). Isolated ARVC can be used for short and long-term cultivation. The isolation and cultivation of ARVC can play a key role in developing new treatment regimens for cardiac diseases.

Isolamento e cultivo de rato adulto Cardiomyocytes

In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

Desenvolvimento e caracterização funcional de células dendríticas Tolerogenic murino

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) handling and signaling pathways as well as high-throughput drug screening and toxicity assays. Herein, we provide a detailed description of the methodologies used to generate high-quality iPSC-CMs that can consistently reproduce molecular and functional characteristics across different cell lines. Additionally, a method is described to reliably assess their functional characterization through the evaluation of Ca2+ handling properties. Low oxygen (O2) conditions, lactate selection, and prolonged time in culture produce high-purity and high-quality ventricular-like cardiomyocytes. Similar to isolated adult rat cardiomyocytes (ARCMs), 3-month-old iPSC-CMs exhibit higher Ca2+ amplitude, faster rate of Ca2+ reuptake (decay-tau), and a positive lusitropic response to &#946;-adrenergic stimulation compared to day 30 iPSC-CMs. The strategy is technically simple, cost-effective, and reproducible. It provides a robust platform to model cardiac disease and for the large-scale drug screening to target Ca2+ handling proteins.

Here we describe and validate a method to consistently generate robust human induced pluripotent stem cell-derived cardiomyocytes and characterize their function. These techniques may help in developing mechanistic insight into signaling pathways, provide a platform for large-scale drug screening, and reliably model cardiac diseases.

Geração de cardiomiócitos derivados de HiPSC semelhantes ao Ventricular e preparações celulares de alta qualidade para caracterização de manuseio de cálcio

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) ...

Functional Characterization of Endogenously Expressed Human RYR1 Variants

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia susceptibility, core myopathies and centronuclear myopathy. Because of the diverse phenotypes linked to RYR1 mutations it is fundamental to characterize their functional effects to classify variants carried by patients for future therapeutic interventions and identify non-pathogenic variants. Many laboratories have been interested in developing methods to functionally characterize RYR1 mutations expressed in patients&#39; cells. This approach has numerous advantages, including: mutations are endogenously expressed, RyR1 is not over-expressed, use of heterologous RyR1 expressing cells is avoided. However, since patients may present mutations in different genes aside RYR1, it is important to compare results from biological material from individuals harboring the same mutation, with different genetic backgrounds. The present manuscript describes methods developed to study the functional effects of endogenously expressed RYR1 variants in: (a) Epstein Barr virus immortalized human B-lymphocytes and (b) satellite cells derived from muscle biopsies and differentiated into myotubes. Changes in the intracellular calcium concentration triggered by the addition of a pharmacological RyR1 activators are then monitored. The selected cell type is loaded with a ratiometric fluorescent calcium indicator and intracellular [Ca2+] changes are monitored either at the single cell level by fluorescence microscopy or in cell populations using a spectrofluorometer. The resting [Ca2+], agonist dose response curves are then compared between cells from healthy controls and patients harboring RYR1 variants leading to insight into the functional effect of a given variant.

Here methods used to study the functional effect of RYR1 mutations endogenously expressed in Epstein Barr Virus immortalized human B-lymphocytes and muscle biopsy derived satellite cells differentiated into myotubes are described.

Caracterização funcional de variantes ryr1 humanas expressas e endógenas

More than 700 variants in the RYR1 gene have been identified in patients with different neuromuscular disorders including malignant hyperthermia ...

Modifications of the Langendorff Method for Simultaneous Isolation of Atrial and Ventricular Myocytes from Adult Mice

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction and electrical activity. Hence, isolating viable, high-quality cardiomyocytes from the heart is the initial and most crucial experimental step. Comparing the various protocols for isolating the cardiomyocytes of adult mice, the Langendorff retrograde perfusion is the most successful and reproducible method reported in the literature, especially for isolating ventricular myocytes. However, isolating quality atrial myocytes from the perfused heart remains challenging, and few successful isolation reports are available. Solving this complicated problem is extremely important because apart from ventricular disease, atrial disease accounts for a large part of heart diseases. Therefore, further investigations on the cellular level to reveal the mechanisms are warranted. In this paper, a protocol based on the Langendorff retrograde perfusion method is introduced and some modifications in the depth of aorta cannulation and the steps that may affect the digestion process to isolate atrial and ventricular myocytes were simultaneously made. Moreover, the isolated cardiomyocytes are confirmed to be amenable to patch clamp investigation.

This protocol describes modifications of the Langendorff method including the depth of aorta cannulation for simultaneous Isolation of atrial and ventricular myocytes from adult mice.

Modificações do Método Langendorff para Isolamento Simultâneo de Miócitos Atrial e Ventricular de Camundongos Adultos

A single cardiomyocyte is a vital tool in the cellular and subcellular level studies of cardiac biology and diseases as a fundamental unit of contraction ...