Vorremmo ringraziare Andrea Portbury per la lettura critica del manoscritto e del NIH (borsa # R01HL061656) per il supporto finanziario.

1. Ablazione del Hearts embrionali <ol><li> Eutanasia di un mouse timed-incinta al giorno embrionale desiderato utilizzando una tecnica di eutanasia approvato. Tutti gli esperimenti sono stati approvati dalla cura degli animali e del Comitato Istituzionale Usa presso la University of North Carolina a Chapel Hill. </li><li> Liberamente spruzzare la femmina con il 70% di etanolo prima di dissezione. Aprire cavità addominale della femmina per recuperare e accise il corno uterino. </li><li> Collocare il corno uterino in una capsula di Petri contenente freddo 1x tampone fosfato salino (PBS) e sciacquare come necessario. Tagliare il corno uterino attraverso la linea mediana per esporre i sacchi embrionali allegati. </li><li> Tagliare un sacco embrionale e recuperare un embrione. Posizionare l&#39;embrione in una seconda capsula di Petri con PBS freddo. Riportare la capsula di Petri con il corno uterino di ghiaccio. </li><li> Decapitare l&#39;embrione per migliorare l&#39;accesso alla parete toracica. Se genotipizzazione è necessario, rimuovere e salvare la coda in una provetta Eppendorf posti su ghiaccio. </li><li> Con l&#39;embrione sul dorso, tagliare aprire la parete toracica per visualizzare il cuore e polmoni. A seconda della fase, la parete toracica può essere trasparente. </li><li> Sollevare delicatamente il cuore con pinze e tagliare i vasi sotto. Quindi, tagliare sopra i grandi vasi di liberare il cuore. Se necessario, rimuovere i tessuti estranei. </li><li> Mettere il cuore in un pozzetto di un piatto da 24 pozzetti riempiti con PBS freddo e porre il recipiente sul ghiaccio. </li><li> Ripetere i passaggi precedenti fino a quando i cuori sono stati rimossi da tutti gli embrioni. </li></ol> 2. Cultura Set-up <ol><li> In una cappa laminare, combinare Matrigel freddo e il terreno di coltura desiderato (ad esempio 10% di siero fetale bovino (FBS) in Dulbecco Modified Eagle Medium (DMEM)) in rapporto 1:1. Non pre-riscaldare il terreno di coltura. </li><li> Mantenendo la Matrigel diluita in ghiaccio, pipetta 500-1000 microlitri della soluzione diluita in un pozzetto di una piastra da coltura da 24 pozzetti che è inoltre mantenuto in ghiaccio. </li><li> Nel cofano, accuratamente raccogliere e posizionare il asportatocuore in un pozzetto della piastra da coltura Matrigel contenente mantenendo il piatto cultura su ghiaccio. </li><li> Se l&#39;orientamento del cuore incorporato è cruciale: Liberamente spruzzare un microscopio invertito e lo spazio circostante con il 70% di etanolo. Nella cappa, posizionare il cuore staccato in un pozzetto della piastra di coltura Matrigel contenente mentre ancora sul ghiaccio. Quindi, posizionare la lastra su un microscopio e di orientarsi velocemente il cuore asportato come Matrigel inizia a solidificare. </li><li> Posizionare il piatto di coltura in un umidificata 37 ° C incubatore (5% di CO 2) e lasciare il Matrigel a solidificare per circa 30 min. </li><li> Aggiungere 1 ml di terreno di coltura pre-riscaldato a ciascun pozzetto contenente un cuore. </li><li> Cuori possono rimanere in coltura per almeno quattro giorni. Cambiare il terreno di coltura ogni due giorni è raccomandato. </li></ol> 3. Fissazione e visualizzazione <ol><li> Eventualmente aggiungere un colorante fluorescente live-cella (ad es Syto-16) per visualizzare il cuore vivo. Fotografia saràlimitata al lato del cuore che è visibile in base alla posizione in cui è stato incorporato. </li><li> Se saranno eseguite le analisi post-coltura (ad esempio l&#39;imaging monte tutto o sezionamento), rimuovere il terreno di coltura e sostituirlo con il freddo il 4% di formaldeide. Porre la capsula in ghiaccio per 30 min. </li><li> Dopo la formaldeide fredda e ghiaccio promuovono dissoluzione del Matrigel, sostituire il fissativo (e Matrigel) con formaldeide fresco e fissare cuori notte a 4 ° C. </li><li> Lavare i cuori con il tampone (ad esempio PBS o Tris) e conservare a 4 ° C fino a quando ulteriori analisi. </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>

Gli autori dichiarano di non avere interessi finanziari in competizione.

