Esta investigación es apoyada por la Fundación Hartwell, la Asociación Americana del Corazón (Desarrollo Científico Subvención # 0830384N) y la Fundación Leducq: 07CVD04 mitral.

1. Preparación <ol><li> Incubar codorniz fértiles o huevos de gallina para la 14 ª etapa - (alrededor de 2 días) o 25 (alrededor de 4,5 días) en 60% de humedad y 37 º C. </li><li> Prepare la solución de Earl estéril de salina balanceada (EBSS) <ol><li> Añadir 100 ml EBSS 10 veces a 600 ml de agua 18 mW </li><li> Añadir 2,2 g de bicarbonato sódico </li><li> Ajustar el pH de la solución a 7,2 </li><li> Llevar la solución a 1000 ml </li><li> Esterilizar la solución al pasar por un filtro de 0,2 micras </li></ol></li><li> Prepare estéril M199 medio de cultivo. <ol><li> Añadir un paquete de polvo de M199 a 700 ml de agua 18 mW. </li><li> Añadir 2,2 g de bicarbonato sódico </li><li> Ajustar el pH de la solución a 7,2. </li><li> Agregar 10 ml (1%) penicilina-estreptomicina </li><li> Llevar la solución a 1000 ml </li><li> Esterilizar la solución al pasar por un filtro de 0,2 micras </li><li> Añadir 1 ml estéril 100X insulina-transferrina-selenio-G suplemento </li><li> Agregar 10 ml (1%) de suero de pollo estéril </li></ol></li><li> Prepare tres dimensiones geles de colágeno a una concentración de colágeno de 1,5 mg / ml. Trabajos anteriores en nuestro laboratorio han demostrado que una concentración de colágeno de más de 1,5 mg / ml proporciona una matriz que es demasiado rígido biomecánicamente para que las células se mueven a través. Las concentraciones de gel de colágeno inferior a 1,5 mg / ml tienen las fibras de colágeno tan pocos que el grupo de células se pueden triturar las fibras a nivel local, produciendo una isla de las células. <ol><li> Agregue el siguiente heladas reactivos a un tubo de centrífuga estéril 15 ml con el fin de: <ul><li> 3X M199: Volumen = volumen total necesario / 3 </li><li> Estéril 18 mW de agua: Volumen = volumen total necesario (M199 volumen + volumen suero de pollo + 0,1 M NaOH + volumen de volumen de colágeno) </li><li> Suero de pollo estéril: Volumen = volumen total necesario * 0.01 </li><li> Rata cola de colágeno I: Volumen = (concentración de gel de colágeno * volumen total necesario) / colágeno concentración de las </li><li> Estéril 0,1 M de NaOH: Volumen = volumen de colágeno * 0,2 Nota: La cantidad de agua, 0,1 M NaOH, y el colágeno añadido variará dependiendo de la concentración de la solución de colágeno. </li></ul></li><li> Mezclar la solución de colágeno y con una pipeta estéril. </li><li> Inmediatamente la transferencia de 0,3 ml de la solución de colágeno en 1,9 cm 2 pozos de crecimiento de la zona. Esta es la solución de colágeno suficiente para cubrir completamente el bien y para proporcionar un gel que es de aproximadamente 3 mm de espesor. Un espesor de gel de al menos 250 micras es necesario para los estudios de la invasión celular. </li><li> Deje que la solución de colágeno a gel por lo menos durante 1 hora a 37 ° C, 5% de CO 2, y 100% de humedad. </li></ol></li></ol> 2. Pre-EMT endocárdica aislamiento de células y Cultura <ol><li> Embrión de aislamiento <ol><li> Coloque una pieza de pulgada de largo de la cinta de laboratorio en el extremo más grande del huevo. Aquí es donde la cámara de aire se encuentra. La cinta se estabiliza la frágil cáscara de huevo de codorniz. </li><li> Suavemente la punción de la zona grabada del huevo en la cámara de aire con la punta de esterilizar tijeras curvas. </li><li> Corte un agujero en la parte grabada de un huevo, que es lo suficientemente grande como para permitir que la yema de pasar a través. Iniciar el agujero en la perforación y corte hacia el exterior en forma de espiral. Asegúrese de cortar a través de la membrana externa, pero evite perforar la yema de huevo. </li><li> Cuando el agujero en la capa está completa, visualizar el embrión de codorniz. El embrión se encuentra normalmente en la parte superior de la yema. </li><li> Comenzar a verter la yema a través del corte agujero en el huevo con una mano. Mantenga una pinza 55 en la otra mano. A medida que el embrión sale del huevo en la parte superior de la yema, deslice suavemente las pinzas # 55 en el embrión y sacarlo de la yema. Nota: Para los huevos de gallina, el aislamiento de embriones consiste en descifrar el huevo de gallina en una superficie fuerte, limpio y romper el huevo en un plato de Petri de 100 mm. El embrión debe estar ubicado en la parte superior de la yema. Deslice suavemente # 55 pinzas en el embrión y sacarlo de la yema. </li><li> Lugar al embrión en un 100 mm placa de Petri que contiene el hielo frío, EBSS estéril. </li></ol></li><li> Etapa los embriones de acuerdo con los criterios de Hamburger y Hamilton 1. Embriones en la etapa 14 - se encuentran en estadio últimos 13, pero aún no en la etapa 15, con 20-21 somitas y una yema de cola. Una característica clave de la 14 ª etapa - los embriones es el ángulo de la cabeza, que es ligeramente superior a 90 º con el cuerpo. </li><li> Quitar el corazón a partir de embriones de codorniz en la etapa 14 - el n º 55 pinzas de corte transversal donde el canal AV y OFT en contacto con la pared torácica. Coloque los corazones en un lado de la placa de Petri. </li><li> Recoge los corazones aislados con una pipeta de transferencia estéril y se colocan en un nuevo plato de 100 mm de Petri llenas de estériles, helada EBSS. </li><html><li> Cortar el OFTs y canales AV transversal de los ventrículos por el n º 55 pinzas. </li><li> La sección de los canales restantes AV y / o tractos de salida (OFTs) longitudinalmente. </li><li> Establecer una pipeta un volumen de 20 uL. Use esta pipeta para aspirar seis longitudinal de corte canales AV y OFTs. </li><li> Expulsar a los canales de AV y OFTs en el gel de colágeno. Aspirar cualquier EBSS el exceso de la superficie del gel. </li><li> Use # 55 pinzas para orientar a los explantes de lo que son planas y por lo