Sarah A. Alfaqeeh es financiado por Kind Saud University College de Odontología, Ministerio de Educación Superior, Reino de Arabia Saudita.

1. Establecer <ol><li> Enfocar los instrumentos de disección (con una piedra de afilar lubricado con aceite mineral) y esterilizar antes de su uso para el cultivo de órganos utilizando un spray de etanol al 70% y la esterilización de calor seco. Afilar las agujas de disección utilizando la electrólisis en 2 M de hidróxido de sodio utilizando un suministro de 12 V. </li><li> Preparar el medio de cultivo utilizado para la disección y la cultura de órganos embrionarios, que consta de modificación F12 Medio Eagle de avanzada Dulbecco (DMEM F12), complementado con 1% GlutaMAX y 1% de penicilina-estreptomicina. </li></ol> 2. La disección de embriones <ol><li> Cull una cronometrada acoplado hembra embarazada que usa un método de programación uno aprobado por el Ministerio del Interior o de otro órgano de gobierno. Diseccionar la piel que rodea el abdomen inferior, y cortar el abdomen con unas tijeras. Localice los dos cuernos uterinos, que se conectan entre sí en la línea media inferior. </li><li> Diseccionar útero y colocar en un tubo lleno con el medio en el hielo. When en hielo, el tejido se puede dejar durante varias horas. </li><li> Extirpar el útero y colocar en una placa de Petri en un medio en el microscopio. Cuando sea posible, las disecciones de embriones se deben realizar en una campana de flujo laminar para minimizar la contaminación. </li><li> Diseccionar los embriones del útero y liberarlos de sus tejidos extraembryonic. </li><li> Decapita a los embriones utilizando una aguja de tungsteno y transferir las cabezas en un plato fresco de medio. </li><li> Aislar las mandíbulas de las cabezas mediante la colocación de una aguja en la cavidad oral y cortar a través hacia la parte posterior de la cabeza (Figuras 1A y 1B). Mandíbulas aisladas pueden ser almacenados temporalmente en un medio en el hielo mientras se espera para picar. </li><li> Para rebanadas mandíbula enteros etapas E11.5-E15.5 son ideales para el cultivo de gérmenes de los dientes. Después E15.5 el hueso en desarrollo en la mandíbula es demasiado difícil para cortar. Rebanadas de dientes todavía se pueden hacer, pero el hueso (hueso alveolar dentario y la formación) primero se debe quitarla disección antes de cortar 9. Retire con cuidado el hueso utilizando agujas de tungsteno y fórceps. </li></ol> 3. Rebanar Embryo <ol><li> Preparar los cortes de tejido mandíbula utilizando un helicóptero estándar tejido tabla (Figura 1C). Utilice una hoja fresca después de aproximadamente 200 mandíbulas, y asegúrese de que se encuentra al ras con el disco de montaje cuando se cae. </li><li> Limpie el disco de montaje y la cuchilla con etanol al 70% antes de su uso. Se debe tener cuidado al usar la máquina, cuando la hoja está muy afilada. </li><li> Ajuste el interruptor a una distancia de corte de entre 200-400 micras, de conformidad con la edad de la muestra, y dependiendo de si se requiere todo el germen del diente. Ajuste la fuerza de la cuchilla en el máximo. </li><li> Transferir una mandíbula con el disco de montaje de plástico con una pipeta o fórceps. Organizar la mandíbula en el disco haciendo que la orientación está aún clara. La lengua en desarrollo puede ser utilizado como un punto de referencia útil para la determinación de la orientación. Para molares uso afRontal / orientación transversal (ver plano de cortar en la Figura 1B), mientras que para los incisivos usar un corte sagital a través de la mandíbula para reflejar la diferente orientación de estos dientes a medida que crecen (ver plano de cortar en la Figura 