Este trabajo fue apoyado por la Unión Europea (PEIF Proyecto 241577 &quot;gran corazón&quot;, 7 º Programa Marco Europeo, CP), Menarini Internacional Luxemburgo Operaciones (AM), Teletón GGP07133 (CP) y Gilead Sciences (AM).

Los protocolos experimentales sobre el tejido humano fueron aprobados por el comité de ética de la Universidad de Careggi Hospital (2006/0024713; renovada mayo de 2009). Cada paciente dio su consentimiento informado por escrito. 1. Soluciones y Preparación del equipo Las soluciones se describen en la Tabla 1. Un diagrama de flujo simplificado del procedimiento de aislamiento de células se encuentra en la Figura 1. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td colspan="2"> Solución </td><td> CP </td><td> DB </td><td> KB </td><td> Tuberculosis </td><td> PS </td><td> EB1 </td><td> EB2 </td></tr><tr><td rowspan="23"> Reactivo (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> glucosa </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> MgCl 2 </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> De 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> La colagenasa Tipo V </td><td></td><td></td><td></td><td></td><td></td><td> 250 &lt;/ Td&gt; <td> 250 </td></tr><tr><td> Proteasa 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> 7,3 NaOH </td><td> 7.1 KOH </td><td> 7.35 NaOH </td><td> 7.2 KOH </td><td> 7,3 NaOH </td><td> 7,3 NaOH </td></tr></tbody></table> . Tabla 1 Las soluciones utilizadas para la recogida de muestras, el aislamiento de células y caracterización funcional de los miocitos CP = solución cardiopléjica.; DB = tampón de disociación; KB = solución de Kraft-Brühe; TB = tampón de Tyrode; PS = solución de la pipeta; EB1 = tampón de enzima 1; EB2 = tampón de enzima 2. <ol><li> Preparar cardioplégica solución (CP). Solución de CP se puede almacenar a 4 ° C durante hasta 1 semana. </li><li> Preparar Ca 2 + libre de tampón de disociación (DB). Esta solución se debe usar en el día. </li><li> Preparar Kraft-Brühe (KB) solución. Solución KB se puede almacenar a 4 ° C durante hasta 1 Week. </li><li> Preparar Ca 2 +-tampón Tyrode libre (TB). Esta solución se debe usar en el día. </li><li> Filtrar todas las soluciones usando filtros de jeringa antes de su uso. </li><li> Preparar el dispositivo de digestión (Figura 2), un recipiente de raspado hecho de dos cepillos enfrentadas de elastómero de silicona, uno de los cuales hace girar por un motor eléctrico. El dispositivo de la digestión es por encargo. Los detalles sobre el dispositivo de la digestión son en la Figura 8; imágenes del dispositivo son en las figuras 2 C y 2D. Lavar la cámara de tejido con etanol al 70% y agua. </li></ol> 2. Recopilación y procesamiento de muestras de miocardio <ol><li> Verter 40 ml de solución cardiplegic (CP) en un tubo de 50 ml y almacenarlo en el hielo para el transporte de muestras desde la sala operativa para el laboratorio de aislamiento de células. </li><li> Recoger la muestra de miocardio ventricular de la sala operatoria inmediatamente después de la escisión, lávelo con heladaSolución de PC y guárdelo en el tubo. Utilice especímenes endocárdicos escindió del tabique ventricular entre superior durante la cirugía a corazón abierto, la ponderación&gt; 100 mg. </li><li> Rápida transferencia de la muestra al área de laboratorio; iniciar el procesamiento de la muestra dentro de los 10 min de la escisión de la muestra. </li><li> Mientras se mantiene la muestra en tampón enfriado con hielo CP, retire con cuidado la capa fibrótica del endocardio con tijeras finas bajo un microscopio estereoscópico; después, cortar el tejido miocárdico para piezas pequeñas (2-3 mm de largo). Dependiendo de tamaño de la muestra de tejido, cortar una cantidad total de miocardio ventricular entre 100 mg y 1 g para cada aislamiento. </li><li> Al terminar de picar carne de tejidos, la transferencia de los trozos de miocardio en el dispositivo de la digestión, con hielo limpio solución CP frío. Evitar llenar todo el volumen entre los dos cepillos de silicio (3-4 ml) con trozos de miocardio, usando no más que 1 g de tejido total. </li></ol> 3. Lavado y digestión de Miocardio Trozos <ol><li> En popaer los trozos se transfieren a la cámara de raspado del dispositivo de digestión, cambiar el tampón CP en la cámara de frío Ca 2 +-tampón de disociación libre (DB). </li><li> Coloque el dispositivo de digestión en un baño termostático, a fin de que la cámara para estar en contacto con el agua caliente en el baño (Figura 1). Ajuste el baño maría a 37,5 ° C y encenderlo, a fin de elevar lentamente la temperatura de la cámara de tejido. Encienda el motor del dispositivo de la digestión, el establecimiento de la velocidad de rotación a 1 revolución / segundo. </li><li> Realizar 3 ciclos de lavado con DB, cambiando la solución en la cámara limpia con DB cada 8 min. La DB se calienta (37 ° C) y oxígeno saturado antes de entrar en contacto con los trozos de miocardio. </li><li> Preparar tampón de enzima 1 (EB1) mediante la adición de 250 U / ml de colagenasa Tipo V y 4 U / ml de proteasa tipo XXIV solución DB. Prepare el tampón de