This work was supported by funding from American Heart Association 12BGIA12040477, NC State University Chancellor&#8217;s Faculty Excellence Program, and National Natural Science Foundation of China H020381370216.

El siguiente protocolo está diseñado para el aislamiento de cardiomiocitos de una camada de cachorros de rata neonatal (10-14 PUP). Si el tamaño de camada es significativamente diferente, el procedimiento puede tener que ser reducido para compensar. Los cachorros deben ser alrededor de 48 ± 6 horas de edad. Todos los procedimientos en este documento han sido aprobados por la Universidad de Carolina del Norte Estado Institucional Cuidado de Animales y el Comité de Uso (IACUC). 1. Preparación celda de aislamiento NOTA: Realice los siguientes pasos el día antes del aislamiento. <ol><li> Autoclave los siguientes: grandes tijeras, tijeras pequeñas, pequeñas pinzas cortantes, grandes pinzas. Use un ciclo de la gravedad durante 60 minutos a un mínimo de 121 ° C y un tiempo de secado de 30 min. </li><li> Colocar 40 ml de solución de tripsina 0,1% en el refrigerador para descongelar O / N. Refrigere de Hank sales equilibradas Solution (HBSS) si no está ya frío. Preparar una solución madre de los medios de comunicación 20% de suero bovino fetal (FBS). <ol><li> En 400 ml de Iscmedios modificado de Dulbecco de OVE (IMDM) añadir 100 ml de FBS, 5 ml L-glutamina (200 mM), 2,5 ml de gentamicina (10 mg ml -1), y 0,9 ml de 2-mercaptoetanol. Esterilizar mediante filtro de vacío. De la tienda a 4 ° C durante un máximo de 3 semanas y esto es escalable para volúmenes más pequeños o más grandes. Diluir 20% de FBS Stock medios de comunicación 1: 1 con IMDM para producir 10% de FBS NRCM medios de comunicación. Sólo hacer en lotes pequeños para evitar el calentamiento y enfriamiento de los medios de comunicación repetida. </li></ol></li></ol> 2. celda de aislamiento Día 1 <ol><li> Realice el siguiente trabajo en una campana de cultivo para evitar la contaminación de las muestras. Para mejor momento comenzar este trabajo de la tarde ya que hay un / incubación O N. </li><li> Preparar espacio de trabajo dentro de una campana de cultivo estéril. Coloque la almohadilla de banco en campana de cultivo. Llene dos vasos de precipitados de 250 ml con 70% de etanol y colocar herramientas quirúrgicas en ellos. Coloque pequeño cubo de hielo en la campana. Asegúrese de que sea lo suficientemente grande como para contener varios tubos y un plato de 150 mm. UV esterilizar el capó for 10 min. Colocar 40 ml de 0,1% de tripsina en hielo. </li><li> Recuperar crías de ratas neonatales de la madre. Para mantener a los animales caliente hasta su uso, colocarlos en un recipiente aislado, como un cubo de hielo envuelto en una plataforma de banca para mantenerlos calientes. </li><li> Llenar una placa de cultivo de 150 mm con 25 ml de HBSS y colocar en hielo. Separar 2-3 gasas y lugar en el área de trabajo. Sosteniendo cachorro por la piel en la parte posterior del cuello, rociar con etanol al 70% y el lugar en el capó. NOTA: Para mantener mejor la esterilidad, esto se puede hacer por otra persona que luego coloca el cachorro limpiado en el capó. </li><li> Mantenga el cachorro por la piel en la parte posterior del cuello. Luego, utilizando las tijeras grandes, en un rápido corte fuerte, la eutanasia por decapitación. Seque la sangre con una gasa. NOTA: Debido a las variaciones en los requerimientos IACUC, puede ser más apropiado para limpiar el cachorro con una mezcla de etanol en lugar de limpiar rociándolo. Asegúrese de consultar con la universidad IACUC como a sus directrices para este paso. </li><li> Continuar reduciendo