Ce travail a été soutenu par la Fondation Margaret M. Alkek au RHF.

Partie 1: Banc de mettre en place <ol><li> Une chambre humide est préparé en plaçant Kimwipe / ddH 2 O dans la chambre de plastique. </li><li> Un tube Falcon de recueillir l&#39;albumine, de la vaisselle pour la culture, des bagues, verre de montre et l&#39;élimination des déchets sont placés sur le banc. </li><li> Plat en pyrex est rempli avec une solution saline 1,4 l (voir note [a]). </li></ol> Partie 2: l&#39;embryon est explanté dans une solution saline <ol><li> Les œufs sont retirés de l&#39;incubateur après 16 heures (stade 4). L&#39;œuf est opene en tapant la coquille avec une pince. Shell morceaux sont enlevés. </li><li> L&#39;albumine fine est recueillie dans le tube Falcon. L&#39;albumine épaisse est retiré avec une pince. </li><li> L&#39;embryon est placé dans une boîte en plastique à l&#39;intérieur du plat saline. L&#39;albumine reste est enlevé avec une pince. </li></ol> Partie 3: l&#39;embryon est centrée sur l&#39;anneau <ol><li> Le sac vitellin est incliné avec une pince afin que les embryons tournée vers le haut. </li><li> Le sac vitellin est coupé au niveau de l&#39;équateur ou en dessous. </li><li> En utilisant une pince fine, la membrane vitelline est rapidement épluchés. La membrane vitelline est orienté de sorte que son côté granulaire (non brillant) tournée vers le haut. </li><li> En utilisant une pince fine, la membrane vitelline est placée sur le verre de montre. </li><li> En utilisant une pince fine, un anneau de verre est appliquée sur le dessus de la membrane vitelline et l&#39;embryon est centré. </li><li> La membrane vitelline est levé autour des bords de l&#39;anneau de verre. L&#39;ensemble est retiré de l&#39;antenne saline. </li></ol> Partie 4: La culture est mis en place sous le microscope <ol><li> La membrane vitelline est levé sur l&#39;anneau de verre à l&#39;aide de pinces fines au microscope. </li><li> En utilisant une pipette Pasteur, la saline est retirée à partir du bord extérieur de l&#39;anneau. </li><li> Avec des ciseaux fins, l&#39;excès de membrane vitelline est enlevée à partir du bord interne de l&#39;anneau de verre. Il est pris soin de ne pas percer la membrane avec pipette ou une pince. </li><li> L&#39;embryon est légèrement rincés avec du sérum physiologique pour enlever les membranes des cellules en vrac et le jaune. </li><li> 200 ul saline est ajoutée au bord externe de l&#39;anneau (ce qui facilitera un transfert ultérieur). </li><li> L&#39;ensemble est recouvert d&#39;un plat de plastique inversé. </li></ol> Partie 5: La culture est transférée à l&#39;incubateur <ol><li> Une boîte de culture Falcon est étiqueté. </li><li> 2.5ml d&#39;albumine mince est ajouté au bas de l&#39;antenne Falcon. </li><li> 200 ul d&#39;albumine mince est ajouté à l&#39;arête intérieure du couvercle du plat Falcon. </li><li> Le plat inversé est retiré de l&#39;assemblée. </li><li> En utilisant une pince fine, l&#39;anneau de verre est glissé le long du bord du verre de montre, et transféré à l&#39;antenne Falcon. </li><li> Tous les salines restante est retirée de la surface intérieure de l&#39;anneau. </li><li> Le plat est couvert Falcon avec couvercle et scellé. </li><li> L&#39;ensemble est placé dans une chambre humide, puis dans l&#39;incubateur. </li><li> Les embryons sont cultivés pendant au moins 24 heures à 38 ° C. </li></ol> Partie 6: culture suivants, l&#39;embryon est fixe, la culture est transférée à l&#39;incubateur <ol><li> Un plat de fixation est remplie de PBS glacé (ou DEPC PBS si des embryons doivent être traitées pour l&#39;hybridation in situ). </li><li> La culture est retiré de l&#39;incubateur et immédiatement placé sur la glace. La boîte de culture est immédiatement remplie de PBS glacé / DEPC PBS. </li><li> L&#39;embryon se détache de la membrane vitelline. L&#39;embryon est transféré dans le plat de fixation, en utilisant l&#39;extrémité émoussée d&#39;une pipette Pasteur. </li><li> L&#39;embryon est coincé à l&#39;aide des extrémités franches pinces fines. Le PBS couvrant le plat de fixation est retiré. Fixateur est ajouté (voir note [b]). </li><li> Selon l&#39;application, l&#39;embryon est fixe pendant 6-8 heures à 4 ° C (cryostat), O / N à 4 ° C (en situs), ou 1 h à température ambiante pour montage immunohistochimie ensemble. </li><li> Le fixateur est enlevé et remplacé par PBS glacé / DEPC PBS. </li><li> Pour les applications en aval telles que l&#39;hybridation in situ ou la coloration immantibody: le système nerveux et le cœur sont perforés à l&#39;aide d&#39;une aiguille extrémité émoussée microcapillaire ou un couteau de microdissection, ce qui permettra d&#39;éviter le piégeage de la sonde / anticorps dans les étapes ultérieures. Notes: [a] Saline est composé de: la solution A (pour 1 l): 121,0 g de NaCl, 15,5 g de KCl, 10,4 g de CaCl 2 .2 H 2 O, 12,7 g de MgCl 2 .6 H 2 O; solution B (pour 1 l) : 2,4 g de Na 2 HPO 4 .2 H 2 O, 0,2 g de NaH 2 PO 4 .2 H 2 O; Autoclave; Avant utilisation, mélanger 120 ml A avec 2700 ml H 2 O; Ajouter 180 ml ​​B. Mélanger [1]; [b] fixateur est préparé juste avant utilisation (4% dans du PBS ou PFA DEPC PBS dans situs). </li></ol> Partie 7: Résultats Représentant Avant de la culture, l&#39;embryon est au stade ligne primitive (HH4). A la fin de la période de la culture, l&#39;embryon a développé au HH10 (longueur 2-3 mm) et est visible dans le centre de la boîte de culture. Il est possible d&#39;étiqueter un groupe de cellules fluorescentes avec carbocyanine DiIjuste avant la culture (0h) et suivre leurs mouvements pendant toute la période de culture. Dans ce cas, les cellules ci-dessous le nœud de Hensen (HN) ont été marqués avec DII. Ces cellules sont indiquées pour contribuer à développer progressivement somites (SOM) et notochorde (n). <img src="/files/ftp_upload/old/903_2008_10_20_2/903_Figure-01.png" alt="" width="780" height="228" /> Partie 8: Dépannage <table style="width: 698px; height: 241px;" border="1" cellspacing="0" cellpadding="0" width="698"><tbody><tr><th> Problème </th><th> Cause </th><th> Remède </th></tr><tr><td> Embryon reste avec du jaune au lieu de venir hors avec membrane (étape 2) </td><td> Mauvaise qualité des œufs </td><td> Demande fraîchement produite oeufs; oeufs magasin à 13 ° C dès réception. Incuber les œufs le même jour que la date d&#39;arrivée. </td></tr><tr><td> Glisser membrane vitelline loin de placement horloger de verre suivants de l&#39;anneau (étape 3) </td><td> Restes d&#39;albumine </td><td> Dans l&#39;étape 2, assurez-vous que tous albumine est éliminé par l&#39;éloignant de la membrane avec une pince inclinée </td></tr><tr><td> Embryon est inaccessible: se trouve sous la membrane (étape 4) </td><td> Mauvais côté de la membrane est à la hausse </td><td> À l&#39;étape 3, assurez-vous du côté de la membrane contenant des granulés jaunes est orientée vers le haut. Le brillant, côté poli doit faire face vers le bas. </td></tr><tr><td> Embryon immergé dans la culture suivante salins / albumine (étape 6) </td><td> Trou dans la membrane; Saline / albumine laissé intérieur de l&#39;anneau avant de la culture </td><td> Dans l&#39;étape 5, assurez-vous que tous albumine / saline est retiré de l&#39;intérieur de l&#39;anneau; l&#39;étape 4, assurez-vous que vous n&#39;avez pas de membrane percer avec une pince (l&#39;utilisation de pinces à bouts francs) </td></tr><tr><td> Embryon désintégré la culture suivante </td><td> L&#39;infection bactérienne </td><td> Stériliser tous les outils et ouvrages en verre; l&#39;utilisation d&#39;antibiotiques / antimycotiques </td></tr></tbody></table>

