1. Protocole Culture Ex-ovo: <ol><li> Incuber les œufs de poule fécondés <ol class="lroman"><li> Œufs de poule fécondés peuvent être stockées à 13 ° C jusqu&#39;à 5 jours avant l&#39;incubation sans initier le développement. Un refroidisseur de vin rouge peut maintenir cette température. </li><li> Incuber les œufs côté émoussé dans un 60% d&#39;humidité constant incubateur avec bascule en continu à 37,5 ° C pendant 72 heures. </li></ol></li><li> Préparer hamacs (figure 1a) <ol class="lroman"><li> Remplir aux ¾ de 9 oz gobelets en plastique avec de l&#39;eau stérile chaude. Un habitué 9 oz coupe a un diamètre de 8 cm de haut. Nous avons constaté que cette taille était le plus approprié pour une application à long temps de culture présentées ici. </li><li> Coupez environ 20 cm s&#39;accrocher envelopper. Apposez circonférentiellement sur le dessus de la tasse en plaçant une bande élastique autour. Plastique doivent être répartis sur le dessus de l&#39;eau. Coupez le plastique excédentaire autour de la bande. Nous avons trouvé s&#39;accrocher envelopper d&#39;être le plus approprié pour cette application car elle empêche l&#39;adhérence du jaune, ce qui entraîne la scission jaune pendant le transport. </li><li> Essuyez le plastique avec Kimwipes (Kimberly-Clark, Inc) mouillé avec 70% d&#39;éthanol. </li></ol></li><li> Retirer les oeufs de l&#39;incubateur après 72 heures d&#39;incubation. </li><li> Stériliser les œufs en essuyant la surface avec 70% d&#39;éthanol Kimwipes mouillée. </li><li> Poser les oeufs à l&#39;horizontale pendant 1-2 minutes pour le bon positionnement de l&#39;embryon (figure 1b). Un carton d&#39;œufs peuvent être utilisés pour la ponte à l&#39;horizontale. Embryons tournera vers le haut après 1-2 minutes. </li><li> Transfert des embryons sur des hamacs de façon aseptique dans une hotte à flux laminaire. <ol class="lroman"><li> L&#39;utilisation d&#39;un bord tranchant (comme le bord d&#39;un seau en métal ou un récipient en verre), appuyez doucement jusqu&#39;à ce que l&#39;œuf il ya une petite dent sur la partie inférieure de l&#39;œuf (figure 1c). Rappelez-vous, l&#39;embryon est placé sur la face supérieure. </li><li> Mettez les pouces dans les côtés opposés de la dent et tirer des obus en dehors horizontalement (figure 1d). </li><li> Placer le jaune d&#39;oeuf avec l&#39;embryon ainsi que l&#39;albumine sur le hamac (figure 1E). L&#39;albumine va fournir un environnement d&#39;amortissement autour du jaune pour absorber les chocs induits par le déplacement du système aussi bien qu&#39;il fournit la nutrition de l&#39;embryon pour des pratiques de culture longue. L&#39;eau sous le hamac d&#39;assurer l&#39;embryon va rester parallèle au sol pendant le transport et servir de conducteur de chaleur lors de l&#39;imagerie si un réchauffeur de circulation d&#39;eau est utilisé. </li><li> L&#39;embryon doit déjà être placé sur le dessus si le transfert est effectué correctement. Si non, utiliser un flou, objet stérile (ie. bout émoussé des ciseaux courbes fermées) et caresse le jaune directionnelle tels que l&#39;embryon tourne vers le haut. </li><li> Placer un diamètre de 10 cm boîte de Petri sur le dessus de la tasse pour sceller l&#39;embryon. </li></ol></li><li> Placer la tasse de la culture dans l&#39;incubateur réglé à 37,5 ° C. Si un incubateur portable est utilisé, placer 1 à 2 sept tasses oz rempli d&#39;eau distillée maintient l&#39;humidité à ~ 60% (figure 1f). </li><li> Si les embryons en culture au-delà de 7 jours embryonnaire, des morceaux coquille écrasée devrait être disséminés sur le pourtour de l&#39;embryon comme une source de calcium pour le développement des os et de la maturation. </li></ol> Les équipements accessoires pour l&#39;extérieur incubateur d&#39;imagerie / manipulation: Un bain d&#39;eau circulant dans la maison a été construite pour maintenir les températures d&#39;incubation tout en imagerie ou de manipulation (figure 3). Un bain d&#39;eau qui peuvent tenir la tasse est connecté via un tube de taille appropriée pour un réservoir d&#39;eau. Un chauffage à résistance chauffe l&#39;eau dans le réservoir qui est réglementé manuellement en suivant la température de l&#39;eau. Une pompe à eau fait circuler l&#39;eau. La représentation picturale illustre la facilité d&#39;imagerie par ultrasongraphy / microscopie florescent (figure 3). <img src="/files/ftp_upload/2154/2125fig1.jpg" alt="Figure 1" /> Figure 1. Préparation du hamac et le transfert d&#39;embryons de poulet aux hamacs. <img src="/files/ftp_upload/2154/2125fig2.jpg" alt="Figure 2" /> Figure 2. Les résultats représentatifs -. Différents embryons de poulet stade adulte moyen technique de culture in ovo ex embryons de HH 17 et suivants peuvent être cultivées par cette technique. <img src="/files/ftp_upload/2154/2125fig3.jpg" alt="Figure 3" /> Figure 3. Schéma et photos représentant du système de circulation de l&#39;eau utilisée en conjonction avec le système de culture ex-ovo de garder les embryons à la température idéale de développement. 