cara anterior del chest a través de la caja torácica. Apriete la piel en el cuello para ayudar a abrir la caja torácica hasta que el corazón es visible. Usando las tijeras pequeñas, retire el corazón y colocar en la placa de cultivo HBSS en hielo. Repita el procedimiento para los otros cachorros. </li><li> Retire cualquier tejido no corazón de las muestras y haga girar la HBSS suavemente para limpiar los corazones. Transferir el tejido del corazón a otra placa de cultivo que contiene frescos HBSS y lavar aún más el tejido. Cortar el tejido más hasta que las piezas son de 1-3 mm 3, y luego traslado a la solución de tripsina. Coloque solución de tripsina en eje de balancín en 4 ° C refrigerador O / N. </li></ol> Aislamiento de células Día 2 NOTA: mientras se aspira en los pasos siguientes, utilizar una pipeta en lugar de una línea de vacío. El tejido se mueve con facilidad, y una línea de vacío va a obstruir o remover el tejido todavía contiene células. Lo mejor es usar una pipeta de aspiración en su lugar. Si el tejido entra en la pipeta, pipetas atrás para extraerla. <ol><li> Haga talícuotas res de 10% NRCM Medios: una con 25 ml y dos con 15 ml. Coloque un tubo de 25 ml de medios de comunicación en 37 ° C baño de agua. Añadir 4 ml de HBSS a 5 diferentes 15 ml tubos cónicos y colocar todo en hielo. Etiqueta de cada 15 ml tubo # 1-5. </li><li> Añadir 40 ml de HBSS a un tubo cónico de 50 ml. Añadir 10 ml de HBSS a la colagenasa, una pipeta de suspender, y combinan las soluciones para crear 50 ml de 1 mg ml -1 solución de colagenasa. Esterilizar usando 0,22 micras filtro de vacío, y almacenar a temperatura ambiente. </li><li> Recuperar tubo de tejido / tripsina corazón. Asegúrese de que la tripsina es clara, y el tejido parece esponjoso. Deje que el tejido se deposite en la esquina de tubo y aspirar todo el líquido posible. </li><li> Añadir 25 ml tibia 10% de FBS NRCM medios de comunicación para el tubo y agitar a mano. Cap y girar en 37 ° C baño de agua durante 2 min. Aspirar el líquido de nuevo. El tejido debe flotar en este paso; Si es así, aspirado desde el fondo del tubo. </li><li> Añadir 10 ml de colagenasa. Gire en baño de agua durante 2 minutos o hasta que la solución está turbia. Quickly aspirar el líquido. Añadir 10 ml de colagenasa reciente al tubo que contiene el tejido y girar en baño de agua durante 2 min. Pipeta para mezclar, solución de transferencia de tubo # 1, y el lugar en el hielo. Puede que no sea posible eliminar todo el líquido. Repetir los pasos 3.2 a 3.6 para los otros tres tubos de 15 ml cónicos. Transferir el líquido restante en el tubo # 5. </li><li> Centrífuga de tubos de 15 ml a 410 rcf durante 8 min. Mientras centrifugación, colocar 15 ml de 10% de tubo medios FBS NRCM en baño de agua. Coloque 40 micras filtro de células en la parte superior de un tubo cónico de 50 ml y mojado con 2 ml de HBSS frío. </li><li> Aspirar el sobrenadante de las células centrifugadas. Añadir 6 ml de HBSS fría a cada tubo y resuspender. Suspensiones de células de la pipeta a través del filtro en 50 ml cónica para retirar cualquier tejido. Enjuague lados de cada tubo de 15 ml con 3 ml de HBSS frío y añadir al colador. Centrifugar las células a 410 rcf durante 10 min. Aspirar el sobrenadante. Resuspender pellet de células en 10 ml 10% de FBS NRCM medios de comunicación y la transferencia de T-75 matraz. Enjuague cónica con medios de 5 ml y añadir a flpreguntar. </li><li> Incubar durante 1 h a 37 ° C y 5% de CO 2. Coloque el pasado 15 ml 10% de tubo medios FBS NRCM en baño de agua. Transferir contenido del T-75 a un nuevo matraz T-175, enjuague T-75 con 15 ml de medio y añadir a