<ol>
	<li>Pannett, P. A., Compton, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cultivation+of+tissues+in+saline.">The cultivation of tissues in saline.</a> Lancet. 206, 381-384 (1924).</li><li>New, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+for+the+cultivation+of+the+chick+embryo+in+vitro.">A new technique for the cultivation of the chick embryo in vitro.</a> J. Embryol. Exp. Morph. 3, 326-331 (1955).</li><li>Alvarez, I. S., Araujo, M., Nieto, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+induction+in+whole+chick+embryo+cultures+by+FGF.">Neural induction in whole chick embryo cultures by FGF.</a> Dev. Biol. 199, 42-54 (1998).</li><li>Psychoyos, D., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+the+organizer+after+radical+ablation+of+Hensen's+node+and+the+anterior+primitive+streak+in+the+chick+embryo.">Restoration of the organizer after radical ablation of Hensen's node and the anterior primitive streak in the chick embryo.</a> Development. 122, 3263-3273 (1996).</li><li>Psychoyos, D., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+and+migratory+routes+of+primitive+streak+cells+in+the+chick+embryo.">Fates and migratory routes of primitive streak cells in the chick embryo.</a> Development. 122, 1523-1534 (1996).</li><li>Voiculescu, O., Papanayotou, C., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+and+temporally+controlled+electroporation+of+early+chick+embryos.">Spatially and temporally controlled electroporation of early chick embryos.</a> Nature Protoc. 3, 419-426 (2008).</li></ol>

