Nous tenons à remercier Andrea Portbury pour la lecture critique du manuscrit et le NIH (subvention n R01HL061656) pour le soutien financier.

1. Exciser les coeurs embryonnaires <ol><li> Euthanasier une souris timed-enceinte au jour embryonnaire désirée à l&#39;aide d&#39;une technique d&#39;euthanasie approuvé. Toutes les expériences ont été approuvées par le soin des animaux et du Comité institutionnel d&#39;utilisation à l&#39;Université de Caroline du Nord à Chapel Hill. </li><li> Généreusement pulvériser la femelle avec de l&#39;éthanol à 70% avant la dissection. Ouvrez la cavité abdominale de la femelle pour récupérer et exciser la corne utérine. </li><li> Placez la corne utérine dans une boîte de Pétri contenant froid 1x saline tamponnée au phosphate (PBS) et rincer si nécessaire. Couper la corne utérine via la ligne médiane pour exposer les sacs embryonnaires joints. </li><li> Couper un sac embryonnaire et récupérer un embryon. Placez l&#39;embryon dans une seconde boîte de Pétri avec du PBS froid. Retourner la boîte de Pétri avec la corne utérine à la glace. </li><li> Décapite l&#39;embryon pour améliorer l&#39;accès à la paroi thoracique. Si génotypage est nécessaire, enlever et sauver la queue dans un tube Eppendorf placé sur la glace. </li><li> Avec l&#39;embryon sur le dos, coupé ouvrir la cage thoracique pour visualiser le cœur et les poumons. Selon le stade, la paroi thoracique peut être transparent. </li><li> Soulevez délicatement le cœur à l&#39;aide des pinces et couper les vaisseaux ci-dessous. Ensuite, coupez au-dessus des grands vaisseaux de libérer le cœur. Si nécessaire, enlever les tissus étrangers. </li><li> Placez le cœur dans un puits d&#39;une plaque de 24 puits rempli de PBS froid et placez le plat sur la glace. </li><li> Répéter les étapes précédentes jusqu&#39;à cœurs ont été supprimés de tous les embryons. </li></ol> 2. Culture Set-up <ol><li> Dans une hotte laminaire, combiner Matrigel froid et le milieu de culture souhaitée (par exemple 10% de sérum de veau fœtal (FBS) en milieu de Eagle modifié par Dulbecco (DMEM)) à un ratio de 1:1. Ne pas préchauffer le milieu de culture. </li><li> Garder le Matrigel dilué sur la glace, pipette 500-1000 ul de la solution diluée dans un puits d&#39;une boîte de culture de 24 puits qui est également maintenu sur la glace. </li><li> Dans la hotte, choisissez soigneusement et placer le exciséecoeur dans un puits de la boîte de culture Matrigel contenant tout en gardant la boîte de culture sur la glace. </li><li> Si l&#39;orientation du cœur embarqué est cruciale: pulvériser généreusement un microscope inversé et l&#39;espace environnant avec 70% d&#39;éthanol. Dans la hotte, mettre le cœur excisé dans un puits de la plaque de culture Matrigel contenant tout en restant sur la glace. Ensuite, placer la plaque sur un microscope et orienter rapidement le coeur d&#39;exciser le Matrigel commence à se solidifier. </li><li> Placez la boîte de culture dans un incubateur humidifié 37 ° C incubateur (5% de CO 2) et laissez le Matrigel à solidifier pendant environ 30 min. </li><li> Ajouter 1 milieu de culture préchauffé ml dans chaque puits contenant un cœur. </li><li> Coeurs peuvent rester en culture pendant au moins quatre jours. Changer le milieu de culture tous les deux jours est recommandée. </li></ol> 3. Fixation et visualisation <ol><li> Si désiré, ajouter un colorant fluorescent des cellules vivantes (par exemple Syto-16) pour visualiser le cœur en direct. Photographie seralimité à la partie du coeur qui est visible en fonction de la position dans laquelle elle a été intégrée. </li><li> Si les analyses post-culture (par exemple, l&#39;imagerie mont tout ou coupe) seront effectuées, éliminer le milieu de culture et le remplacer par le froid 4% de formaldéhyde. Placer le plat sur la glace pendant 30 min. </li><li> Après le formaldéhyde froid et de la glace favorisent la dissolution du Matrigel, remplacer le fixateur (et Matrigel) avec le formaldéhyde fraîche et fixer des coeurs pendant une nuit à 4 ° C. </li><li> Laver les cœurs avec tampon (par exemple PBS ou Tris) et conserver à 4 ° C jusqu&#39;à ce que des analyses plus poussées. </li></ol>