T-175. Incubar durante 1 hora. </li></ol> 4. Criopreservación <ol><li> Coloca medio de congelación en el hielo. Traslado medio celular de T-175 a 50 ml tubo cónico, y recoger 10 l de suspensión celular para celular recuento usando azul tripán. Centrifugar a 410 rcf durante 5 min. Mientras centrifugación, contar las células utilizando un contador de células. </li><li> Aspirar los medios de cultivo y las células volver a suspender en medio de congelación a una concentración de 2 a 4.000.000 de células por ml. Colocar 1 ml de suspensión celular en vial criogénico, luego ubicar los viales una velocidad controlada de refrigeración congelación contenedor, y el lugar en el congelador a -80ºC O / N. Dentro de 1-2 días, transferir viales en nitrógeno líquido. </li></ol> 5. Las células de descongelación <ol><li> Escudo plato en la fibronectina mediante la disolución de 250 l60; de la fibronectina (concentración madre de 1 mg ml -1) en 9,75 ml de agua (concentración final 0,025 mg ml -1). Agregue suficiente solución para cubrir la placa de cultivo y se incuba a 37 ° C durante al menos 30 minutos. <ol><li> En cuatro de 15 ml tubos cónicos, hacer las mezclas DMSO / medios de la siguiente añadiendo DMSO al 20% de Medios. Añadir (5%) 9,5 ml de medio con 500 l de DMSO. Añadir (2,5%) 9,75 ml de medio con 250 l de DMSO. Añadir 10 ml de medio (hacer dos de ellos). </li></ol></li><li> Calentar las mezclas de los medios mencionados en baño a 37 ° C. Eliminar las células de nitrógeno líquido y colocar en hielo seco. Cuando mezclas de medios de comunicación son cálidos, descongelar las células en 37 ° C baño de agua hasta que estén descongelados. No deje las células en un baño de agua durante demasiado tiempo, ya que puede causar daño a las células. </li><li> Transferir contenido del vial criogénico al 5% tubo DMSO / media. Centrifugar a 410 rcf durante 8 min. Aspirar el sobrenadante dejando sedimento celular. Añadir el contenido de 2,5% tubo DMSO / media a teléfonos de pellets y resuspend. Centrifugar a 410 rcf durante 8 min. Aspirar el sobrenadante dejando sedimento celular. </li><li> Añadir el contenido de un 0% tubo DMSO / media a la celda de pellets y resuspender. Centrifugar a 410 rcf durante 8 min. Aspirar el sobrenadante dejando sedimento celular. </li><li> Añadir el contenido de segunda 0% DMSO / medios de comunicación para sedimento celular y resuspender. Tome pequeña muestra para el recuento celular. Centrifugar a 410 rcf durante 8 min. Mientras centrifugación células recuento en un hemocitómetro usando azul tripán para marcar las células muertas. Cantidad de células Record, así como la viabilidad. </li><li> Aspirar el sobrenadante dejando sedimento celular. Resuspender en 10% NRCM medios (opcional: añadir 100 mM bromodesoxiuridina (BrdU)) y células de la placa en placas recubiertas de fibronectina. </li></ol> 6. Cultivo Celular <ol><li> Células de la placa en unos 100 mil células por cm 2. Ajustar la densidad de placas basado en el uso previsto de la cultura. Preparar 2% NRCM medios de comunicación mediante la dilución de 20% de medios en IMDM en una proporción de 1:10. El siguiente es un resumen de una cultura típica. &lt;/ Li&gt; <li> El día 1, enjuagar las células muertas de distancia con IMDM tibia, agregue 10% NRCM (opcional 100 mM BrdU). El día 2, enjuagar las células muertas de distancia con IMDM tibia, agregue 10% NRCM. Día 3-añadir 2% NRCM. El día 4 añadir 2% NRCM. El día 5 aseguran que las células son confluentes y paliza. Mantener las células en cultivo por alrededor de una semana. En ese momento, los fibroblastos pueden comenzar a superar significativamente los NRMCR. </li></ol>