2. Les demandes sélectionnées: Microinjection I. Cette culture de l&#39;embryon est adapté pour des applications où les solutions de micro-injection doivent être injectés au système vasculaire de l&#39;imagerie de contraste à base (c.-à-microscopie à fluorescence, la micro-topographie, etc calculé) (figure 4). Ci-dessous est le protocole de micro-injection: <ol><li> Préparer le Appara d&#39;injectionTus <ol class="lroman"><li> Mode tiré 0,75 tubes en verre ID capillaire dans les micro-aiguilles embout biseauté en utilisant un microforge. Taille de la pointe requis dépend de la veine cible de diamètre / débit désiré. </li><li> Raccorder la seringue à 0,03 tube de silicone ID pouces. </li><li> Chargez le tube avec la solution à injecter et à connecter le micro-aiguille à la tubulure. </li><li> Placez le micro-aiguille sur le micromanipulateur et une seringue pour porte-seringue ou une pompe à seringue. </li></ol></li><li> Ajouter de l&#39;eau stérile dans la tasse à augmenter ainsi l&#39;embryon. Nous avons connu que pour une bonne pénétration de l&#39;aiguille dans un vaisseau sanguin (ie. navire vitelline), la pénétration horizontale est nécessaire. Cela nécessite l&#39;embryon d&#39;être sur le même plan que les bords de la tasse. <ol class="lroman"><li> Sortez la bande de caoutchouc tenant le plastique sous l&#39;embryon. </li><li> Soulever un côté de la plastique doucement et ajouter de l&#39;eau stérile pour la tasse. </li><li> Fixez la bande de caoutchouc sur le plastique de nouveau. </li></ol></li><li> Injecter la solution à un navire embryonnaire. Dans notre expérience, quand la solution est injectée à un navire vitelline, micro-aiguilles nécessaires pour être dimensionnée e environ 1 / 10 du diamètre du vaisseau pour maintenir la perfusion efficace sans causer des saignements. <ol class="lroman"><li> Pomper la solution à la micro-aiguille assurant pas de formation de bulles d&#39;air. </li><li> Aligner l&#39;extrémité de la micro-aiguille sur le navire sélectionné. Pour vaisseaux vitellins, en sélectionnant une zone de fourche donnera superficie maximale de pénétrer dans le navire. </li><li> Poussez l&#39;aiguille vers le navire. Le navire va d&#39;abord se rétracter. Une fois que l&#39;aiguille pénètre, commencer la perfusion. La couleur du sang qui coule devrait changer avec la perfusion. Si le changement de couleur ne se voit pas, l&#39;aiguille peut avoir pénétré dans le navire. Dans ce cas, lentement se rétractent la micro-aiguille de pompage tout en solution jusqu&#39;à changer de couleur dans le vaisseau est vu. Une fois la perfusion terminée, retirer la micro-aiguille hors du navire. </li></ol></li></ol><img src="/files/ftp_upload/2154/2125fig4.jpg" alt="Figure 4" /> Figure 4: microinjection d&#39;un colorant fluorescent dans le système vasculaire de l&#39;embryon ex-ovo-chiches cultivés. II. Procédure microchirurgicale - Gauche Ligature auriculaire Cette configuration de la culture ex-ovo est idéale pour réaliser des applications chirurgicales pour les embryons de poulet car elle offre une accessibilité totale à l&#39;embryon. Voici le protocole d&#39;une technique par exemple, a quitté la ligature auriculaire (LAL) (figure 5). La procédure est réalisée environ 24 heures dans la période de culture (HH24): <ol><li> Préparer pronation noeuds ~ 0,5 mm de diamètre à l&#39;aide de suture en nylon 10-0 chirurgicale. </li><li> Avec une pince fine, localement enlever les membranes choriales et allantoïdien sur l&#39;embryon et soulever l&#39;embryon de l&#39;arrière pour faire pivoter verticalement de telle sorte que c&#39;est le côté gauche de l&#39;embryon est visible. </li><li> En utilisant une pince fine, enlevez le péricarde sur l&#39;atrium gauche. </li><li> Alignez les noeuds sur l&#39;oreillette gauche et le serrer. Le nœud doit être lié de manière à interdire ~ 75% de la circulation sanguine d&#39;origine pour le côté gauche. Couper les bords du noeud avec microciseaux et retirez et jetez de suture excès. </li><li> Tournez l&#39;embryon revenir à son orientation d&#39;origine (à droite sur le dessus). </li><li> Contrôle Sham impliquera la même procédure mais la suture sera juste passé à travers et non liées. L&#39;efficacité du traitement du labo (75% de constriction) sera confirmée à 24 heures post-chirurgie, et les embryons disqualifiés seront exclus. </li></ol><img src="/files/ftp_upload/2154/2154fig5.jpg" alt="Figure 5" /> Figure 5:. Un contrôle et une gauche d&#39;embryon de poulet ligaturé auriculaire La flèche indique l&#39;oreillette gauche, qui est d&#39;environ 75% plus petit dans l&#39;embryon ligaturé.

