Wir möchten Andrea Portbury für die kritische Durchsicht des Manuskripts und die NIH (grant # R01HL061656) für finanzielle Unterstützung danken.

1. Herausschneiden der embryonalen Herzen <ol><li> Euthanize eine Zeitfahr-schwangeren Maus an der gewünschten embryonalen Tag mit einem zugelassenen Euthanasie Technik. Alle Experimente wurden von der Institutional Animal Care und Use Committee an der University of North Carolina in Chapel Hill genehmigt. </li><li> Liberally sprühen die weiblichen mit 70% Ethanol vor Dissektion. Öffnen Sie den weiblichen Bauchhöhle abzurufen und Verbrauchsteuern die Uterushorns. </li><li> Legen Sie die Uterushorns in eine Petrischale mit kaltem 1x Phosphat-gepufferte Kochsalzlösung (PBS) und spülen Sie nach Bedarf. Schneiden Sie die Uterushorns über der Mittellinie, um die angeschlossenen embryonalen Säcke freizulegen. </li><li> Schneiden Sie einen Fruchtblase und Abrufen eines Embryos. Setzen Sie den Embryo in einer zweiten Petrischale mit kaltem PBS. Bringen Sie die Petrischale mit dem Uterushorn zu Eis. </li><li> Enthaupte des Embryos, um den Zugriff auf die Brustwand zu verbessern. Wenn Genotypisierung notwendig ist, zu entfernen und speichern den Schwanz in ein Eppendorf-Röhrchen auf Eis gelegt. </li><li> Mit demEmbryo auf dem Rücken, schnitt den Brustwand auf das Herz und die Lunge zu visualisieren. Je nach Stadium kann die Brustwand transparent sein. </li><li> Heben Sie das Herz mit einer Pinzette und schneiden Sie die Schiffe unten. Dann, vor den großen Gefäßen geschnitten, um das Herz zu befreien. Wenn nötig, entfernen Sie überflüssige Gewebe. </li><li> Legen Sie das Herz in eine Vertiefung einer 24-Well-Schale mit kaltem PBS gefüllt und die Schale auf Eis. </li><li> Wiederholen Sie die vorherigen Schritte, bis alle Herzen von Embryonen herausgeschnitten haben. </li></ol> 2. Kultur Set-up <ol><li> In einer laminaren Haube verbinden kaltem Matrigel und die gewünschte Kulturmedium (z. B. 10% fötalem Rinderserum (FBS) in Dulbecco modifiziertem Eagle Medium (DMEM)) im Verhältnis 1:1. Nicht vorwärmen das Kulturmedium. </li><li> Keeping the verdünnter Matrigel auf Eis, Pipette 500-1000 ul der verdünnten Lösung in eine Vertiefung einer 24-Well-Kulturschale, die auch auf Eis gehalten. </li><li> In der Haube, sorgfältig abholen und legen Sie den ausgeschnittenenHerz in einer Vertiefung der Matrigel enthaltenden Kulturschale während die Kulturschale auf Eis. </li><li> Wenn Ausrichtung des eingebetteten Herz ist entscheidend: Großzügig sprühen einem inversen Mikroskop und den umgebenden Raum mit 70% Ethanol. In der Haube, legen Sie das ausgeschnittene Herz in einer Vertiefung der Matrigel-haltigen Kultur Platte, während immer noch auf Eis. Legen Sie dann die Platte auf einem Mikroskop und schnell orientieren die ausgeschnitten Herz als das Matrigel zu verfestigen beginnt. </li><li> Legen Sie die Kulturschale in einem befeuchteten 37 ° C Inkubator (5% CO 2) und lassen Sie das Matrigel für ca. 30 min zu festigen. </li><li> 1 ml vorgewärmten Kulturmedium in jede Vertiefung, die ein Herz. </li><li> Herzen können in Kultur für mindestens vier Tage bleiben. Ändern das Kulturmedium alle zwei Tage wird empfohlen. </li></ol> 3. Fixierung und Visualisierung <ol><li> Falls gewünscht, einen fluoreszierenden Farbstoff lebenden Zellen (zB Syto-16), um die Live-Herz visualisieren. Fotografie wirdbegrenzt auf die Seite des Herzens, die sichtbar ist, basierend auf der Position, in der sie eingebettet ist. </li><li> Wenn Post-Kultivierung Analysen (z. B. ganze Berg Bildgebung oder Schneiden) durchgeführt werden, entfernen Sie das Kulturmedium und ersetzen mit kaltem 4% Formaldehyd. Die Schale auf Eis für 30 min. </li><li> Nach dem kalten Eis Formaldehyd und fördern Auflösung des Matrigel, ersetzen Sie das Fixativ (und Matrigel) mit frischem Formaldehyd und beheben Herzen über Nacht bei 4 ° C. </li><li> Waschen Sie die Herzen mit Puffer (zB PBS oder Tris) und bei 4 ° C bis zur weiteren Analyse. </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>