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The authors have nothing to disclose.

Este protocolo permite a los NRMCR a ser aislados, criopreservadas y descongeladas. Descongelación de las células es una parte crucial del procedimiento. Una serie de diluciones de DMSO se utiliza para eliminar lentamente DMSO de las células 14. Es importante que la extracción de DMSO realizarse rápidamente ya que las células son particularmente sensibles a morir inmediatamente después de la descongelación. Si se requieren células más o menos para la descongelación, los volúmenes de las soluciones...

El cultivo celular de los cardiomiocitos es una herramienta fundamental en la investigación cardiaca moderna. Cardiomiocitos de rata neonatal (NRMCR) son de uso general ya que el aislamiento y la cultura es más fácil que la de los cardiomiocitos de rata adulta 1. El método NRCM todavía tiene varias limitaciones, incluyendo un procedimiento de aislamiento de largo y la proliferación celular limitado en el plato. Hay numerosos protocolos para el aislamiento de NRMCR con la mayoría requiriendo generalmente 4-48 hr de trabajo 2-6. Además, las células se aíslan con frecuencia de 1 a cachorros de 2 días de edad de rata 2,4-7; el momento del nacimiento puede ser impredecible y el conflicto con otros trabajos en el laboratorio. Los aislamientos pueden ser ineficiente y derrochador si se necesita sólo una pequeña cantidad de células para los experimentos. La mayoría de los esfuerzos en mejorar el enfoque del flujo de trabajo en la reducción del tiempo de aislamiento, sin embargo, esto no resuelve el problema de medir el tiempo del nacimiento de las crías. Como alternativas, muchos laboratorios utilizancardiomiocitos derivados de células madre embrionarias (CES) o las células madre pluripotentes inducidas (iPSCs). Sin embargo, el proceso de reprogramación y / o diferenciación puede ser muy lento y costoso también. No puede haber otros problemas al utilizar estas células como modelos miocitos in vitro. Ambos cardiomiocitos derivados Esc- y iPSC- han demostrado que presentan diferencias en la electrofisiología de cardiomiocitos primarios 8-10. NRMCR disociadas son capaces de ser almacenado durante varios días utilizando refrigeración 11, sin embargo, esto no permiten el almacenamiento a largo plazo. El nitrógeno líquido se utiliza típicamente para preservar las células de un mayor período de tiempo, sino que requiere un crioprotector tal como sulfóxido de dimetilo (DMSO). Investigaciones anteriores han demostrado que la concentración ideal entre 5-10% de DMSO en los medios de congelación permite la criopreservación de NRMCR, pero aun así la viabilidad sigue siendo baja 12. Aunque DMSO ayuda a proteger las células durante gratuitazing, puede ser tóxico para las células a concentraciones por encima de 1,5% 13. Estudios anteriores han demostrado que la eliminación lentamente DMSO de las células, puede mejorar la viabilidad celular 14. Hemos tratado de mejorar la eficiencia de los ensayos basados ​​en células NRCM por criopreservar las células después del aislamiento. Esto permite que las células se descongelaron y se utilizan cuando sea necesario, reduciendo la frecuencia de aislamientos y el consumo de los animales. Usando este método, se muestra que es posible criopreservar NRMCR y descongelar para su uso en un momento posterior. Después de descongelar las células mantienen una viabilidad aceptable y producen culturas NRCM que son positivas para α-actinina sarcomérica (α-SA) y el contrato de forma espontánea.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>IMDM (+25mM HEPES +L-glutamine)</td>
			<td>Gibco</td>
			<td>12440-046</td>
			<td>With added 25mM HEPES and L-glutamine</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Hyclone</td>
			<td>SH30070.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Gibco</td>
			<td>15710-064</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (+Ca +Mg)</td>
			<td>Corning</td>
			<td>21-020-CV</td>
			<td>With added calcium and magnesium, pH 7.1-7.4</td>
		</tr>
		<tr>
			<td>Trypsin 0.25%</td>
			<td>Gibco</td>
			<td>25300-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.05%</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostor CS5</td>
			<td>BioLife Solutions</td>
			<td>205102</td>
			<td>Freezing media</td>
		</tr>
		<tr>
			<td>Cryogenic Vial</td>
			<td>Corning</td>
			<td>430659</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase</td>
			<td>Sigma</td>
			<td>C1889-50MG</td>
			<td></td>
		</tr>
		<tr>
			<td>40&#956;m Cell Strainer</td>
			<td>Greiner Bio-One</td>
			<td>542040</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilizing Vacuum Filter (0.22&#956;m)</td>
			<td>Corning</td>
			<td>431118</td>
			<td></td>
		</tr>
		<tr>
			<td>50mL Conical</td>
			<td>Corning</td>
			<td>430828</td>
			<td></td>
		</tr>
		<tr>
			<td>15mL Conical</td>
			<td>Corning</td>
			<td>430790</td>
			<td></td>
		</tr>
		<tr>
			<td>trypan blue</td>
			<td>Cellgro</td>
			<td>25-900-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Mr Frosty Freezing Container</td>
			<td>ThermoScientific</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Millicell EZ&#160;SLIDES</td>
			<td>Millipore</td>
			<td>PEZGS0416</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-sarcomeric actinin antibody</td>
			<td>Sigma</td>
			<td>A7811</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Corning</td>
			<td>356008</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromodeoxyuridine</td>
			<td>BD Biosciences</td>
			<td>51-7581KZ</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and cryopreservation of neonatal rat cardiomyocytes

