1. Ex-ovo Cultura Protocolo: <ol><li> Incubar huevos de gallina fertilizados <ol class="lroman"><li> Fecundados los huevos de gallina se puede almacenar a 13 ° C hasta 5 días antes de la incubación sin iniciar el desarrollo. Un enfriador de vino tinto puede mantener esta temperatura. </li><li> Incubar los huevos lado romo en un 60% de humedad constante incubadora con la oscilación continua a 37,5 ° C durante 72 horas. </li></ol></li><li> Preparar hamacas (Figura 1a) <ol class="lroman"><li> Llene ¾ de 9 oz vasos de plástico con agua estéril tibia. Un habitual 9 oz taza tiene un diámetro máximo de 8 cm. Hemos encontrado que este tamaño era el más adecuado para su aplicación a largo tiempo en la cultura que aquí se presenta. </li><li> Cortar unos 20 cm de film transparente. Ponga circunferencialmente en la parte superior de la copa mediante la colocación de una banda elástica alrededor. Plástico debe ser esparcida en la superficie del agua. Corte el exceso de plástico alrededor de la banda. Encontramos film transparente, para ser más adecuado para esta aplicación ya que evita la adherencia de la yema de huevo, lo que resulta en la división de la yema durante el transporte. </li><li> Limpie el plástico con Kimwipes (Kimberly-Clark, Inc.) humedecido con etanol al 70%. </li></ol></li><li> Eliminar los huevos de la incubadora después de 72 horas de incubación. </li><li> Esterilizar los huevos limpiando la superficie con etanol al 70% Kimwipes mojada. </li><li> Poner los huevos en horizontal durante 1-2 minutos para el posicionamiento adecuado del embrión (Figura 1b). Un cartón de huevos se pueden utilizar para poner los huevos en posición horizontal. Los embriones se gire a la cima después de 1-2 minutos. </li><li> La transferencia de embriones en hamacas asépticamente en una campana de flujo laminar. <ol class="lroman"><li> El uso de un borde afilado (como el borde de un cubo de metal o un vaso de vidrio), toque suavemente el huevo hasta que haya una pequeña abolladura en la parte inferior del huevo (Figura 1c). Recuerde, el embrión se coloca en el haz. </li><li> Pon los pulgares en los lados opuestos de la abolladura y tire las cáscaras aparte horizontalmente (Figura 1d). </li><li> Coloque la yema de huevo con embrión, así como la albúmina en la hamaca (Figura 1). Albúmina proporcionará un ambiente de amortiguación alrededor de la yema de absorber las crisis inducidas por el sistema en movimiento, así como proporciona la nutrición al embrión para las prácticas de la cultura de largo. Agua por debajo de la hamaca se asegurará de que el embrión se quedará paralelo al suelo durante el transporte y servir como un conductor de calor durante la exposición, si un calentador de circulación del agua se utiliza. </li><li> El embrión ya se debe colocar en la parte superior si la transferencia se realiza correctamente. Si no, utilice un enfoque, objeto estéril (es decir, la punta roma de tijeras curvas cerradas) y acariciar direccionalmente la yema de tal manera que el embrión se gira a la cima. </li><li> Coloque un diámetro de 10 cm placa de Petri en la parte superior de la copa para sellar el embrión. </li></ol></li><li> Lugar en la Copa cultura en el conjunto de la incubadora a 37,5 ° C. Si una incubadora portátil se utiliza, la colocación de 1 a 2 sept tazas oz con agua destilada mantiene la humedad en el ~ 60% (Figura 1f). </li><li> Si los embriones más allá del día de cultivo embrionario 7 de la cáscara de huevo trituradas piezas deben ser esparcidas alrededor de la periferia del embrión como fuente de calcio para el desarrollo óseo y la maduración. </li></ol> Equipo auxiliar para los fuera de la incubadora de imagen / manipulación: Un baño de agua circulante fue construido en casa para mantener las temperaturas de incubación, mientras que la manipulación de imágenes o (Figura 3). Un baño de agua que puede caber la taza está conectada mediante un tubo de tamaño apropiado para un depósito de agua. Un calentador de resistencia calienta el agua en el embalse que se regula manualmente mediante la temperatura del agua. Una bomba de agua hace circular el agua. Representación pictórica muestra la facilidad de imágenes a través de ultrasongraphy / microscopía florescent (Figura 3). <img src="/files/ftp_upload/2154/2125fig1.jpg" alt="Figura 1" /> Figura 1. Preparación de la hamaca y la transferencia de embriones de pollo para hamacas. <img src="/files/ftp_upload/2154/2125fig2.jpg" alt="Figura 2" /> Figura 2. Los resultados representativos -. Diferentes embriones de pollo etapa crecido a través de la cultura técnica de ex ovo embriones de HH 17 en adelante se pueden cultivar a través de esta técnica. <img src="/files/ftp_upload/2154/2125fig3.jpg" alt="Figura 3" /> Figura 3. Imágenes esquemáticas y representante del sistema de circulación de agua que se utiliza en conjunción con el sistema de cultivo ex-ovo para mantener los embriones a la temperatura ideal de desarrollo. 2. Las solicitudes seleccionadas: I. La microinyección Este cultivo de embriones es adecuado para aplicaciones donde las soluciones de microinyección que ser inyectado a la vasculatura de contraste de imagen basado en (es decir, la microscopía fluorescente, micro-topografía computarizada, etc) (Figura 4). A continuación se muestra el protocolo de microinyección: <ol><li> Preparar la inyección Apparatus <ol class="lroman"><li> Moda sacó 0,75 tubos de vidrio ID capilar en microagujas punta biselada con un microforge. Tamaño de la punta requerida depende de la vena blanco diámetro / caudal deseado. </li><li> Conecte la jeringa al tubo de 0.03 pulgadas de silicona ID. </li><li> Cargar el tubo con la solución a inyectar y conectar la microaguja de la tubería. </li><li> Coloque la microaguja en el micromanipulador y una jeringa para soporte de jeringa o bomba de jeringa. </li></ol></li><li> Añadir agua destilada en el vaso así lograr que el embrión. Hemos experimentado que para la penetración adecuada de la aguja en un vaso sanguíneo (es decir, buques vitelina), la penetración horizontal, es necesario. Esto requiere que el embrión para estar en el mismo plano que los bordes de la copa. <ol class="lroman"><li> Saque la cinta de goma de plástico debajo de la celebración de la del embrión. </li><li> Levantar un lado del plástico suave y añadir un poco de agua estéril a la taza. </li><li> Asegure la banda de goma en el plástico nuevo. </li></ol></li><li> Se inyecta la solución a un buque de embriones. En nuestra experiencia, cuando la solución se inyecta en un vaso vitelino, microagujas necesario ª tamaño aproximadamente 1 / 10 del diámetro del vaso para mantener la perfusión eficaz sin causar sangrado. <ol class="lroman"><li> Bomba de la solución a la microaguja garantizar la no formación de burbujas de aire. </li><li> Alinee la punta de la microaguja en el recipiente seleccionado. Para los buques vitelina, la selección de un área tenedor dará área máxima de penetrar en el buque. </li><li> Empuje la aguja hacia el buque. El primer buque se retraerá. Una vez que la aguja penetra, iniciar la perfusión. El color de la sangre que fluye debe cambiar con la perfusión. Si el cambio de color no se ve, la aguja puede haber penetrado de la embarcación. En ese caso, poco a poco retraer el microagujas y al mismo tiempo de bombeo de la solución hasta que el cambio de color en el vaso se ve. Una vez que la perfusión se ha completado, retire la microaguja de la embarcación. </li></ol></li></ol><img src="/files/ftp_upload/2154/2125fig4.jpg" alt="Figura 4" /> Figura 4: La microinyección de un tinte fluorescente en la vasculatura de un embrión de pollo ex ovo cultivadas. II. Procedimiento microquirúrgico - Ligadura auricular izquierda Esta configuración de la cultura ex-ovo es ideal para realizar aplicaciones quirúrgicas de embriones de pollo, ya que proporciona un acceso completo al embrión. A continuación se muestra el protocolo de una técnica de ejemplo, la ligadura de la izquierda auricular (LAL) (Figura 5). El procedimiento se realiza aproximadamente 24 horas en el período de cultivo (HH24): <ol><li> Prepare encima de la cabeza nudos ~ 0,5 mm de diámetro con sutura de nylon 10-0 quirúrgica. </li><li> Con unas pinzas finas, localmente eliminar las membranas coriónica y alantoideo sobre el embrión y levantar el embrión de la parte posterior para rotar verticalmente de manera que el lado izquierdo del embrión es visible. </li><li> Con unas pinzas finas, eliminar el pericardio en la aurícula izquierda. </li><li> Alinear el nudo en la aurícula izquierda y apretarlo. El nudo debe estar vinculada con el fin de prohibir el ~ 75% del flujo de sangre original en el lado izquierdo. Cortar los bordes del nudo con micro tijeras y retire con cuidado y deseche el exceso de sutura. </li><li> Gire el embrión de regreso a su orientación original (a la derecha en la parte superior). </li><li> Sham controles supondrá el mismo procedimiento, pero sólo la sutura se pasa a través y no atados. El tratamiento eficaz LAL (75 constricción%) se confirmó a las 24 horas posteriores a la cirugía, y los embriones descalificado será excluido. </li></ol><img src="/files/ftp_upload/2154/2154fig5.jpg" alt="Figura 5" /> Figura 5:. Un control y un auricular izquierda embrión de pollo liga Flecha muestra la aurícula izquierda, que es aproximadamente el 75% más pequeño en el embrión de liga.