<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>

L&#39;accès optique et d&#39;expérimentation dans les embryons aviaire est difficile en raison de contraintes de la coquille d&#39;œuf. Fenêtrage limite considérablement le nombre d&#39;accès à des approches d&#39;injection et de microchirurgie 8 microvaisseaux. En conséquence seuls les embryons précoces peuvent être manipulés et de l&#39;observation en continu n&#39;est pas possible. Début ex-ovo-cultures utilisant des boîtes de Petri ont été d&#39;un usage limité à cause du contrô...

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		<th>Material Name</th>
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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>
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<tr>
<td>Model GB1, Avery Incubators, Hugo CO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Saran Wrap</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<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>
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<td>9 oz plastic cup</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>100 mm diameter Petri dish</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>1602N thermal air</td>
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<td>GQF Manufacturing CO, Savannah GA</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>
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<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>
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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>
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<td>Fine 55-forceps</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Sarasota FL</td>
<td>&nbsp;</td>
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<td>10-0 nylon surgical suture</td>
<td>&nbsp;</td>
<td>Ethicon</td>
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<td>Tubing</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>1mm OD</td>
</tr>
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<td>3 mL syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
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<tr>
<td>200 &mu;L pipetter and pipette tips</td>
<td>&nbsp;</td>
<td>VWR</td>
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an ex-ovo chicken embryo culture system suitable for imaging and microsurgery applications