tanto el lado del lumen se enfrenta el gel de colágeno. Trate de no perforar el gel de colágeno con las pinzas. </li><li> Después de 2 horas a 37 ° C y 5% de CO 2, quite el miocardio de los explantes por el n º 55 pinzas. Las células endocárdicas debe quedarse atrás en la superficie del gel de colágeno. Por otra parte, el miocardio se puede dejar en el explante. Con la quita de miocardio, las células del endocardio con no someterse a EMT, sin intervención. Con el miocardio presentan un subconjunto de las células endocárdicas, se transformará en mesénquima, pero el grado de transformación puede ser modulada a factores externos. Nota: Aunque el miocardio se quedará en el explante se debe permitir por lo menos 2 horas para insertarse en el gel antes de medio, se añade. </li><li> Añadir 0,4 ml de medio M199 explantes cada uno, así que lo contiene. </li><li> Cultivo de las células a 37 ° C, 5% de CO 2, y 100% de humedad. </li></ol> 3. Después de la EMT AV aislamiento Cojín de células mesenquimales y Cultura <ol><li> Romper el huevo de gallina en una superficie fuerte, limpio y romper el huevo en un plato de Petri de 100 mm. El embrión debe estar ubicado en la parte superior de la yema. Levante el embrión con # 5 pinzas por el acaparamiento de las membranas que rodean al embrión y luego colocar el embrión en un 100 mm placa de Petri llena, heladas EBSS estéril. </li><li> Etapa los embriones de acuerdo con los criterios de Hamburger y Hamilton 1. Características de una etapa de 25 embriones son diferentes codo y articulaciones de la rodilla y la pigmentación ocular leve. </li><li> Aislar a los corazones de todos los embriones HH25 y colocar el corazón en un lado de la placa de Petri. </li><li> Recoge los corazones aislados con una pipeta de transferencia estéril y se colocan en un nuevo plato de 100 mm de Petri llenas de estériles, helada EBSS. </li><li> Aislar a la región AV de todos los corazones a la vez. Coloque la AV en un lado de la placa de Petri. Agitar suavemente la región AV para quitar toda la sangre, que pueden afectar la viabilidad celular. </li><li> Recoge los aislados AV con una pipeta de transferencia estéril y se colocan en un nuevo plato de 100 mm de Petri llenas de estériles, helada EBSS. </li><li> Secuencialmente aislar a los cojines de la AV. Asegúrese de que no hay presencia de miocardio en los cojines de AV. </li><li> Recoger los cojines aislados con una pipeta de transferencia estéril y se colocan en un tubo de centrífuga estéril 15 ml. </li><li> Girar los cojines de la parte inferior del tubo de centrífuga de 15 g durante 3 minutos. </li><li> Utilice una punta estéril 1.000 ml para eliminar la mayor cantidad de sobrenadante EBSS como sea posible sin molestar a los cojines. </li><li> Añadir 1 ml de 0,25% de tripsina-EDTA que ha sido pre-calentado a 37 ° C. Permita que los cojines para la incubación de la tripsina hasta que se disuelva por completo, lo cual es normalmente alrededor de 3-5 minutos. </li><li> Añadir 100 ml de suero de pollo al tubo de centrífuga para saciar la tripsina. </li><li> Que sedimenten las células a 160 xg durante 5 minutos. Eliminar el sobrenadante con una punta estéril, l 1000. </li><li> Resuspender las células en 2 ml de medio M199 y mezclar con la pipeta. Tomar 10 l de la suspensión celular a contar con hemocitómetro y determinar el número total de células que han sido aislados </li><li> Determinar el número de geles de colágeno se hará en función del número de células aisladas. Las células necesitan para ser recubierto con una densidad de 2 x 10 5 células / ml con un volumen de 250 l de gel. </li><li> Que sedimenten las células a 160 xg durante 5 minutos. Eliminar el sobrenadante con una punta estéril, l 1000. </li><li> Utilizando el protocolo mencionados, añadir 3X M199, agua, suero de pollo, el colágeno, y 0,1 M de NaOH, en ese orden, para el pellet de células. Mezclar por pipeteo suave. </li><li> Añadir 250 l de cada área de crecimiento de 1,9 cm 2 también. </li><li> Deje que la solución de colágeno a gel por lo menos durante 1 hora a 37 ° C, 5% de CO 2, y 100% de humedad. </li><li> Añadir 0,4 ml de medio de cultivo e incubar durante la noche. </li><li> Para continuar con el cultivo de los geles en condiciones de estrés, liberar a los geles de los lados de la cultura y con una punta de la pipeta estéril 200 mL. Culturas limitado mecánicamente son geles que se mantienen unidos a los lados de la cultura también. </li></ol> 4. Resultados representante <img src="/files/ftp_upload/2159/2159fig1.jpg" alt="Figura 1" /> Figura 1:. Ejemplos de embriones (A) embrión HH14-codorniz, y (B) HH25 embrión de pollo se muestran aquí. fig2.jpg &quot;alt =&quot; Figura 2 &quot;/&gt; Figura 2: HH14-válvula de explantes endocárdica después de 2 horas de cultivo (A) con el presente miocardio, o (B) con el miocardio eliminado.. <img src="/files/ftp_upload/2159/2159fig3.jpg" alt="Figura 3" /> Figura 3:. HH14 válvulas endocárdicas explantes (A) Con la presente miocardio, muchas de las células se someten a la transformación del endocardio y mesenquimales tienen una forma de huso, fenotipo mesenquimal después de 48 horas de cultivo. (B) Cuando el miocardio se elimina todas las células de mantener un adoquín en forma de fenotipo endocárdica después de 48 horas en la cultura. <img src="/files/ftp_upload/2159/2159fig4.jpg" alt="Figura 4"/> Figura 4:. Células HH25 las células mesenquimales de la válvula en un gel de colágeno se redondean inmediatamente después de la siembra. <img src="/files/ftp_upload/2159/2159fig5.jpg" alt="Figura 5"/> Figura 5:. HH25 células mesenquimales en cultivo (A) Después de 48 horas de cultivo en un gel de colágeno limitado el HH25 células mesenquimales están empezando a extenderse. (B) HH25 células en un gel de colágeno y libre de estrés siete días después de la siembra y 6 días después de la liberación han compactado el gel a aproximadamente el 50% de la superficie original.