Die Autoren erklären, dass sie keine finanziellen Interessen haben.

Die aktuelle Kultur-System stellt erhebliche Vorteile für die embryonale Maus Herz Studien. Diese Kultur-System bewahrt Kontraktilität und die koronare Plexus, mit begrenzten Zeichen der Nekrose, auch nach vier Tagen in Kultur. Ferner stellt die halbfeste Matrix genug Unterstützung, um die dreidimensionale Morphologie der Entwicklung von Herz zu halten, während eine Flexibilität bis zum Vertragsabschluss und auch beschichtete Kügelchen in Position zu halten, während der Kultur. Trotz dieser Unterstützung ist die...

In den letzten Jahren hat sich die transgene Maus die vorherrschende Modellsystem zur Untersuchung der Entwicklung Herzfehler. Aber auch andere Modell Organismen, wie zum Beispiel dem Zebrafisch, erwiesen erhebliche Vorteile gegenüber der Maus haben. Drei wichtige Vorteile der Zebrafisch sind die externen Eiablage zur Erleichterung des Zugangs zu den Embryonen, die optische Transparenz der Embryonen, die einen einfachen Visualisierung der kardialen Entwicklung ermöglicht, und die Leichtigkeit der Anwendung niedermolekulare Behandlungen zu modulieren Entwicklung des Embryos 1. Somit würde die Entwicklung einer Technik, die ex utero Wachstum eines embryonalen Organ erlaubt umgehen, wenigstens teilweise die Grenzen forschungsaktuell Studium Entwicklungsprozesse in transgenen Mäusen erfahren. Ex vivo kardialen Kultur-Systeme sowohl in der Küken und Maus-Embryo entwickelt, ermöglichen die Behandlung mit kleinen Molekülen und Analyse der verschiedenen Regionen des Herzens kommunizieren 2-6. Für ganze Maus Herz kultur, herzen von Embryonen bis zu embryonalen genommen (E) 12.5 Alter kann in Kulturmedium mit oder ohne rocking 2,3,5 platziert werden. Mit dieser Technik haben embryonalen Herzen erfolgreich auf die Gleichwertigkeit der E13.5 inkubiert und Herzen mit rocking kultiviert wurden so lange wie drei Tage (ab E10.5) 3 erhalten. Es wurden jedoch keine Studien die erfolgreiche Kultur der Herzen von älteren Embryonen berichtet. Ebenso wurden Experimente rescue dem Aufbringen des therapeutischen Mittels global zu dem Kulturmedium 2 begrenzt. Ein Stück Kultur, bei dem Herzen ausgeschnitten, eingebettet sind, und im Schnitt mit einem Vibratom hat auch für jüngere Herzen wie E12.5 Maus Herzen und Hamburger-Hamilton Stufe 36 (etwa E16 in der Maus) Küken Herzen ausgenutzt 2,4,6 und ältere Herzen, wie postnatalen und adulten Maus heARTS und erwachsenen menschlichen Herzen 7,8. Während die embryonalen Analysen typischerweise 150 um dicke Abschnitte 2,4 genutzt haben, können eine große Schnittdicke als 500 um betragen ohne Nachweis von Sauerstoffmangel 8. Diese Scheibe Kulturen haben so lange wie zwei Monate in Kultur gehalten wurden, mit den meisten Scheiben Aufrechterhaltung Kontraktilität