la enzima 2 (EB2) mediante la adición de 250 U / ml de colagenasa tipo V a la solución de base de datos. Calentamiento (37 ° C) y oxygenate EB1 y EB2. </li><li> Realizar dos ciclos de 12 min de la digestión en el dispositivo de la digestión de rotación con 100% EB1 oxigenada (a 37 ° C). En cada ciclo, usar ~ 3 ml de EB1. Eliminar la solución por aspiración de la pipeta y desecharla después de cada ciclo. </li><li> Preparar 6 tubos de 15 ml para la recogida de células y ~ 80 ml de solución KB frío (4 ° C) para eluir los tampones. </li><li> Lleve a cabo un primer ciclo de digestión de 15 minutos con 3 ml 100% EB2 oxigenada a 37 ° C. Después de que el ciclo de digestión, recoger la solución que contiene los primero miocitos disociadas en un tubo de 15 ml y diluir la suspensión celular con 12 ml de solución KB frío. Guarde la plana tubo a temperatura ambiente. </li><li> Diluir la solución EB2 restante con una cantidad igual de DB con el fin de reducir a la mitad la concentración de colagenasa V para los siguientes ciclos de digestión. </li><li> Lleve a cabo otros ciclos 5 12 min de digestión con 3 EB2 ml a 37 ° C; después de cada uno de ellos recoger de los miocitos que contiene tampón en un tubo cónico de 15 ml y sediluir con 12 ml de solución de KB. Guarde los tubos de células que contiene 6 a temperatura ambiente durante 30 min. </li></ol> 4. Resuspensión celular y Ca 2 + Readaptación <ol><li> Añadir 1 mg de albúmina / ml de suero bovino (BSA) a 20 ml de tampón de Ca 2 +-Tyrode libre (TB). Se filtra la solución. </li><li> Centrifugar los seis tubos cónicos de miocitos que contiene a 100 xg durante 5 minutos a la fuerza para resolver los miocitos. Eliminar el sobrenadante y resuspender las células en cada tubo con una cantidad variable (1-3 ml, dependiendo del rendimiento) de BSA que contiene la TB a TA. </li><li> Aumente gradualmente concentración de Ca2 + en el tampón de células que contiene añadiendo pequeñas alícuotas de 100 mmol / L de solución de CaCl2. En los primero y segundo pasos concentración de Ca2 + se eleva hasta 50 mmol / L y 100 mmol / L, respectivamente. Los siguientes pasos Ca 2 + de adición se llevan a cabo cada 5 min y la concentración se eleva por 100 mol / L en cada paso tOA concentración final de 0,9 mmol / L. </li><li> Evaluar el rendimiento del procedimiento de aislamiento. Transferir 0,5 ml de solución de los miocitos que contiene en la cámara de fondo de vidrio de un microscopio. Evalúe 15 campos microscópicos aumento de 10x objetivo y calcular el porcentaje de miocitos sanos (por ejemplo, células con forma de bastón con estrías claras y sin inclusiones importantes, Figura 2). El rendimiento esperado es de alrededor de 20%. </li></ol> 5. Evaluación funcional de los cardiomiocitos aislados. El siguiente protocolo es un ejemplo de la evaluación funcional de los cardiomiocitos humana incluyendo registros simultáneos de los potenciales de acción y intracelular de Ca 2 + flujos. <ol><li> Preparar la solución de la pipeta (PS) para los experimentos de patch clamp en la configuración de parche perforado. La solución puede ser almacenada a -20 ° C durante hasta 3 meses. </li><li> Añadir 1,8 mmol / L CaCl2 al Ca 2 + tampón Tyrode libre (TB). Usí esta solución para superfusion de los cardiomiocitos durante los experimentos de patch clamp / fluorescencia. </li><li> Transferencia de 1 ml de suspensión celular a un tubo de 1,5 ml y añadir 10 mmol / L de Fluoforte y 10 l Powerload Concentrado. Incubar durante 30 minutos a temperatura ambiente. A continuación, ajuste el tubo en posición vertical y salir de la celda que conformarse con 5 min; resuspender las células en Ca 2 + que contiene la tuberculosis. </li><li> Transferir 0,25 ml de suspensión de células a una pequeña (0,5 ml), microscopio de temperatura controlada montado cámara de registro, superfused por gravedad con un sistema de microperfusor se calentó a una velocidad de flujo de 0,3 ml / min (temperatura: 37 ± 0,5 ° C). </li><li> El uso de un extractor de micropipeta, preparar pipetas patch clamp con un diámetro de la punta de 3 a 5 micras y una resistencia de 3 a 4,5 M cuando se llena con PS. </li><li> Añadir anfotericina B a un lote de PS (250 mg / ml) y utilizarla para rellenar los electrodos. </li><li> Seleccione una celda en forma de barra con estrías claras, libre de inclusiones, form el sello giga y esperar 5 a 10 minutos, hasta que la resistencia de acceso es inferior al 20 MΩ. </li><li> Obtener los potenciales de acción en el modo de pinza de corriente utilizando pulsos cortos (&lt;3 ms) a diferentes frecuencias de estimulación (0,2 Hz, 0,5 Hz y 1 Hz, 1 min en cada frecuencia). Durante la fase de grabación, encender iluminación de campo brillante a 492 ± 3 nm y detectar Fluoforte fluorescencia a 505-520 nm. Adquirir la fluorescencia y la membrana señales potenciales utilizando Digidata 1440A y software de ordenador pClamp 10.0. Repita la secuencia de la grabación varias veces si es necesario; sin embargo, mantener el tiempo de grabación total por debajo de 15 min para cada celda. </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>