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is difficult and time consuming. The most commonly used cells are neonatal rat cardiomyocytes (NRCMs), which require isolation every time cells are needed. The birth of the rats can be unpredictable. Cryopreservation is proposed to allow for cells to be stored until needed, yet freezing/thawing methods for primary cardiomyocytes are challenging due to the sensitivity of the cells. Using the proper cryoprotectant, dimethyl sulfoxide (DMSO), cryopreservation was achieved. By slowly extracting the DMSO while thawing the cells, cultures were obtained with viable NRCMs. NRCM phenotype was verified using immunocytochemistry staining for &#945;-sarcomeric actinin. In addition, cells also showed spontaneous contraction after several days in culture. Cell viability after thawing was acceptable at 40-60%. In spite of this, the methods outlined allow one to easily cryopreserve and thaw NRCMs. This gives researchers a greater amount of flexibility in planning experiments as well as reducing the use of animals.

Después del aislamiento de los cardiomiocitos de rata neonatal, las células se congelaron en nitrógeno líquido, y se pueden almacenar durante al menos varios meses. Típicamente después de la descongelación, la viabilidad se determinó por análisis de azul de tripano será de entre 40-60%. Aunque esto es más bajo que otros tipos de células, con la siembra y la cultura propia de las células se proliferan (figura 1). Con el fin de verificar la contractilidad, las células pueden ser imágenes ut...

Watch this Scientific Journal Video about Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes at JoVE.com

Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

El aislamiento y criopreservación de rata neonatal cardiomiocitos

Cell culture has become increasingly important in cardiac research, but due to the limited proliferation of cardiomyocytes, culturing cardiomyocytes is ...

isolation-and-cryopreservation-of-neonatal-rat-cardiomyocytes

Research

JoVE Journal

Developmental Biology

12.4K vistas.  College of Veterinary Medicine, North Carolina State University. The isolation of neonatal rat cardiomyocytes is a time consuming and unpredictable procedure. This study describes methods for cryopreservation and thawing of neonatal rat cardiomyocytes that allows for more efficient use of cells. The thawed NRCMs can be used for various experiments without the need for performing isolations each time.

Video: Isolation and Cryopreservation of Neonatal Rat Cardiomyocytes

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Aislamiento de células neurales madre / progenitoras de la región periventricular de la rata de Adultos y de la médula espinal humana

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Investigación

Biología del Desarrollo

Isolation and Characterization of Satellite Cells from Rat Head Branchiomeric Muscles

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent problems in speech, swallowing, and sucking. In vitro culture systems that allow the study of satellite cells (myogenic stem cells) from head muscles are crucial to develop new therapies based on tissue engineering to promote muscle regeneration after surgery. These systems will offer new perspectives for the treatment of cleft palate patients. A protocol for the isolation, culture and differentiation of satellite cells from head muscles is presented. The isolation is based on enzymatic digestion and trituration to release the satellite cells. In addition, this protocol comprises an innovative method using extracellular matrix gel coatings of millimeter size, which requires only low numbers of satellite cells for differentiation assays.