während dieser Zeit 9. Im Vergleich zu Studien in isolierten Herzmuskelzellen, erlauben diese Scheibe Kulturen der Co-Kultur von Herzmuskelzellen mit ihren benachbarten Zelltypen und eine nützliche Methode zur ex vivo Analyse. Jedoch erfordern diese Kulturen aufwendiger Satzes als einfach dadurch ein Herz in Kulturmedium (zB Einbetten des lebenden Herzen zum Schneiden auf einem Vibratom), und eine Analyse ist offensichtlich auf den Teil des Herzens innerhalb des Abschnitts begrenzt. Angesichts der oben beschriebenen Einschränkungen für die Kultivierung embryonaler Maus Herzen und der Fülle von transgenen Mäusen available für Studie haben wir eine Ex-vivo-Maus Herz Kultur-System ähnlich wie ein ex vivo Lungen-Kultur-System von Weaver entwickelt zu werden, ermöglicht et al. Unsere Kultur 10 System langfristig Kultur und Visualisierung der Umbau Koronarzirkulation innerhalb ganzen embryonalen Maus Herzen. Darüber hinaus ermöglicht die Verwendung von Matrigel Perlen in in der Nähe des Herzens gehalten werden, wodurch eine lokalisierte Behandlung mit therapeutischen Mitteln. Diese Versuche können in verschiedenen Entwicklungsstadien Zeitpunkten durchgeführt werden, um die Wirkung einer bestimmten Behandlung auf ein Verfahren wie koronare Bildung zu vergleichen. Da kleine Moleküle durch Matrigel diffundieren kann, kann dieses Kultursystem auch zur Kultur präpariert Regionen des Herzens in der Nähe zueinander, um zu bestimmen, ob bestimmte Zell-Zell-Kontakte, die für bestimmte Entwicklungsprozesse oder ob parakrine Signalisierung von einem Bereich zum anderen ist es verwendet werden erforderlich. Diese Kultur-Systemist relativ einfach und im Gegensatz zur Scheibe Kultur-System, nutzt grundlegende Kultur Reagenzien und Set-ups, die leicht verfügbar in den meisten Labors sind. Kurz gesagt, werden herausgeschnitten embryonalen Herzen in verdünnter Matrigel, das eine semi-festen Träger enthält, kultiviert. Diese Unterstützung ausreichend ist, um die dreidimensionale Morphologie des Herzens befindet und ermöglicht gleichzeitig das Herz kontrahiert. Mit diesem System können ganze Herzen von älteren Maus-Embryonen (E14.5-E16.5) in Kultur für bis zu vier Tage gehalten werden. Die gesamte koronare Plexus aufrechterhalten wird, anders als in den Schnittkulturen, so dass alle Signal-Signale, die von verschiedenen Regionen des Herzens auftreten vorhanden bleiben. Darüber hinaus können in lebenden Zellen Fluoreszenzfarbstoffe durchdringen die Matrigel bis zur Visualisierung der Live-Herzen zu ermöglichen, und Protein-konjugierten Perlen können in der Nähe des Herzens platziert werden, um eine lokalisierte Signalisierung Quelle bereitzustellen. Zusammen machen diese Vorteile diese Technik eine ideale Methode zur Untersuchung Entwicklungsprozesse im Embryoic Maus Herz.