Los autores declaran que no tienen intereses financieros en competencia.

Hemos descrito y validado un método para aislar miocitos viables a partir de muestras quirúrgicas de miocardio ventricular humano. A partir de los protocolos descritos anteriormente que había sido utilizado con éxito para células aisladas a partir de muestras quirúrgicas auriculares, la técnica para permitir la separación de los miocitos viables individuales de miocardio ventricular enferma fue desarrollado y afinado. Los primeros informes mostraron que el aislamiento de los cardiomiocitos individuales de trozos...

La disección de las propiedades electrofisiológicas del miocardio ha progresado notablemente después el desarrollo de técnicas para el solo aislamiento de miocitos cardiacos. También se han hecho posible los avances recientes en la comprensión de la excitación cardíaca contracción de acoplamiento (EC-Acoplamiento) por la capacidad de aislar los cardiomiocitos viables individuales que conservan todas las propiedades fisiológicas del tejido intacto. Métodos de fijación Patch se emplean rutinariamente para estudiar la función y la modulación farmacológica de las corrientes de iones sarcolema cardíaco. Grabaciones de la dinámica del calcio intracelular con Ca 2 + tintes sensibles también se llevan a cabo regularmente en los miocitos cardíacos individuales de una variedad de modelos sanos y enfermos, proporcionar datos vitales sobre la fisiología de la CE-acoplamiento, así como en las alteraciones patológicas de la intracelular de Ca 2 + homeostasis que conlleva un deterioro mecánico y aumento de la carga de las enfermedades cardíacas arritmogénica. Información de estos estudios es fundamental para la comprensión de los efectos mecánicos y electrofisiológicos de las drogas en el ámbito clínico. Sin embargo, hay diferencias entre especies específicas en las corrientes transmembrana y en las proteínas CE-acoplamiento que dan cuenta de las características específicas de potencial de acción cardíaco y la mecánica cardíaca. Por lo tanto, mientras que los estudios de miocitos aislados de mamíferos no humanos han dilucidado las propiedades biofísicas y funciones fisiológicas de los canales iónicos transmembrana específicos y proteínas CE-acoplamiento, que no proporcionan necesariamente modelos pertinentes de miocitos cardiacos humanos. Por lo tanto, el aislamiento de miocitos viables de miocardio humano es esencial para entender completamente la fisiopatología de las enfermedades cardíacas y validar nuevos enfoques terapéuticos. Tejido auricular humano es fácilmente disponible como apéndices auriculares son a menudo descartados durante los procedimientos quirúrgicos. Estudios cuantitativos iniciales de los potenciales de acción cardiaco humanos adultos y cu iónicarrents emplean células auriculares enzimáticamente aislados 1-4. Las grabaciones de los potenciales o corrientes a partir de células humanas adultas ventriculares aislados de acción han sido posteriormente reportado 3,5-10. La mayoría de estos estudios se han utilizado células obtenidas de corazones explantados y utilizados ya sea colagenasa de perfusión de un segmento de la arteria coronaria o la exposición de cantidades relativamente grandes de tejido extirpado a la colagenasa para obtener células aisladas. Estos estudios permiten una caracterización detallada de un número de corrientes de iones transmembrana de los cardiomiocitos ventriculares humanos de corazones sanos y de pacientes con insuficiencia cardiaca terminal. Grabaciones de L-tipo Ca 2 + actual (I Ca-L) 5-7, corriente hacia el exterior de potasio transitoria (I) 8, la corriente de entrada del rectificador de potasio (I κ1) 8, los diferentes componentes de la corriente de potasio rectificadora retardada (I κ ) 9 han sido reportados. Avances y refinación deel procedimiento de aislamiento 10, permite una caracterización clara de la base iónica del aumento del