<ol>
	<li>Tu, S., Chi, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+models+in+cardiac+development+and+congenital+heart+birth+defects.">Zebrafish models in cardiac development and congenital heart birth defects.</a> Differentiation. 84, 4-16 (2012).</li><li>Lavine, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+growth+factor+signals+regulate+a+wave+of+Hedgehog+activation+that+is+essential+for+coronary+vascular+development.">Fibroblast growth factor signals regulate a wave of Hedgehog activation that is essential for coronary vascular development.</a> Genes Dev. 20, 1651-1666 (2006).</li><li>Red-Horse, K., Ueno, H., Weissman, I. L., Krasnow, 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=Coronary+arteries+form+by+developmental+reprogramming+of+venous+cells.">Coronary arteries form by developmental reprogramming of venous cells.</a> Nature. 464, 549-553 (2010).</li><li>Stuckmann, I., Evans, S., Lassar, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythropoietin+and+retinoic+acid,+secreted+from+the+epicardium,+are+required+for+cardiac+myocyte+proliferation.">Erythropoietin and retinoic acid, secreted from the epicardium, are required for cardiac myocyte proliferation.</a> Dev. Biol. 255, 334-349 (2003).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+FGF-BMP+signaling+axis+regulates+outflow+tract+valve+primordium+formation+by+promoting+cushion+neural+crest+cell+differentiation.">The FGF-BMP signaling axis regulates outflow tract valve primordium formation by promoting cushion neural crest cell differentiation.</a> Circ. Res. 107, 1209-1219 (2010).</li><li>Pillekamp, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+and+characterization+of+a+mouse+embryonic+heart+slice+preparation.">Establishment and characterization of a mouse embryonic heart slice preparation.</a> Cellular Physiology and Biochemistry: international journal of experimental cellular physiology, biochemistry, and pharmacology. 16, 127-132 (2005).</li><li>Habeler, W., Peschanski, M., Monville, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+heart+slices+for+cell+transplantation+and+physiological+studies.">Organotypic heart slices for cell transplantation and physiological studies.</a> Organogenesis. 5, 62-66 (2009).</li><li>Janssen, P. M., Lehnart, S. E., Prestle, J., Hasenfuss, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+contractile+characteristics+of+human+myocardium+in+multi-day+cell+culture.">Preservation of contractile characteristics of human myocardium in multi-day cell culture.</a> J. Mol. Cell Cardiol. 31, 1419-1427 (1999).</li><li>Brandenburger, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+slice+culture+from+human+adult+ventricular+myocardium.">Organotypic slice culture from human adult ventricular myocardium.</a> Cardiovasc. Res. 93, 50-59 (2012).</li><li>Weaver, M., Dunn, N. R., Hogan, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmp4+and+Fgf10+play+opposing+roles+during+lung+bud+morphogenesis.">Bmp4 and Fgf10 play opposing roles during lung bud morphogenesis.</a> Development. , 127-2695 (2000).</li><li>Viragh, S., Challice, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+the+epicardium+and+the+embryonic+myocardial+circulation+in+the+mouse.">The origin of the epicardium and the embryonic myocardial circulation in the mouse.</a> Anat. Rec. 201, 157-168 (1981).</li><li>Kirby, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cardiac Development. , (2007).</li><li>Arima, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenic+morphogenesis+driven+by+dynamic+and+heterogeneous+collective+endothelial+cell+movement.">Angiogenic morphogenesis driven by dynamic and heterogeneous collective endothelial cell movement.</a> Development. 138, 4763-4776 (2011).</li></ol>

Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

Le système de culture actuelle pose des avantages significatifs pour les études cardiaques embryonnaires de souris. Ce système de culture préserve la contractilité du myocarde et le plexus coronaire, avec des signes limités de nécrose, même après quatre jours de culture. En outre, la matrice semi-solide fournit un soutien suffisant pour maintenir la morphologie tridimensionnelle du coeur en développement, tout en permettant une flexibilité de contrat et également pour maintenir des perles revêtues en place ...

Ces dernières années, la souris transgénique a été le système de modèle prédominant pour l&#39;étude des malformations cardiaques de développement. Toutefois, d&#39;autres organismes modèles tels que le poisson-zèbre, se sont avérés avoir des avantages significatifs par rapport à la souris. Trois avantages majeurs du poisson zèbre sont la pose externe des œufs, pour faciliter l&#39;accès à des embryons; la transparence optique des embryons, ce qui permet une visualisation facile du développement cardiaque, et la facilité d&#39;application de petites traitements moléculaires pour moduler le développement de l&#39;embryon 1. Ainsi, le développement d&#39;une technique de culture qui a permis la croissance des ex utero d&#39;un organe embryonnaire serait contourner, au moins en partie, les limitations actuellement rencontrées par les chercheurs qui étudient les processus de développement chez les souris transgéniques. Systèmes de culture cardiaques ex vivo ont été développés à la fois le poussin et embryon de souris qui permettent un traitement avec de petites molécules et des analyses de façon difrentes régions du cœur communiquent 2-6. Pour la culture de cœur de souris ensemble, coeurs prélevées sur des embryons jusqu&#39;à embryonnaire (E) 12.5 de l&#39;âge peut être placé dans un milieu de culture avec ou sans balancement 2,3,5. Grâce à cette technique, les cœurs embryonnaires ont été incubées avec succès à l&#39;équivalence des E13.5, et les cœurs cultivés avec rocking ont été maintenus aussi longtemps que trois jours (à partir de E10.5) 3. Cependant, aucune étude n&#39;a rapporté la culture réussie des cœurs d&#39;embryons âgés. De même, les expériences de sauvetage ont été limitées à l&#39;application de l&#39;agent thérapeutique au niveau mondial pour le milieu de culture 2. Un système de culture de tranche, dont les cœurs sont excisées, intégrées, et sectionné à l&#39;aide d&#39;un vibratome, a également été utilisé pour les deux cœurs jeunes, comme E12.5 cœurs de souris et Hamburger-Hamilton stade 36 (environ E16 chez la souris) cœurs chiches 2,4,6, et plus cœurs, comme post-natal et adulte souris heArts et les coeurs humains adultes 7,8. Bien que les analyses embryonnaires sont généralement utilisés sections épaisses de 150 um 2,4, épaisseur de coupe peut être un excellent jusqu&#39;à 500 um sans preuve d&#39;un manque d&#39;oxygène 8. Ces cultures de tranche ont été maintenus aussi longtemps que deux mois en