This protocol describes the isolation of satellite cells from branchiomeric head muscles of a 9 week-old rat. The muscles originate from different branchial arches. Subsequently, the satellite cells are cultured on a spot coating of millimeter size to study their differentiation. This approach avoids the expansion and passaging of satellite cells.

Aislamiento y caracterización de las células satélite de Músculos Rata Head Branchiomeric

Fibrosis and defective muscle regeneration can hamper the functional recovery of the soft palate muscles after cleft palate repair. This causes persistent ...

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative strategies for treatment of heart disease. This conversion has been achieved by forced expression of defined factors such as Gata4 (G), Mef2c (M) and Tbx5 (T). Traditionally, iCMs are generated by a cocktail of viruses expressing these individual factors. However, reprogramming efficiency is relatively low and most of the in vitro G,M,T-transduced fibroblasts do not become fully reprogrammed, making it difficult to study the reprogramming mechanisms. We recently have shown that the stoichiometry of G,M,T is crucial for efficient iCM reprogramming. An optimal stoichiometry of G,M,T with relative high level of M and low levels of G and T achieved by using our polycistronic MGT vector (hereafter referred to as MGT) significantly increased reprogramming efficiency and improved iCM quality in vitro. Here we provide a detailed description of the methodology used to generate iCMs with MGT construct from cardiac fibroblasts. Isolation of cardiac fibroblasts, generation of virus for reprogramming and evaluation of the reprogramming process are also included to provide a platform for efficient and reproducible generation of iCMs.

We describe here a protocol for the generation of iCMs using retrovirus-mediated delivery of Gata4, Tbx5 and Mef2c in a polycistronic construct. This protocol yields a relatively homogeneous population of reprogrammed cells with improved efficiency and quality and is valuable for future studies of iCM reprogramming.

Mejora de la generación de cardiomiocitos inducida uso de un constructo Polycistronic Expresando relación óptima de Gata4, MEF2C y Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

Aislamiento de las células murinas embrionarias Hemogenic endoteliales

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks&#39; Balanced Salt Solution (HBSS) at 37 &#176;C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 - 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca2+) and magnesium (Mg2+)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 - 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

El aislamiento efectivo de los islotes funcionales de páncreas de ratón neonatales

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories ...

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

La visualización de las variaciones del ciclo celular y la determinación de la nucleación en cardiomiocitos postnatales

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

Bottlenose Dolphin (Tursiops truncatus) Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to laboratories for studies to further our understanding of marine mammal reproduction. Heterologous IVF, a replacement for homologous IVF, could provide a means to test the sperm fertility potential; to study gamete physiology and early embryo development; and to avoid the use of valuable dolphin oocytes, which are difficult to obtain. Here, we present protocols that have been successfully used to collect and cryopreserve dolphin spermatozoa. The collection of semen is performed by manual stimulation on trained dolphins. Cryopreservation is accomplished using a TRIS egg-yolk based extender with glycerol. In addition, we present a protocol that describes heterologous IVF using dolphin spermatozoa and bovine oocytes and that verifies the hybrid nature of the resulting embryo using PCR. Heterologous fertilization raises questions on fertilization and can be used as a tool to study gamete physiology and early embryo development. In addition, the success of heterologous IVF demonstrates the potential of this technique to test dolphin sperm fertilizing capacity, which is worth further examination.

Bottlenose Dolphin Tursiops truncatus Spermatozoa: Collection, Cryopreservation, and Heterologous In Vitro Fertilization

Here, we present protocols that have been successfully used for dolphin spermatozoa collection, cryopreservation, and heterologous IVF performance using bovine oocytes.

Tonina Delfín (Tursiops truncatus) espermatozoides: recolección, criopreservación y fecundación In Vitro heteróloga

The use of cryopreserved dolphin spermatozoa facilitates the exchange of genetic material between aquatic parks and makes spermatozoa accessible to ...