potencial arritmogénica en la insuficiencia cardíaca terminal, que comprende la acción potencial de prolongación 11, retrasó después de despolarizaciones 12 y aumentó divertido corriente 13 que conduce a la despolarización diastólica y latidos prematuros. Miocitos cardíacos adultos normalmente están aislados de animales pequeños por perfusión retrógrada de todo el corazón con diferentes mezclas de enzimas, una técnica que produce altos rendimientos de Ca 2 +-células tolerantes 14. Aislamiento de miocitos cardiacos a partir de fragmentos de tejido es inherentemente menos éxito probablemente a causa de la limitación del acceso de las enzimas a miocitos individuales en comparación con la alcanzada por la perfusión de las arterias coronarias. Debido a la limitada disponibilidad de los corazones de donantes no utilizadas, la única forma práctica de obtener células ventriculares humanos normales sobre una base regular es por digestio enzimátican de los fragmentos muy pequeños a menudo tejidos extirpados durante los procedimientos quirúrgicos electivos. El único modelo de enfermedad humana que se ha caracterizado a fondo a nivel celular es la insuficiencia cardíaca terminal, debido a la accesibilidad a los corazones trasplantados. Sin embargo, la insuficiencia cardíaca terminal se produce en una minoría de los pacientes y a menudo implica una vía común de la remodelación severa de las células del miocardio, que es relativamente independiente de la causa subyacente 15. La capacidad de evaluar la función de los cardiomiocitos individuales de los pacientes en una etapa anterior no no de la enfermedad es crucial para entender la patofisiología específica de diferentes enfermedades hereditarias o adquiridas. La miocardiopatía hipertrófica (MCH) es un ejemplo elocuente. HCM es un frecuente (1/500 personas) condición cardiaca hereditaria caracterizada por hipertrofia cardiaca, aumento de alteraciones de riesgo y contráctiles arritmogénicas debido a la obstrucción del tracto de salida y la disfunción diastólica 16. Los cardiomiocitos de corazones HCM undergo unos complejos procesos de remodelación que implican cambios en la estructura celular (hipertrofia, desorganización miofibrilar) y CE-Acoplamiento 17. Sin embargo, la mayoría de la información de la disfunción de los miocitos en HCM ha venido de modelos animales transgénicos. Dado que sólo una minoría de pacientes con MCH evoluciona hacia la insuficiencia cardíaca terminal y requiere de un trasplante cardíaco, el corazón de HCM son muy rara vez están disponibles para el aislamiento de células con los métodos estándar. Sin embargo, al menos el 30% de los pacientes con MCH presentan síntomas obstructivos debido al flujo de sangre del tracto de salida alteración hipertrofia septal masiva durante la sístole (HCM) 18. La opción terapéutica disponible más eficaz para el alivio de la obstrucción en la MCH es la miectomía septal quirúrgica: durante este procedimiento quirúrgico, una porción de tamaño variable del tabique superior se elimina por vía aórtica trans. Esta porción de tabique hipertrofiado es, por tanto, disponible para el aislamiento de células del tejido fresco. Un método para el aislamiento de ventricula humanar miocitos de pequeñas muestras endomiocárdicas individuales, transvenosos biopsia se ha desarrollado y publicado previamente 19. Hemos implementado un método para aislar miocitos septales individuales de las muestras de miocardio ventricular de los pacientes sometidos a cirugía cardíaca, incluyendo pacientes con HCM sometidos miectomía septal y los pacientes sometidos a procedimientos de reemplazo de válvulas. Además de una descripción detallada del protocolo de aislamiento, electrofisiológicos representativa y Ca 2 + fluorescencia se presentan las mediciones, lo que demuestra la viabilidad de los miocitos ventriculares aislados humanos y la viabilidad de patch clamp e intracelulares de Ca 2 + Estudios.