<ol>
	<li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> J Morphol. 88, 49-92 (1951).</li><li>Runyan, R. B., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasion+of+mesenchyme+into+three-dimensional+collagen+gels:+A+regional+and+temporal+analysis+of+interaction+in+embryonic+heart+tissue.">Invasion of mesenchyme into three-dimensional collagen gels: A regional and temporal analysis of interaction in embryonic heart tissue.</a> Developmental Biology. 95, 108-108 (1983).</li><li>Butcher, J. T., Norris, R. A., Hoffman, S., Mjaatvedt, C. H., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periostin+promotes+atrioventricular+mesenchyme+matrix+invasion+and+remodeling+mediated+by+integrin+signaling+through+Rho/PI+3-kinase.">Periostin promotes atrioventricular mesenchyme matrix invasion and remodeling mediated by integrin signaling through Rho/PI 3-kinase.</a> Developmental Biology. 302, 256-256 (2007).</li></ol>

El método para aislar células del endocardio de la etapa HH14 - corazones desarrollado originalmente por Runyan y Markwald proporciona un entorno controlado, en el entorno in vitro para estudiar los factores que inician y modular embrionarias EMT 2. HH14 - explantes cultivados sin endocardio miocardio no someterse a EMT, sin intervención biomecánicas o bioquímicas. Si el miocardio se deja en el explante se dará la señal de un subconjunto de las células endocárd...