Comprendre les relations entre facteurs génétiques et microenvironnementales ce disque normal et malformés développement embryonnaire est fondamentale pour la découverte de nouvelles stratégies thérapeutiques. Les progrès de la technologie d&#39;imagerie ont permis étude quantitative de l&#39;organisation et la maturation du plan du corps, mais à un stade ultérieur la morphogenèse embryonnaire est moins claire. Embryons de poulet sont un système de vertébrés attractif modèle animal pour cette application en raison de sa facilité de culture et de la manipulation chirurgicale. Embryons précoces peuvent être cultivées pendant un court moment sur ​​les anneaux de papier filtre, qui permet un accès complet optique pour la structuration et le destin cellulaire des études 1,2. Etudier les processus de développement avancés tels que la morphogenèse cardiaque sont traditionnellement effectuées par une fenêtre de la coquille 3-5, mais cette technique limite l&#39;accès optique due à la taille de fenêtre. Nous avons déjà développé une méthode simple pour la culture des embryons toute l&#39;ex-ovo-sur hexagonale pèsent bateaux pour un maximum de 10 jours, ce qui a permis d&#39;imagerie haute résolution via l&#39;échographie 6,7. Ces cultures ont été difficiles à transporter, limitant les types d&#39;outils d&#39;imagerie disponibles pour des expériences en direct. Nous présentons ici un système de culture améliorée sans coquille avec un bon rapport coût-efficacité, chambre environnementale portables. Les œufs étaient fissurés sur un hamac créé par une membrane de polyuréthane (s&#39;accrocher envelopper) apposé circonférentiellement pour une tasse en plastique partiellement rempli d&#39;eau stérile. Les dimensions de la circonférence et la profondeur du hamac sont à la fois critique pour maintenir la tension de surface, tandis que la mécanique du hamac et eau sous aidé à atténuer les vibrations induites par le transport. Un faible encombrement circulant bain d&#39;eau a également été développé pour permettre un contrôle continu de la température lors de l&#39;expérimentation. Nous démontrons la capacité de la culture des embryons de cette manière pendant au moins 14 jours sans défaut ou retard morphogénique et emploient ce système en microchirurgie plusieurs applications d&#39;imagerie.

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

Dans cet article, nous présentons une méthodologie simple pour permettre à long terme<em&gt; Ex-ovo-</em&gt; Culture embryon aviaire. Cette technique est idéale pour l&#39;expérimentation longitudinale complète nécessitant l&#39;accessibilité et / ou optiques de transport stériles des embryons aviaires.

Une Ex-ovo- Poulet embryonnaire du système de culture approprié pour les applications d&#39;imagerie et de microchirurgie

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 vues.  Cornell University. Dans cet article, nous présentons une méthodologie simple pour permettre à long terme Ex-ovo- Culture embryon aviaire. Cette technique est idéale pour l'expérimentation longitudinale complète nécessitant l'accessibilité et / ou optiques de transport stériles des embryons aviaires.

Vidéo: 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.

Analyses Bioelectric d&#39;un système ostéo-intégrés Implant Intelligent Design pour amputés

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 ...

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Biologie

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.

Poussin<em&gt; Ovo ex</em&gt; Culture et<em&gt; Ovo ex</em&gt; CAM Test: Comment il fonctionne vraiment

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.

La xénotransplantation de cellules souches humaines dans l&#39;embryon de poulet

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.

La méthode de culture des embryons rongeurs entier en utilisant le système des rotateurs de type flacon de culture

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.

Fermé protocole Culture Cell System utilisant des navires HyperStack avec la technologie des matériaux perméables au gaz

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.

Transfert de gènes dans des embryons de poulet âgés par<em&gt; Ex ovo</em&gt; Électroporation

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.

Souris développement embryonnaire dans tout un système de culture d&#39;embryons sans sérum

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; Système de culture à étudier la thyroïde développement

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.

Préparation du Rat Serum Convient pour la culture d&#39;embryons de mammifères entier

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.

Mise en place d’un pH extracellulaire acide système de Culture

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