<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

Los cardiomiocitos de corazones enfermos son sometidos a procesos de remodelación de complejos que implican cambios en la estructura celular, contracción de acoplamiento excitación y corrientes iónicas de membrana. Esos cambios son probable que sea responsable del aumento del riesgo arritmogénica y las alteraciones contráctiles conducen a la disfunción sistólica y diastólica en pacientes cardíacos. Sin embargo, más información sobre las alteraciones de la función de los miocitos en enfermedades cardíacas ha llegado a partir de modelos animales. Aquí describimos y validar un protocolo para aislar miocitos viables desde pequeñas muestras quirúrgicas de miocardio ventricular de los pacientes sometidos a operaciones de cirugía cardiaca. El protocolo se describe en detalle. Mediciones electrofisiológicas y de calcio intracelulares se presentan para demostrar la viabilidad de un número de mediciones de células individuales en cardiomiocitos ventriculares humanos obtenidos con este método. El protocolo informó élre pueden ser útiles para futuras investigaciones sobre las bases celulares y moleculares de las alteraciones funcionales del corazón humano en la presencia de diferentes enfermedades cardiacas. Además, este método puede ser usado para identificar nuevas dianas terapéuticas a nivel celular y para probar la eficacia de nuevos compuestos en cardiomiocitos humanos, con valor de traslación directa.

Se empleó el método descrito anteriormente para caracterizar las anormalidades funcionales de cardiomiocitos aislados del tabique interventricular de pacientes con miocardiopatía hipertrófica (MCH) que se sometieron a la operación miectomía, en comparación con los pacientes no quirúrgicos que fallan no hipertróficas 21. Los resultados que figuran en esta sección se derivan de que el trabajo 21 y se muestra aquí como un ejemplo de cómo esta técnica se puede utilizar para caracterizar la...

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

Los conocimientos actuales sobre las bases celulares de las enfermedades cardiacas se basa principalmente en estudios sobre modelos animales. Aquí describimos y validar un nuevo método para obtener cardiomiocitos viables individuales de pequeñas muestras quirúrgicas de miocardio ventricular humano. Miocitos ventriculares humanos pueden ser utilizados para estudios electrofisiológicos y las pruebas de drogas.

Aislamiento y caracterización funcional de Human ventricular cardiomiocitos a partir de muestras quirúrgicas recientes

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 vistas.  University of Florence.  Los conocimientos actuales sobre las bases celulares de las enfermedades cardiacas se basa principalmente en estudios sobre modelos animales. Aquí describimos y validar un nuevo método para obtener cardiomiocitos viables individuales de pequeñas muestras quirúrgicas de miocardio ventricular humano. Miocitos ventriculares humanos pueden ser utilizados para estudios electrofisiológicos y las pruebas de drogas. 

Video: 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).

Paciente celulares derivadas Cultura y aislamiento de CD133<sup&gt; +</sup&gt; Putativos células madre del cáncer de melanoma

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

Investigación

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.

El aislamiento de las células madre del cáncer de muestras de cáncer 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.

Colección, Aislamiento, y citometría de flujo análisis de las muestras endocervicales 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.

Hipertensión pulmonar tromboembólica crónica y Evaluación de la función ventricular derecha en el 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.

Evaluación del retiro de reserva e intracelular calcio diastólico calcio de retículo sarcoplásmico en cardiomiocitos ventriculares aisladas

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.

Aislamiento y cultivo de cardiomiocitos de rata adulta

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.

Desarrollo y caracterización funcional de células dendríticas tolerogénicas 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.

Generación de cardiomiocitos derivados de HiPSC ventricular y preparaciones celulares de alta calidad para la caracterización del manejo del calcio

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.

Caracterización funcional de variantes de RYR1 humanas expresadas endógenamente

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.

Modificaciones del método de Langendorff para el aislamiento simultáneo de miocitos auriculares y ventriculares de ratones 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 ...