2A). </li><li> Eliminar el exceso de medio de todo el tejido usando papel de filtro con el fin de secar ligeramente la mandíbula en el disco de montaje. </li><li> Inmediatamente después del secado, coloque el disco en la mesa circular de acero inoxidable del helicóptero. Encienda la máquina y pulse Iniciar. La tabla atraviesa automáticamente de izquierda a derecha, mientras que al mismo tiempo el brazo de cortar llevar la cuchilla se eleva y se dejó en hasta 200 golpes / min. </li><li> Apague la máquina una vez que el tejido ha sido completamente cortado. Asegúrese de que el brazo de la cuchilla está en la posición elevada y extraiga el disco. No ponga los dedos debajo del brazo cuchilla o dejar la máquina desatendida cuando cortar. </li><li> Tome el disco de montaje y añade inmediatamente medio to las rebanadas en el disco con el fin de evitar el secado excesivo. Desplazar cuidadosamente el tejido en rodajas de la parte inferior del disco mediante una aguja de tungsteno y luego pipeta en una pequeña placa de Petri de medio (Figura 2A). </li><li> Separe las rebanadas con una aguja y luego seleccionar los cortes de tejidos que contienen el tejido de interés bajo un microscopio estereoscópico (Figuras 1D, 1E, y 2B). Coloque las rebanadas seleccionados en placas de cultivo con medio fresco. </li></ol> 4. Slice Cultura <ol><li> Preparar platos de órganos-centrales pocillo de cultivo listos para el cultivo mediante la adición de aproximadamente 2 ml de agua en autoclave para el pocillo exterior para prevenir la deshidratación, y mediante la colocación de una rejilla de metal a través del centro del plato (Figuras 3A y 3B). </li><li> Preparar rejillas de metal cortando tiras de aproximadamente 4 cm x 1,5 cm a partir de láminas de malla de acero inoxidable. Use una perforadora para hacer un agujero en el centro y doblarlos lados para crear un extremo escalonada. La rejilla debe encajar plana a través de la pozo central, situada paralela a la parte inferior del plato (Figuras 3A y 3B). Autoclave las rejillas para esterilizar antes de su uso. </li><li> Cortar una pieza de tereftalato de polietileno transparente (PET) de la membrana (tamaño de poro 0,4 micras) suficientemente grande para cubrir el orificio de la rejilla. Coloque la membrana cortada en el plato con el sector seleccionado. </li><li> Coloque la rebanada en la parte superior de la membrana y luego levante con cuidado la membrana de la media, con una rodaja de aplanado en el centro. </li><li> Coloque el filtro en la rejilla sobre el agujero circular en el centro, y añadir aproximadamente 1 ml de medio de cultivo para el pozo, hasta el nivel de la membrana. </li><li> Para manipulaciones agregan proteínas o inhibidores de molécula pequeña para el medio (por ejemplo 8). </li><li> Fotografiar la rebanada por un récord de la morfología en el día 0 de la cultura. </li><li> Colocar las cápsulas en una atmósfera húmeda de 5% de CO <sUB&gt; 2 a 37 ° C en una incubadora. </li><li> Fotografía las culturas, a intervalos regulares durante un promedio de 7 días. Cambie el medio de cultivo cada 48 a 72 horas. Para la fotografía ver las rebanadas con una fuente de luz desde abajo (Figuras 2C-E). </li></ol> 5. Tracing Lineage Si se necesita el rastreo linaje, colorantes lipófilos tales como Dil o DiO pueden ser inyectados en el germen del diente antes de la etapa 4, cultivo de cortes. <ol><li> Tire de agujas capilares de vidrio utilizando un extractor de aguja. </li><li> Rompa la punta de la aguja con las pinzas y coloque la punta hacia abajo en la solución de colorante. Dejar reposar unos minutos, mientras que el medio de contraste llena la punta por acción capilar. </li><li> Coloque la aguja se