culture, avec la plupart des tranches de maintenir la contractilité tout au long de cette période 9. Par rapport aux études dans les cardiomyocytes isolés, ces cultures de tranche permettent la co-culture de cardiomyocytes avec leurs types de cellules voisines et fournissent une méthode utile pour l&#39;analyse ex vivo. Cependant, ces cultures nécessitent plus élaborée set-up que de simplement placer un coeur dans un milieu de culture (par exemple intégrer le cœur vivant de sectionnement sur ​​un vibratome), et toute analyse est évidemment limitée à la partie du cœur au sein de la section. Compte tenu des limites décrites ci-dessus pour la culture des cœurs embryonnaires de souris et la richesse des souris transgéniques available pour l&#39;étude, nous avons développé un système ex vivo de souris de coeur de culture similaire à un système de culture du poumon ex vivo développé par Weaver, et 10 Notre système de culture al. permet culture à long terme et la visualisation de la rénovation circulation coronaire au sein de l&#39;ensemble cœurs embryonnaires de souris. En outre, l&#39;utilisation de Matrigel permet perles pour être maintenus en place à proximité du centre, offrant ainsi un traitement localisé avec des agents thérapeutiques. Ces expériences peuvent être réalisées à différents moments du développement de comparer l&#39;effet d&#39;un traitement donné sur un processus tels que la formation de l&#39;artère coronaire. Parce que les petites molécules peuvent diffuser à travers le Matrigel, ce système de culture peut également être utilisé pour la culture des régions disséquées du cœur près de l&#39;autre afin de déterminer si des contacts entre cellules spécifiques sont nécessaires pour certains processus de développement ou si la signalisation paracrines d&#39;une région à l&#39;autre est nécessaire. Ce système de cultureest relativement simple et, contrairement au système de culture de tranche, fait appel à des réactifs de culture de base et les set-ups qui sont facilement disponibles dans la plupart des laboratoires. En bref, cœurs embryonnaires excisés sont cultivées en Matrigel dilué, qui fournit un support semi-solide. Ce support est suffisante pour maintenir la morphologie tridimensionnelle du coeur, tout en permettant la contraction du coeur. Grâce à ce système, coeurs entiers de plus d&#39;embryons de souris (E14.5-E16.5) peuvent être maintenues en culture pendant quatre jours. L&#39;ensemble du plexus coronaire est maintenue, contrairement aux cultures de tranche, donc aucun repère de signalisation qui se produisent dans différentes régions du cœur restent présents. En outre, les colorants fluorescents des cellules vivantes peuvent traverser le Matrigel pour permettre la visualisation du cœur live, et des perles en protéines conjugués peuvent être placés à proximité du coeur de fournir une source de signalisation localisée. Ensemble, ces avantages font de cette technique une méthode idéale pour étudier les processus de développement dans l&#39;embryonic cœur de la souris.