<ol>
	<li>Zamir, E. A., Czirok, A., Cui, C., Little, C. D., Rongish, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesodermal+cell+displacements+during+avian+gastrulation+are+due+to+both+individual+cell-autonomous+and+convective+tissue+movements.">Mesodermal cell displacements during avian gastrulation are due to both individual cell-autonomous and convective tissue movements.</a> Proc Natl Acad Sci U S A. 103, 19806-19811 (1980).</li><li>Ehrman, L. A., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+regulation+in+the+heart+forming+region+of+avian+embryos.">Lack of regulation in the heart forming region of avian embryos.</a> Dev Biol. 207, 163-175 (1999).</li><li>Clark, E. B., Hu, N., Rosenquist, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+conotruncal+constriction+on+aortic-mitral+valve+continuity+in+the+stage+18,+21+and+24+chick+embryo.">Effect of conotruncal constriction on aortic-mitral valve continuity in the stage 18, 21 and 24 chick embryo.</a> Am J Cardiol. 53, 324-327 (1984).</li><li>deAlmeida, A., McQuinn, T., Sedmera, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+ventricular+preload+is+compensated+by+myocyte+proliferation+in+normal+and+hypoplastic+fetal+chick+left+ventricle.">Increased ventricular preload is compensated by myocyte proliferation in normal and hypoplastic fetal chick left ventricle.</a> Circ Res. 100, 1363-1370 (2007).</li><li>Nakamura, H., Funahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+DNA+into+chick+embryos+by+in+ovo+electroporation.">Introduction of DNA into chick embryos by in ovo electroporation.</a> Methods. 24, 43-48 (2001).</li><li>Butcher, J. T., McQuinn, T. C., Sedmera, D., Turner, D., 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=Transitions+in+early+embryonic+atrioventricular+valvular+function+correspond+with+changes+in+cushion+biomechanics+that+are+predictable+by+tissue+composition.">Transitions in early embryonic atrioventricular valvular function correspond with changes in cushion biomechanics that are predictable by tissue composition.</a> Circ Res. 100, 1503-1511 (2007).</li><li>McQuinn, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+ultrasonographic+imaging+of+avian+cardiovascular+development.">High-frequency ultrasonographic imaging of avian cardiovascular development.</a> Dev. Dyn. 236, 3503-3513 (2007).</li><li>Korn, M. J., Cramer, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Windowing+chicken+eggs+for+developmental+studies.">Windowing chicken eggs for developmental studies.</a> J Vis Exp. , (2007).</li><li>Auerbach, R., Kubai, L., Knighton, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+procedure+for+the+long-term+cultivation+of+chicken+embryos.">A simple procedure for the long-term cultivation of chicken embryos.</a> Dev Biol. 41, 391-394 (1974).</li></ol>