<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

Developmental Studien in der Maus durch die Unzugänglichkeit des Embryos während der Schwangerschaft behindert. So, um Protokolle zu isolieren und Kultur einzelner Organe von Interesse sind unerlässlich, um ein Verfahren zur Visualisierung von Veränderungen sowohl in der Entwicklung und ermöglicht neuartige Behandlungsstrategien bieten. Um die langfristige Kultur der embryonalen Herzen im späten Stadien der Schwangerschaft zu fördern, haben wir ein Protokoll, in dem die ausgeschnittene Herz in einem semi-solid, verdünnter Matrigel kultiviert wird. Dieses Substrat bietet genug Unterstützung, um die dreidimensionale Struktur zu erhalten, ist aber flexibel genug, um anhaltende Kontraktion zu ermöglichen. Kurz Herzen sind aus dem Embryo herausgeschnitten und in einem Gemisch aus kaltem Matrigel 1:1 mit Wachstumsmedium. Nach dem verdünnten Matrigel erstarrt, wird das Wachstum mittel-bis der Kulturschale gegeben. Herzen so spät wie embryonalen Tag 16,5 herausgeschnitten lebensfähig waren für vier Tage nach der Präparation. Analyse der koronaren Plexus zeigt, dass dieses Verfahren nicht der Fall iststören koronaren vaskulären Entwicklung. So stellen wir eine neue Methode für die langfristige Kultur von embryonalen Herzen.

Unter Verwendung dieser Technik das Herz seine dreidimensionale Morphologie erhält und lebensfähig bleibt, wie durch fortgesetzte Kontraktionen (Movie 1). Diese Kontraktionen sind durchweg mehr im Vordergrund als in den Vorhöfen in die Herzkammern. Nach Kultur, kann Herzen fixiert und entweder für Immunohistochemie oder Histologie Marker-Expression oder Strukturen untersuchen verarbeitet werden. 1A zeigt den Boden der Kammern und der großen Arterien eines embryonalen Maus Herz, das...

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

Developmental Studien in der Maus durch die Unzugänglichkeit des Embryos während der Schwangerschaft behindert. Um die langfristige Kultur der embryonalen Herzen im späten Stadien der Schwangerschaft zu fördern, haben wir ein Protokoll, in dem die ausgeschnittene Herz in einem semi-solid, verdünnter Matrigel kultiviert wird.

A Novel<em&gt; Ex vivo</em&gt; Kultur Verfahren zur embryonalen Maus Herz

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

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Research

JoVE Journal

Biology

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

13.2K Aufrufe.  University of North Carolina at Chapel Hill. Developmental Studien in der Maus durch die Unzugänglichkeit des Embryos während der Schwangerschaft behindert. Um die langfristige Kultur der embryonalen Herzen im späten Stadien der Schwangerschaft zu fördern, haben wir ein Protokoll, in dem die ausgeschnittene Herz in einem semi-solid, verdünnter Matrigel kultiviert wird.

Serie: 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.

Method for Culture of Early Hühnerembryonen 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 ...

Forschung

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.

In utero und ex vivo Elektroporation für die Genexpression in Maus retinalen Ganglienzellen

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.

Embryonalen Maus-Lung Kultur, A-System, die molekularen Mechanismen des Branching Bewerten

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.

Ein System für<em&gt; Ex vivo</em&gt; Kultivierung von embryonalen Pankreas

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.

Eine neue Methode für die Kultur und polarisierte Stimulation der menschlichen Darmschleimhaut Explantate

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.

Isolierung von embryonalen ventrikuläre Endothelzellen

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.

Maus embryonalen Entwicklung in einem Serum-freien 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 ...

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-Kultur von embryonalen Maus-Haut-und-Live-Darstellung von Migration Melanoblasten

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.

Proteinisolierung aus den Entwicklungs embryonalen Mäuseherzklappen Region

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

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

Die c-fos-Protein Immunohistologische Nachweis: Ein nützliches Werkzeug als Marker der Mittelwegen beteiligt sind in spezifischen physiologischen Antworten<em&gt; In Vivo</em&gt; und<em&gt; Ex Vivo</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...