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<td>Extra fine Bonn scissors, curved</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14085-08</td>
<td>&nbsp;</td>
<tr>
<td>Dumont tweezers #55</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>14099</td>
<td>&nbsp;</td>
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<td>Dumont tweezers #5</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Inc.</td>
<td>14098</td>
<td>&nbsp;</td>
<tr>
<td>Sterile transfer pipettes</td>
<td>&nbsp;</td>
<td>Samco Scientific </td>
<td>2041S</td>
<td>&nbsp;</td>
<tr>
<td>4-well culture plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>176740</td>
<td>&nbsp;</td>
<tr>
<td>Sterile disposable filter units, PES, 0.2 &mu;m pore size membrane</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>566-0020</td>
<td>&nbsp;</td>
<tr>
<td>Sterile 15 mL centrifuge tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>430828</td>
<td>&nbsp;</td>
<tr>
<td>Sterile petri dishes, 100 mm x 15 mm</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>25384-342</td>
<td>&nbsp;</td>
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<td>Laboratory tape</td>
<td>&nbsp;</td>
<td>VWR International</td>
<td>89097-920</td>
<td>&nbsp;</td>
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<td>Rat-tail collagen I</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>354236</td>
<td>&nbsp;</td>
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<td>10X Earl's Balanced Salt Solution</td>
<td>&nbsp;</td>
<td>Quality Biological, Inc.</td>
<td>119-064-131</td>
<td>&nbsp;</td>
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<td>M199 powder</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>31100-035</td>
<td>&nbsp;</td>
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<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
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<td>Insulin-Transferrin-Selenium-G supplement (100X)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>41400-045</td>
<td>&nbsp;</td>
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<td>Chicken serum</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>16110-082</td>
<td>&nbsp;</td>
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<td>0.25% Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25200-056</td>
<td>&nbsp;</td>
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<td>Sodium bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S5761</td>
<td>&nbsp;</td>
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<td>Sodium hydroxide solution, 1.0 N</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>S2770</td>
<td>&nbsp;</td>
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<td>Fertile quail eggs (Coturnix coturnix)</td>
<td>&nbsp;</td>
<td>Lake Cumberland Game Bird Farm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fertile chicken eggs (Gallus gallus)</td>
<td>&nbsp;</td>
<td>Cornell University Poultry Farm</td>
<td>&nbsp;</td>
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isolation and culture of avian embryonic valvular progenitor cells

Formación adecuada y la función de las válvulas del corazón embrionario es crítica para la progresión del desarrollo. El corazón embrionario temprano es un tubo en forma de U del endocardio rodeado de miocardio. El miocardio secreta gelatina cardíaca, una matriz rica en ácido hialurónico gelatinosa, en el nódulo auriculoventricular (AV) y el tracto de salida (OFT) del lumen. En la etapa HH14 valvulogenesis comienza cuando un subconjunto de las células endocárdicas, recibir señales de miocardio, endocardio someterse a la transformación de mesenquimales (EMT), e invadir la gelatina cardíaca. En la etapa HH25 los cojines son totalmente valvular mesenchymalized, y esto es lo que finalmente mesénquima forma el aparato valvular y del tabique del corazón. Comprensión de los mecanismos que inician y modulan el proceso de diferenciación celular y la EMT son importantes debido a su conexión con graves defectos congénitos del corazón. En este estudio se presentan los métodos para aislar pre-EMT endocárdica y después de la EMT células mesenquimales, que son los dos diferentes fenotipos de células del cojín prevalvular. Pre-EMT endocardial células pueden ser cultivadas con o sin el miocardio. Después de la EMT células mesenquimales colchón AV pueden ser cultivadas en el interior mecánicamente restringido o sin estrés geles de colágeno. Estas 3D en modelos in vitro imitar los principales eventos morfogénicos valvular y son útiles para la deconstrucción de los mecanismos de valvulogenesis etapa temprana y tardía.