<table border="1">
<tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>REAGENTS</td>
 </tr>
 <tr>
 <td>Timed-pregnant mice</td>
 <td></td>
 <td></td>
 <td>To be dissected at the embryonic stage of interest</td>
 </tr>
 <tr>
 <td>PBS (1x)</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Cellgro </td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>Sigma-Aldrich </td>
 <td>F2442 </td>
 <td></td>
 </tr>
 <tr>
 <td>Growth factor-reduced Matrigel</td>
 <td>BD Bioscience </td>
 <td>356231 </td>
 <td></td>
 </tr>
 <tr>
 <td>Syto-16</td>
 <td>Invitrogen </td>
 <td>S7578 </td>
 <td>Used as directed in 13 </td>
 </tr>
 <tr>
 <td>24-well culture plate</td>
 <td>Fisher Scientific</td>
 <td>07-200-84</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>EQUIPMENT</td>
 </tr>
 <tr>
 <td>Stereoscopic microscope</td>
 <td>Nikon </td>
 <td>SMZ645</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell culture incubator</td>
 <td>Thermo </td>
 <td>3110 </td>
 <td></td>
 </tr>
</tbody>
 </table>

a novel ex vivo culture method for the embryonic mouse heart

Des études de développement chez la souris sont entravés par l&#39;inaccessibilité de l&#39;embryon pendant la gestation. Ainsi, les protocoles d&#39;isoler et de culture différents organes d&#39;intérêt sont indispensables pour fournir une méthode de deux stratégies de traitement novatrices visualisation changements dans le développement et permettant. Afin de promouvoir la culture à long terme du cœur embryonnaire à un stade avancé de gestation, nous avons développé un protocole dans lequel le cœur excisé est cultivé dans un semi-solide, Matrigel dilué. Ce substrat fournit une aide suffisante pour maintenir la structure en trois dimensions, mais est suffisamment souple pour permettre la contraction continue. En bref, les coeurs sont excisés de l&#39;embryon et placés dans un mélange de Matrigel froid dilué 1:1 avec le milieu de croissance. Après le Matrigel dilué se solidifie, le milieu de croissance est ajouté à la boîte de culture. Coeurs excisées aussi tard que jour embryonnaire 16,5 étaient viables pendant quatre jours après dissection. Analyse du plexus coronaire montre que cette méthode ne tient pasperturber le développement vasculaire coronaire. Ainsi, nous présentons une nouvelle méthode de culture à long terme des cœurs embryonnaires.

Grâce à cette technique, le cœur conserve sa morphologie tridimensionnelle et reste viable, comme indiqué par les contractions continues (Film 1). Ces contractions sont toujours plus importante dans les oreillettes que dans les ventricules. Après la culture, coeurs peuvent être fixés et traités soit pour l&#39;immunohistochimie ou histologie pour examiner l&#39;expression de marqueurs spécifiques ou des structures. Figure 1A montre la base des ventricules et les grandes artère...

Watch this Scientific Journal Video about A Novel Ex vivo Culture Method for the Embryonic Mouse Heart at JoVE.com

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Des études de développement chez la souris sont entravés par l&#39;inaccessibilité de l&#39;embryon pendant la gestation. Afin de promouvoir la culture à long terme du cœur embryonnaire à un stade avancé de gestation, nous avons développé un protocole dans lequel le cœur excisé est cultivé dans un semi-solide, Matrigel dilué.

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 ...

a-novel-ex-vivo-culture-method-for-the-embryonic-mouse-heart

Research

JoVE Journal

Biology

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

13.3K vues.  University of North Carolina at Chapel Hill. Des études de développement chez la souris sont entravés par l'inaccessibilité de l'embryon pendant la gestation. Afin de promouvoir la culture à long terme du cœur embryonnaire à un stade avancé de gestation, nous avons développé un protocole dans lequel le cœur excisé est cultivé dans un semi-solide, Matrigel dilué.

Vidéo: A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

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

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

Method for Culture of Early Chick Embryos ex vivo New Culture

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

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 ...

Recherche

Biologie

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

En électroporation in vivo et ex utero pour l&#39;expression génique dans les cellules ganglionnaires de la rétine de la souris

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

Culture poumon embryonnaire de la souris, un système pour évaluer les mécanismes moléculaires des Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Un système d&#39;<em&gt; Ex vivo</emLa culture&gt; de pancréas embryonnaire

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

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

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

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

Une nouvelle méthode pour la Culture et polarisé stimulation des explants de muqueuse intestinale de l&#39;homme

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

Isolation of Embryonic Ventricular Endothelial Cells

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

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

Isolement des cellules endothéliales embryonnaires ventriculaires

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

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 ...

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 ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

Protein Isolation du développement embryonnaire Souris Coeur Région Valve

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Mesure de la rigidité de<em&gt; Ex Vivo</em&gt; Souris aortes Utiliser Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

A Novel

Résumé

Explorer plus de vidéos

Chapitres dans cette vidéo

Méthode pour la culture d'embryons de poulet Early ex vivo (Nouvelle Culture)

En électroporation in vivo et ex utero pour l'expression génique dans les cellules ganglionnaires de la rétine de la souris

Culture poumon embryonnaire de la souris, un système pour évaluer les mécanismes moléculaires des Branching

Un système d'

Une nouvelle méthode pour la Culture et polarisé stimulation des explants de muqueuse intestinale de l'homme

Isolement des cellules endothéliales embryonnaires ventriculaires

Souris développement embryonnaire dans tout un système de culture d'embryons sans sérum

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

Protein Isolation du développement embryonnaire Souris Coeur Région Valve

Mesure de la rigidité de