La méthode de culture New 2 peut être utilisé pour une large variété d&#39;applications, allant de greffes du facteur de croissance contenant des billes 3, de monter toute hybridation in situ et immunohistochimie toute monture 4. Culture sur une période de 24 heures permet la surveillance continue du développement embryonnaire dans des applications comme laps de temps le mouvement des cellules d&#39;analyse 5 ou de surveillance de la GFP contenant des constructions électr...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Eggs</td>
<td>Animal</td>
<td>Charles River Laboratories</td>
<td>Premium Fertile</td>
<td>&nbsp;</td>
<tr>
<td>Stereomicroscope</td>
<td>Microscope</td>
<td>Leica Microsystems</td>
<td>MZ9.5 or similar</td>
<td>&nbsp;</td>
<tr>
<td>Marsh Automatic Incubator</td>
<td>Tool</td>
<td>Lyon</td>
<td>RX</td>
<td>&nbsp;</td>
<tr>
<td>Hybridization Incubator</td>
<td>Tool</td>
<td>Robbins Scientific</td>
<td>M1000</td>
<td>&nbsp;</td>
<tr>
<td>Pyrex dish (2)</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Watchmaker&#x2019;s glass 50mm</td>
<td>Tool</td>
<td>VWR</td>
<td>66112-060</td>
<td>&nbsp;</td>
<tr>
<td>Glass rings</td>
<td>Tool</td>
<td>Physical Plant facility</td>
<td>&nbsp;</td>
<td>cut 4 mm thick sections of glass tubing (27 mm outer diam, 25 mm inner diam). Do not fine polish.</td>
</tr>
<tr>
<td>Curved Forceps (1)</td>
<td>Surgery</td>
<td>Electron Microscopy Sciences</td>
<td>72991-4C</td>
<td>&nbsp;</td>
<tr>
<td>Forceps (2)</td>
<td>Surgery</td>
<td>Fine Science Tools</td>
<td>11002-13</td>
<td>blunt ended using sharpening Stone and 100ul mineral oil</td>
</tr>
<tr>
<td>Sharpening Stone Dan&#x2019;s Black Arkansas</td>
<td>Surgery</td>
<td>Electron Microscopy Sciences</td>
<td>62082-00</td>
<td>&nbsp;</td>
<tr>
<td>Fine scissors</td>
<td>Surgery</td>
<td>Fine Science Tools</td>
<td>14161-10</td>
<td>&nbsp;</td>
<tr>
<td>Plastic dishes</td>
<td>Tool</td>
<td>Falcon</td>
<td>353001</td>
<td>&nbsp;</td>
<tr>
<td>Rubber Bulb</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>70980</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur Capillary Pipette</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>70950-12</td>
<td>round edge under flame</td>
</tr>
<tr>
<td>Microcapillary tube</td>
<td>Surgery</td>
<td>Sigma</td>
<td>P1049-1PAK</td>
<td>Pull using vertical micropipette puller; blunt end with fine forceps</td>
</tr>
<tr>
<td>Microdissecting knife</td>
<td>Surgery</td>
<td>Fine Science Tools</td>
<td>10056-12</td>
<td>Use to puncture cavities prior to in situ hybridization</td>
</tr>
<tr>
<td>Minuten pins 0.2mm diam</td>
<td>Surgery</td>
<td>Fine Science Tools</td>
<td>26002-20</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184 Silicon Elastomer Curing Agent and Base</td>
<td>Reagent</td>
<td>Dow Corning</td>
<td>0001986475</td>
<td>Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C</td>
</tr>
<tr>
<td>Diethylpyrocarbonate (depc)</td>
<td>Reagent</td>
<td>Acros Organics</td>
<td>10025025</td>
<td>Add 1ml depc to 1l PBS; shake; autoclave</td>
</tr>		</tbody>
		</table>
		</div>

method for culture of early chick embryos ex vivo (new culture)