introduce en un soporte y conectar mediante un tubo a un inyector o la boca de aspiración. </li><li> Coloque la punta de la aguja en el medio de cultivo tocar el área de la rebanada debido a etiquetar. Aplicar presión de aire para desplazar una pequeña cantidad de colorante fuera de la una agujad sobre el tejido. </li><li> Retire la aguja y comprobar si el etiquetado en virtud de la fluorescencia. </li><li> Rebanadas con éxito etiquetados pueden ser transferidos a filtros y se cultivaron como se describe en el paso 4. </li></ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+mesoderm+to+the+developing+dental+papilla.">Contribution of mesoderm to the developing dental papilla.</a> The International journal of developmental biology. 55, 59-64 (2011).</li><li>Buchtova, M., 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=Initiation+and+patterning+of+the+snake+dentition+are+dependent+on+Sonic+hedgehog+signaling.">Initiation and patterning of the snake dentition are dependent on Sonic hedgehog signaling.</a> Developmental biology. 319, 132-145 (2008).</li><li>Buchtova, M., Stembirek, J., Glocova, K., Matalova, E., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+regression+of+the+dental+lamina+underlies+the+development+of+diphyodont+dentitions.">Early regression of the dental lamina underlies the development of diphyodont dentitions.</a> Journal of dental research. 91, 491-498 (2012).</li><li>Cho, S. W., 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=The+primary+enamel+knot+determines+the+position+of+the+first+buccal+cusp+in+developing+mice+molars.">The primary enamel knot determines the position of the first buccal cusp in developing mice molars.</a> Differentiation; research in biological diversity. 75, 441-451 (2007).</li><li>Sakano, M., 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=Cell+dynamics+in+cervical+loop+epithelium+during+transition+from+crown+to+root:+implications+for+Hertwig's+epithelial+root+sheath+formation.">Cell dynamics in cervical loop epithelium during transition from crown to root: implications for Hertwig's epithelial root sheath formation.</a> Journal of periodontal research. , (2012).</li><li>Fisher, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+development+in+vitro+of+the+whole+and+halved+lower+molar+tooth+germ+of+the+mouse.">Morphological development in vitro of the whole and halved lower molar tooth germ of the mouse.</a> Archives of oral biology. 16, 1481-1496 (1971).</li><li>Coin, R., Schmitt, R., Lesot, H., Vonesch, J. L., Ruch, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+halved+embryonic+lower+first+mouse+molars:+correlation+with+the+distribution+pattern+of+non+dividing+IDE+cells,+the+putative+organizers+of+morphogenetic+units,+the+cusps.">Regeneration of halved embryonic lower first mouse molars: correlation with the distribution pattern of non dividing IDE cells, the putative organizers of morphogenetic units, the cusps.</a> The International journal of developmental biology. 44, 289-295 (2000).</li><li>Kavanagh, K. D., Evans, A. R., Jernvall, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+evolutionary+patterns+of+mammalian+teeth+from+development.">Predicting evolutionary patterns of mammalian teeth from development.</a> Nature. 449, 427-432 (2007).</li><li>Munne, P. M., Tummers, M., Jarvinen, E., Thesleff, I., Jernvall, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tinkering+with+the+inductive+mesenchyme:+Sostdc1+uncovers+the+role+of+dental+mesenchyme+in+limiting+tooth+induction.">Tinkering with the inductive mesenchyme: Sostdc1 uncovers the role of dental mesenchyme in limiting tooth induction.</a> Development. 136, 393-402 (2009).</li></ol>