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their potential applications in cardiac research, including drug discovery, disease modeling, tissue engineering, and regenerative medicine. However, cells produced by existing protocols display a range of immaturity compared with native adult ventricular cardiomyocytes. Many efforts have been made to mature hPSC-CMs, with only moderate maturation attained thus far. Therefore, an engineered system, called biowire, has been devised by providing both physical and electrical cues to lead hPSC-CMs to a more mature state in vitro. The system uses a microfabricated platform to seed hPSC-CMs in collagen type I gel along a rigid template suture to assemble into aligned cardiac tissue (biowire), which is subjected to electrical field stimulation with a progressively increasing frequency. Compared to nonstimulated controls, stimulated biowired cardiomyocytes exhibit an enhanced degree of structural and electrophysiological maturation. Such changes are dependent upon the stimulation rate. This manuscript describes in detail the design and creation of biowires.

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

La maduración de madre humanas cardiomiocitos derivado de células en Biowires uso de la estimulación eléctrica

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in the elderly and end-stage renal disease (ESRD) patients, who commonly suffer from hyperphosphatemia and hypercalcemia. At present, there are no medication therapies that can stop its progression. The mechanisms that underlie this pathological process remain unclear. The aortic valve leaflet is composed of a thin layer of valve endothelial cells (VECs) on the outer surfaces of the aortic cusps, with valve interstitial cells (VICs) sandwiched between the VECs. The use of a rat model enables the in vitro study of ectopic calcification based on the in vivo physiopathological serum phosphate (Pi) and calcium (Ca) levels of patients who suffer from hyperphosphatemia and hypercalcemia. The described protocol details the isolation of a pure rat VIC population as shown by the expression of VIC markers: alpha-smooth muscle actin (&#945;-SMA) vimentin and tissue growth factor beta (TGF&#946;) 1 and 2, and the absence of cluster of differentiation (CD) 31, a VEC marker. By expanding these VICs, biochemical, genetic, and imaging studies can be performed to study and unravel the key mediators underpinning CAVD.

This protocol describes the isolation, culture, and calcification of rat-derived valve interstitial cells, a highly physiological in vitro model of calcific aortic valve disease (CAVD). Exploitation of this rat model facilitates CAVD research in exploring the cell and molecular mechanisms that underlie this complex pathological process.

Células intersticiales de la válvula de aislamiento y caracterización de rata principal: un nuevo modelo para el estudio de calcificación de la válvula aórtica

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in ...

Handling and Treatment of Male European Eels (Anguilla anguilla) for Hormonal Maturation and Sperm Cryopreservation

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and maturation. As of yet, weekly injections of human chorionic gonadotropin (hCG) have proved to maturate males after just 5-6 weeks of treatment, producing high volumes of high-quality sperm during several weeks. In addition, sperm cryopreservation protocols using different extenders, cryoprotectants and cooling and thawing times have been previously described for European eel. Here, we show that Tanaka&#180;s extender solution can be directly used for fertilization or for cryopreservation, making unnecessary the usage of different types of solutions and dilutions. Furthermore, the use of methanol as a cryoprotectant makes this protocol easy to use as methanol has low toxicity and does not activate the sperm. The sperm does not need to be cryopreserved immediately after the addition of the cryoprotectant, and it can be used long after being thawed. Moreover, sperm motility is still high after thawing although it is lower than that of fresh sperm. The aim of this work is to show the best available protocol for European eel handling, maturation, and sperm cryopreservation.

Handling and Treatment of Male European Eels Anguilla anguilla for Hormonal Maturation and Sperm Cryopreservation

Protocols for European eel maturation and sperm cryopreservation have been improved over the last years. This article describes the best protocol available using human chorionic gonadotropin (hCG) for inducing maturation and methanol as cryoprotectant.

Manejo y tratamiento del hombre europeo anguilas (Anguilla anguilla) de maduración Hormonal y criopreservación de esperma

During the last years, several research groups have been working on the development and improvement of new protocols for the European eel handling and ...