L&#39;embryon de poulet est un outil précieux dans l&#39;étude du développement embryonnaire précoce. Sa transparence, l&#39;accessibilité et la facilité de manipulation, en font un outil idéal pour étudier la formation et la structuration du cerveau, du tube neural, somite et primordia cœur. Applications de la culture d&#39;embryons de poulets comprennent l&#39;électroporation de constructions d&#39;ADN ou d&#39;ARN afin d&#39;analyser la fonction des gènes, des greffes de perles du facteur de croissance revêtement tels que FGF et BMP, ainsi que monter toute l&#39;hybridation in situ et immunohistochimie. Cette vidéo montre les différentes étapes de la culture d&#39;embryon de poulet; d&#39;abord, l&#39;embryon est explanté dans une solution saline. Ensuite, l&#39;embryon est centrée sur un anneau de verre. Les membranes entourant l&#39;embryon sont levées le long des murs de l&#39;anneau. L&#39;anneau est ensuite placé dans une boîte de culture contenant un pool d&#39;albumine. La boîte de culture est scellé et placé dans une chambre humide, où l&#39;embryon est cultivé pendant au moins 24 heures. Enfin, l&#39;embryon est retiré de l&#39;anneau, fixées et traitées pour d&#39;autres applications. Un guide de dépannage est également présentée.

Watch this Scientific Journal Video about Method for Culture of Early Chick Embryos ex vivo New Culture at JoVE.com

Method for Culture of Early Chick Embryos ex vivo New Culture

Cette vidéo montre la nouvelle culture, une méthode par laquelle les embryons de poulet sont cultivées en dehors de l&#39;œuf jusqu&#39;à 24 heures. Cette méthode permet d&#39;étudier le développement précoce (ligne primitive à 14 soms.), Une période correspondant à E7-9 chez la souris. Les applications de cette technique comprennent l&#39;électroporation, l&#39;hybridation in situ et immunohistochimie.

Méthode pour la culture d&#39;embryons de poulet Early ex vivo (Nouvelle 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 ...

method-for-culture-of-early-chick-embryos-ex-vivo-new-culture

Research

JoVE Journal

Biology

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

15.5K vues.  Institute of Biosciences and Technology - Texas A&M Health Science Center. Cette vidéo montre la nouvelle culture, une méthode par laquelle les embryons de poulet sont cultivées en dehors de l'œuf jusqu'à 24 heures. Cette méthode permet d'étudier le développement précoce (ligne primitive à 14 soms.), Une période correspondant à E7-9 chez la souris. Les applications de cette technique comprennent l'électroporation, l'hybridation in situ et immunohistochimie.

Vidéo: Method for Culture of Early Chick Embryos ex vivo New Culture

Double Whole Mount in situ Hybridization of Early Chick Embryos

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  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Double Mont entier hybridation in situ d&#39;embryons de poulet début

Recherche

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 ...

In vivo-like Organotypic Murine Retinal Wholemount Culture

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages.
Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used.
By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5.
In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.

In vivo-like Organotypic Murine Retinal Wholemount Culture

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

In vivo-like organotypiques Culture Wholemount murin rétiniennes

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

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 ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Culture ex vivo des cellules humaines primaires épithéliales des trompes de Fallope

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

Suivi des déformations des tissus morphogénétiques dans l&#39;embryon de poulet Early

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale expansion, may become cumbersome, laborious and time-consuming when large numbers of cells are required. To address this need, the performance of a new multi-layered cell culture vessel to facilitate easy scale up of cells from single layered T-flasks will be discussed. The flasks tested are available in 3- and 5-layer format and enable culture and complete recovery of three and five times the number of cells respectively, compared to T-175 flasks. A key feature of the BD Multi-Flask is a mix/equilibration port that allows rapid in-vessel mixing as well as uniform distribution of cells and reagents within and 
between layers of each vessel and consistently produce cells that can be cultured in an environment that is congruent to T-175 flasks.
The design of these Multi-Flasks also allows for convenient pipette access for adding reagents and cells directly into the flasks as well as efficient recovery of valuable cells and reagents and reduces risk of contamination due to pouring. For applications where pouring is preferred over pipetting, the design allows for minimal residual liquid retention so as to reduce wastage of valuable cells and reagents.

Cells play an instrumental and increasing role in research, and the discovery and development of new therapeutics. With this increasing need for greater number of cells we need more efficient and effective ways for growing and harvesting attachment dependent cells. A Multilayered flask with the right features can serve this purpose.

Scale-Up de la culture des cellules de mammifères en utilisant un nouveau flacon multicouches

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale ...

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; Méthode de la culture pour le embryonnaire Souris Coeur

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

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

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

La méthode la Culture Slice pour la suite du développement des germes de dents dans explantation Culture

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

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

Ex vivo Culture de la peau de souris embryonnaires et Live-imagerie de la migration mélanoblaste

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...