Acceso óptico y la experimentación en embriones de aves es un reto debido a las limitaciones de la cáscara del huevo. Ventanas limita de manera significativa el número de acceso para métodos de inyección y de microcirugía 8 microvasos. Como resultado de ello sólo embriones pueden ser manipulados y la observación continua no es posible. Los primeros ex-ovo culturas utilizando placas de Petri eran de uso limitado debido al control inadecuado de la tensión superficial sobre el embrión impedid...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Fertile white Leghorn chicken eggs</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Model GB1, Avery Incubators, Hugo CO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Saran Wrap</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Kimwipes</td>
<td>&nbsp;</td>
<td>Kimberly-Clark, Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Rubber bands</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Warm sterile water</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>9 oz plastic cup</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>100 mm diameter Petri dish</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>1602N thermal air</td>
<td>&nbsp;</td>
<td>GQF Manufacturing CO, Savannah GA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Fluorescein-conjugated dextran (2 MDa, 1% w/v in phosphate buffered saline)</td>
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<td>Sigma Aldrich Inc.</td>
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<td>Microforge</td>
<td>&nbsp;</td>
<td>Glassworx, Inc, St Louis MO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Glass capillary tubes (0.75 mm ID)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Micromanipulator</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Sarasota FL</td>
<td>Model M3301L</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent microscopy</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>Z20</td>
<td>&nbsp;</td>
<tr>
<td>Fine 55-forceps</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Sarasota FL</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>10-0 nylon surgical suture</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Tubing</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>1mm OD</td>
</tr>
<tr>
<td>3 mL syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>200 &mu;L pipetter and pipette tips</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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an ex-ovo chicken embryo culture system suitable for imaging and microsurgery applications