Watch this Scientific Journal Video about Isolation and Culture of Avian Embryonic Valvular Progenitor Cells at JoVE.com

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

En este artículo se proporciona un método para aislar y cultivar la codorniz o pollo HH14<sup&gt; -</sup&gt; Válvula de células del endocardio y HH25 células mesenquimales amortiguador de la válvula.

Aislamiento y cultivo de células embrionarias progenitoras aviar valvular

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

isolation-and-culture-of-avian-embryonic-valvular-progenitor-cells

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Biology

10.1K vistas.  Cornell University. En este artículo se proporciona un método para aislar y cultivar la codorniz o pollo HH14 - Válvula de células del endocardio y HH25 células mesenquimales amortiguador de la válvula.

Video: Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons 

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the cerebellar neuronal types (granule cells, Purkinje cells and inhibitory interneurons), granule neurons are by far the most numerous and are the most abundant type of neurons in the mammalian brain. In rodents, cerebellar granule neurons are generated during the first two post-natal weeks from progenitor cells in the outermost layer of the cerebellar cortex, the external granule layer (EGL). The protocol presented here describes techniques to enrich and culture granule neurons and their progenitor cells from post-natal mouse cerebellum. We will describe procedures to obtain cultures of increasing purity3,4, which can be used to study the differentiation of proliferating progenitor cells into granule neurons5,6. Once the progenitor cells differentiate, the cultures also provide a homogenous population of granule neurons for experimental manipulation and characterization of phenomena such as synaptogenesis, glutamate receptor function7, interaction with other purified cerebellar cells8,9 or cell death7.

Isolation and Culture of Post-Natal Mouse Cerebellar Granule Neuron Progenitor Cells and Neurons

Here we present a method to isolate and culture cerebellar granule neuron progenitor cells and cerebellar granule neurons from postnatal mouse.

Aislamiento y cultivo de Post-Natal células de ratón cerebelosa gránulo Progenitor neuronas y las neuronas

The cerebellar cortex is a well described structure that provides unique opportunities for studying neuronal properties and development1,2. Of the ...

Investigación

Biología

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

La cultura y el mantenimiento de células estaminales embrionarias humanas

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

El aislamiento y la expansión a gran escala de células humanas adultas Colonia progenitoras endoteliales formación

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

El aislamiento y la derivación de células embrionarias de ratón Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adultas y embrionarias del músculo esquelético Cultura Microexplant y aislamiento de células madre de músculo esquelético

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation of Valvular Endothelial Cells

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system. These thin, fibrous tissues are subjected to significant mechanical stresses as they open and close several billion times over a lifespan. The incredible endurance of these tissues is due to the resident valvular endothelial (VEC) and interstitial cells (VIC) that constantly repair and remodel in response to local mechanical and biological signals. Only recently have we begun to understand the unique behaviors of these cells, for which in vitro experimentation has played a key role. Particularly challenging is the isolation and culture of VEC. Special care must be used from the moment the tissue is removed from the host through final plating. Here we present protocols for direct isolation, side specific isolation, culture, and verification of pure populations of VEC. We use enzymatic digestion followed by a gentle swab scraping technique to dislodge only surface cells. These cells are then collected into a tube and centrifuged into a pellet. The pellet is then resuspended and plated into culture flasks pre-coated with collagen I matrix. VEC phenotype is confirmed by contact inhibited growth and the expression of endothelial specific markers such as PECAM1 (CD31), Von Willebrand Factor (vWF), and negative expression of alpha-smooth muscle actin (&alpha;-SMA). The functional characteristics of VEC are associated with high levels of acetylated LDL. Unlike vascular endothelial cells, VEC have the unique capacity to transform into mesenchyme, which normally occurs during embryonic valve formation1. This can also occur during significantly prolonged post confluent in vitro culture, so care should be made to passage at or near confluence. After VEC isolation, pure populations of VIC can then be easily acquired.

We provide a method for isolating and culturing pure populations of heart valve endothelial cells (VEC). VEC can be isolated from either side of the cusp or leaflet and immediately following, underlying interstitial cell (VIC) isolation is straightforward.

Aislamiento de células endoteliales valvular

Heart valves are solely responsible for maintaining unidirectional blood flow through the cardiovascular system.  These thin, fibrous tissues are ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

Aislamiento y cultivo de células endoteliales pulmonares de ratones recién nacidos

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Aislamiento y cultivo de células de la zona nefrogénica del riñón de embriones de ratón

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Aislamiento y cultivo primario de células hepáticas de ratas

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Aislamiento de células endoteliales embrionarias ventriculares

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...