Los autores declaran que no tienen intereses financieros en competencia.

Este método de la cultura de los dientes tiene la ventaja de que el acceso a la germen del diente es excelente, lo que permite linaje precisa la localización y la colocación de los granos en el epitelio o el mesénquima. Por lo tanto, las regiones definidas del germen dentario en desarrollo pueden ser dirigidos específicamente. Durante el cultivo de la morfología cambiante de la germen del diente se puede seguir, y el efecto de las manipulaciones evaluarse rápidamente. El método, sin ...

Para un número de experimentos es importante ser capaz de cultivo de tejidos ex vivo para seguir el desarrollo. Cultura de desarrollar tejido proporciona acceso a determinados períodos de desarrollo y permite la manipulación de los genes por la adición de factores al medio de cultivo, o en perlas cargadas, y por el uso de la transfección y la electroporación 1. Para muchos experimentos es importante ser capaz de visualizar el tejido a medida que crece, por ejemplo, para seguir el destino de las células de linaje etiquetado como el tejido se somete a la morfogénesis. Esto puede ser particularmente problemático para los tejidos que se desarrollan profunda dentro del embrión, que no son evidentes cuando un bloque de tejido del embrión se cultiva. Los dientes son buenos ejemplos de esto, a medida que se desarrollan dentro del proceso nasal mandíbula, maxilar y frontal. Cuando se cultiva toda la mandíbula las estructuras superficiales de los dientes se pueden ver pero los cambios en la morfología sólo pueden ser analizados después de la sección de tejido fijado <shasta&gt; 2. Hemos adaptado una técnica de cultivo de cortes en directo lo que nos permite seguir el desarrollo de gérmenes del diente y el acceso a las diferentes partes del diente durante el desarrollo. Las culturas técnica de las rodajas de germen del diente en la interfase gas-líquido, utilizando un método modificado Trowell 3. Estos cultivos de cortes han sido muy útiles en directamente después de la morfogénesis del diente, y permitiendo linaje rastreo de componentes distintos, tales como el nudo del esmalte y la papila dental y el folículo 1,4-7. La técnica no se limita a los embriones de ratón y con éxito se ha utilizado para la cultura en vivo rebanadas de cerdo y la serpiente 8,9 tejido dental. Además de la ventaja de ser capaces de visualizar el desarrollo del diente, el método rebanada también tiene la ventaja de que las rodajas delgadas de tejido han aumentado el acceso a los nutrientes del medio y el aire de la incubadora. Esto se traduce en un mejor crecimiento de los gérmenes de los dientes, que coinciden con el desarrollo in vivo y espectáculo La invasión masde iones de las células endoteliales en la papila 7. En contraste, los gérmenes de los dientes en la mandíbula culturas enteros desarrollan más lento que los in vivo y el centro de la cultura es a menudo necrótico en cultivos a largo plazo. En la cultura rebanada, el diente se desarrolla dentro de un trozo de la mandíbula, y su interacción con el hueso circundante y el desarrollo de otros tejidos puede ser monitoreado. En nuestro método, el tejido se corta recta después de la disección sin necesidad de integrar en un medio de soporte 10,11 y no hay necesidad para cualquier sistema para sujetar el tejido a la guillotina. El método es, por tanto, no invasiva y rápida, permitiendo que muchos mandíbulas que se seccionaron en una sesión.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR</td>
			<td>101077Y</td>
			<td>100% ethanol was diluted in distilled H2O to 70%.</td>
		</tr>
		<tr>
			<td>DMEM F12</td>
			<td>Gibco</td>
			<td>12634-010</td>
			<td>Advanced Dulbecco&#39;s Modified Eagle Medium F12</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Sigma</td>
			<td>P0781</td>
			<td></td>
		</tr>
		<tr>
			<td>DiI (Molecular probes)</td>
			<td>Vybrant</td>
			<td>V-22885</td>
			<td>Cell-labeling solution</td>
		</tr>
		<tr>
			<td></td>
			<td>Invitrogen</td>
			<td></td>
			<td>Cell tracker CM-DiI, C-7000</td>
		</tr>
		<tr>
			<td>DiO (Molecular probes)</td>
			<td>Vybrant</td>
			<td>V22886</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Geminator 500</td>
			<td>Thomas</td>
			<td>Thomas No. 3885A20</td>
			<td>Dry-heat sterilization</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>McIlwain tissue chopper</td>
			<td>Ted Pella, Inc.</td>
			<td>10180</td>
			<td>Standard table</td>
		</tr>
		<tr>
			<td>Organ culture dish (Center-Well Organ Culture Dish)</td>
			<td>Falcon</td>
			<td>353037</td>
			<td></td>
		</tr>
		<tr>
			<td>Membranes (Cell Culture Inserts, 0.4 &#956;m pore size)</td>
			<td>BD Falcon</td>
			<td>353090</td>
			<td>PET track-etched membrane, 6-well format</td>
		</tr>
		<tr>
			<td>Metal grids (Stainless Steel - AISI 304 - Mesh)</td>
			<td>Goodfellow</td>
			<td>FE228710</td>
			<td>(Fe/Cr18/Ni10)</td>
		</tr>
		<tr>
			<td>AutoFlow Direct Heat CO2 Incubator</td>
			<td>Nuaire</td>
			<td>NU-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Picospritzer III</td>
			<td>Intracel Ltd</td>
			<td>051-0500-900 0-100 psi</td>
			<td>Single channel picospritzer III</td>
		</tr>
		<tr>
			<td>Glass capillary with filament 1 mm</td>
			<td>WPI</td>
			<td>TW100F-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten wire 0.1 mm</td>
			<td>Goodfellow</td>
			<td>W005138</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten wire 0.38 mm</td>
			<td>Goodfellow</td>
			<td>W005155</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirator tubes</td>
			<td>Sigma</td>
			<td>A5177</td>
			<td>Used for mouth aspiration lineage tracers</td>
		</tr>
	</tbody>
</table>