Entender las relaciones entre factores genéticos y microambientales que impulsan el desarrollo embrionario normal y malformaciones es fundamental para el descubrimiento de nuevas estrategias terapéuticas. Los avances en tecnología de imagen han permitido a la investigación cuantitativa de la organización y la maduración de la estructura corporal, pero la morfogénesis embrionaria en una etapa posterior es menos clara. Embriones de pollo es un animal vertebrado atractivo sistema de modelo para esta aplicación debido a su facilidad de la cultura y la manipulación quirúrgica. Principios de los embriones pueden ser cultivadas por un corto tiempo en los anillos de papel de filtro, que permite el acceso completo óptico de patrones celulares y 1,2 destino estudios. El estudio de procesos avanzados de desarrollo tales como la morfogénesis cardíaca se realiza tradicionalmente a través de una ventana de la cáscara del huevo 3-5, pero esta técnica limita el acceso óptico debido a tamaño de la ventana. Anteriormente hemos desarrollado un método simple para la cultura embriones enteros ex ovo en el hexagonal pesar barcos de hasta 10 días, lo que permitió imágenes de alta resolución a través de la ecografía 6,7. Estos cultivos fueron difíciles de transportar, lo que limita los tipos de herramientas de imágenes disponibles para los experimentos en vivo. Presentamos aquí una mejora de caparazón sistema de cultivo con un costo-efectivo, cámara ambiental portátil. Los huevos estaban rotas en una hamaca, creada por una membrana de poliuretano (film transparente) fijado circunferencialmente a una taza de plástico llena parcialmente con agua estéril. Las dimensiones de la circunferencia y la profundidad de la hamaca fueron fundamentales para mantener la tensión de la superficie, mientras que la mecánica de la hamaca y el agua por debajo ayudado a amortiguar las vibraciones inducidas por el transporte. Un tamaño pequeño baño de agua circulante también fue desarrollado para permitir un control continuo de la temperatura durante la experimentación. Se demuestra la capacidad de cultivar los embriones de esta manera por lo menos 14 días sin defectos o retraso morfogénica y emplear este sistema en varias de microcirugía y aplicaciones de imágenes.

Watch this Scientific Journal Video about An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications at JoVE.com

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

En este artículo se presenta una metodología sencilla para que a largo plazo<em&gt; Ex-ovo</em&gt; Cultivo de embriones de aves. Esta técnica es ideal para la experimentación longitudinal que requiere la plena accesibilidad ópticos y / o transporte de estériles en embriones de aves.

Un Ex-ovo</emPollo> Embrión de cultivo apropiado para aplicaciones de imagen y sistema de Microcirugía

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

an-ex-ovo-chicken-embryo-culture-system-suitable-for-imaging

Research

JoVE Journal

Biology

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

22.9K vistas.  Cornell University. En este artículo se presenta una metodología sencilla para que a largo plazo Ex-ovo Cultivo de embriones de aves. Esta técnica es ideal para la experimentación longitudinal que requiere la plena accesibilidad ópticos y / o transporte de estériles en embriones de aves.

Video: An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform routine activities, but prosthetic suspensions using traditional socket technology can prove to be cumbersome and uncomfortable for a person with limb loss.  Moreover, for those with high proximal amputations, limited residual limb length may prevent exoprosthesis attachment all together. Osseointegrated implant technology is a novel operative procedure which allows firm skeletal attachment between the host bone and an implant. Preliminary results in European amputees with osseointegrated implants have shown improved clinical outcomes by allowing direct transfer of loads to the bone-implant interface.  Despite the apparent advantages of osseointegration over socket technology, the current rehabilitation procedures require long periods of restrictive load bearing prior which may be reduced with expedited skeletal attachment via electrical stimulation.  The goal of the osseointegrated intelligent implant design (OIID) system is to make the implant part of an electrical system to accelerate skeletal attachment and help prevent periprosthetic infection. To determine optimal electrode size and placement, we initiated proof of concept with computational modeling of the electric fields and current densities that arise during electrical stimulation of amputee residual limbs.  In order to provide insure patient safety, subjects with retrospective computed tomography scans were selected and three dimensional reconstructions were created using customized software programs to ensure anatomical accuracy (Seg3D and SCIRun) in an IRB and HIPAA approved study.  These software packages supported the development of patient specific models and allowed for interactive manipulation of electrode position and size. Preliminary results indicate that electric fields and current densities can be generated at the implant interface to achieve the homogenous electric field distributions   required to induce osteoblast migration, enhance skeletal fixation and may help prevent periprosthetic infections.  Based on the electrode configurations experimented with in the model, an external two band configuration will be advocated in the future.

There is a need to develop alternative prosthesis attachment due to limb loss attributed to vascular occlusive diseases and trauma. The goal of the work is to introduce an osseointegrated intelligent implant design system to increase skeletal fixation and reduce periprosthetic infection rates for patients needing osseointegrated technology.

Los análisis bioeléctricos de una osteointegrados Sistema Inteligente diseño de los implantes para Amputados

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform ...

Investigación

Biología

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

Pollo Ex ovo Cultura y Ex ovo CAM Ensayo: cómo funciona realmente

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

Xenotrasplante de células madre humanas en el embrión de pollo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

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 ...

Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice. HYPERStack vessels allow closed system scale up from the current stacked plate products and delivers >2.5X more cells in the same volumetric footprint. The HYPERStack vessels function via gas permeable material which allows gas exchange to occur, therefore eliminating the need for internal headspace within a vessel. The elimination of headspace allows the compartment where cell growth occurs to be minimized to reduce space, allowing more layers of cell growth surface area within the same volumetric footprint.
For many applications such as cell therapy or vaccine production, a closed system is required for cell growth and harvesting. The HYPERStack vessel allows cell and reagent addition and removal via tubing from media bags or other methods.
This protocol will explain the technology behind the gas permeable material used in the HYPERStack vessels, gas diffusion results to meet the metabolic needs of cells, closed system cell growth protocols, and various harvesting methods.

An Introduction into the technology, protocol and handling of the Corning HYPERStack Vessels and accessories used for high yield adherent cell culture. The protocol will show how to use the closed system vessels for increasing cell harvesting over current stacked plate products.

Sistema de célula cerrada Cultura protocolo mediante buques HyperStack con la tecnología de materiales permeables al gas

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice.   ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

Transferencia de genes en embriones de pollo por los mayores<em&gt; Ex ovo</em&gt; La electroporación

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

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 ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

Un<em&gt; Ex vivo</em&gt; Sistema de Cultura para estudiar el desarrollo de la tiroides

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Preparación de suero de rata Adecuado para mamíferos Whole cultivo de embriones

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Establecimiento de un pH extracelular ácido sistema de cultivo

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

Un Ex-ovo Embrión de cultivo apropiado para aplicaciones de imagen y sistema de Microcirugía