the slice culture method for following development of tooth germs in explant culture

Cultura explante permite, por tanto, la manipulación de desarrollar órganos en puntos específicos de tiempo y es un método importante para el biólogo del desarrollo. Para muchos órganos es difícil acceder a los tejidos en desarrollo para permitir el monitoreo durante el cultivo ex vivo. El método de cultivo de cortes permite el acceso a los tejidos para que los movimientos morfogenéticos pueden ser seguidos y poblaciones específicas de células pueden ser objeto de manipulación o rastreo de linaje. En este trabajo se describe un método de cultivo de cortes que ha sido muy exitoso para el cultivo de los gérmenes de los dientes en una amplia gama de especies. El método proporciona un excelente acceso a los gérmenes de los dientes, que se desarrollan a un ritmo similar al observado in vivo, rodeado de los otros tejidos de la mandíbula. Esto permite interacciones de tejido entre el diente y el tejido circundante a controlar. Aunque este documento se concentra en los gérmenes de los dientes, el mismo protocolo se puede aplicar a seguir el desarrollo de un númerode otros órganos, como las glándulas salivales, el cartílago de Meckel, glándulas nasales, lengua y orejas.

Con el fin de seguir el movimiento de la primera folículo dental molar, 250 micras rodajas frontales fueron tomadas a través de una mandíbula utilizando el método descrito anteriormente. El folículo dental es la capa de mesénquima que rodea el epitelio del esmalte exterior (OEE) del diente en desarrollo, y se ha mostrado previamente para tomar parte en la formación de tejido del periodonto 6. Mandíbulas fueron disecados en E14.5, la etapa de desarrollo de los dientes cap. En la rebanada el contorno de...

Watch this Scientific Journal Video about The Slice Culture Method for Following Development of Tooth Germs In Explant Culture at JoVE.com

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Aquí detallamos un método de cultivo de gérmenes dentales en rodajas mandíbula usando un cortador de tejidos. Este método permite un acceso único a los dientes durante el desarrollo, que proporciona una excelente oportunidad para la manipulación y el linaje de rastreo, no está disponible utilizando métodos de cultivo más tradicionales.

El método de cultivo de la rebanada por Siguiendo desarrollo de gérmenes de dientes en Explante Cultura

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

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Research

JoVE Journal

Biology

14.0K vistas.  King's College London.  Aquí detallamos un método de cultivo de gérmenes dentales en rodajas mandíbula usando un cortador de tejidos. Este método permite un acceso único a los dientes durante el desarrollo, que proporciona una excelente oportunidad para la manipulación y el linaje de rastreo, no está disponible utilizando métodos de cultivo más tradicionales. 

Video: The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

Cultura Cortar organotípicos de los cerebros de ratas E18

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Investigación

Biología

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.  

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

Preparación de balas de cañón de genes y transfección biolística de neuronas en cultivo Cortar

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Método para la Cultura de primeros embriones del polluelo ex vivo (Nueva Cultura)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

A neuronales y astrocitos Co-cultivo de ensayo para el análisis de alto contenido de neurotoxicidad

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

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 ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

El método de cultivo de embriones de roedores enteros utilizando el sistema de tipo de botella rotador Cultura

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food.
Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out.
To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area.
We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.

We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.

Un nuevo método para la Cultura y polarizado estimulación de explantes mucosa intestinal humana

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

A Novel<em&gt; Ex vivo</em&gt; Método de la cultura para el corazón de embriones de ratón

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Embriones de ratón con el desarrollo en todo un sistema de cultivo de embriones sin suero

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.