Il sistema attuale cultura pone vantaggi significativi per gli studi embrionali cuore del mouse. Questo sistema di coltura preserva contrattilità miocardica e il plesso coronario, con segnali limitati di necrosi, anche dopo quattro giorni di coltura. Inoltre, la matrice semi-solido fornisce supporto sufficiente a mantenere la morfologia tridimensionale del cuore di sviluppo pur consentendo flessibilità per contratto e anche per tenere perline rivestite in posizione durante cultura. Nonostante questo sostegno, questa m...

Negli ultimi anni, il topo transgenico è stato il sistema modello per lo studio predominante difetti cardiaci sviluppo. Tuttavia, altri organismi modello, come il pesce zebra, hanno dimostrato di avere vantaggi significativi rispetto al mouse. Tre vantaggi principali del zebrafish sono la posa esterna delle uova, per facilità di accesso alle embrioni; la trasparenza ottica degli embrioni, che permette una facile visualizzazione di sviluppo cardiaco, e la facilità di applicazione piccole trattamenti molecolari per modulare sviluppo dell&#39;embrione 1. Pertanto, lo sviluppo di una cultura tecnica che ha permesso ex crescita in utero di un organo embrionale sarebbe bypassare, almeno in parte, le limitazioni attualmente sperimentato dai ricercatori che studiano i processi dello sviluppo in topi transgenici. Sistemi di coltura ex vivo cardiaci sono stati sviluppati sia nella pulcino ed embrione di topo che permettono il trattamento con piccole molecole e analisi di come differenti regioni del cuore comunicano 2-6. Per tutta la cultura del cuore del mouse, cuori prelevati da embrioni fino a embrionale (E) 12,5 di età può essere posizionato in terreno di coltura con o senza dondolo 2,3,5. Usando questa tecnica, cuori embrionali sono state incubate con successo per l&#39;equivalenza di E13.5 e cuori coltura con dondolo sono stati mantenuti fino a tre giorni (partendo E10.5) 3. Tuttavia, nessuno studio ha riportato la cultura di successo di cuori da embrioni anziani. Allo stesso modo, gli esperimenti di salvataggio è stata limitata ad applicare l&#39;agente terapeutico globalmente al mezzo di coltura 2. Un sistema di coltura fetta, in cui i cuori sono asportati, incorporati e sezionato con un vibratome, è stato anche utilizzato per entrambi i cuori più giovani, come E12.5 i cuori del mouse e Hamburger-Hamilton stadio 36 (circa E16 nel topo) pulcino cuori 2,4,6, e più cuori, come il post-natale e di topo adulto hearts e cuori umani adulti 7,8. Mentre le analisi embrionali sono tipicamente utilizzate sezioni spesse 150 micron 2,4, spessore della sezione può essere un grande come 500 micron senza evidenza di privazione di ossigeno 8. Queste culture fetta sono state mantenute fino a due mesi di cultura, con la maggior parte delle fette mantenendo contrattilità in tutto questo periodo 9. Rispetto agli studi in cardiomiociti isolati questi culture fetta consentono il co-coltura di cardiomiociti con i loro tipi di cellule adiacenti e forniscono un metodo utile per l&#39;analisi ex vivo. Tuttavia, queste colture richiedono più elaborato set-up che semplicemente posizionando un cuore in mezzo di coltura (per es incorporamento cuore vivo per il sezionamento su un vibratome), e ogni analisi è ovviamente limitata alla porzione del cuore all&#39;interno della sezione. Date le limitazioni sopra descritte per la coltura di embrioni cuori del mouse e la ricchezza di topi transgenici available per lo studio, abbiamo sviluppato un sistema di coltura del mouse cuore ex vivo simile a un sistema di coltura polmone ex vivo sviluppato da Weaver, et 10 Il nostro sistema di cultura al. permette cultura a lungo termine e la visualizzazione della circolazione coronarica rimodellamento all&#39;interno interi embrionali cuori del mouse. Inoltre, l&#39;utilizzo di Matrigel permette perline per essere tenute in posizione vicina al cuore, fornendo così un trattamento localizzato con agenti terapeutici. Questi esperimenti possono essere eseguiti a differenti tempi di sviluppo per confrontare l&#39;effetto di un dato trattamento su un processo come la formazione di arteria coronaria. Perché le piccole molecole possono diffondere attraverso Matrigel, questo sistema di coltura può essere utilizzato anche per la cultura regioni sezionati del cuore vicino all&#39;altro per determinare se specifici contatti cellula-cellula sono necessarie per alcuni processi di sviluppo o se segnalazione paracrina da una regione all&#39;altra è necessario. Questo sistema di colturaè relativamente semplice e, a differenza del sistema di coltura fetta, fa uso di reagenti cultura di base e set-up che sono facilmente reperibili nella maggior parte dei laboratori. In breve, cuori embrionali escisse sono coltivate in Matrigel diluito, che fornisce un semi-solido supporto. Questo supporto è sufficiente a mantenere la morfologia tridimensionale del cuore permettendo anche la contrazione del cuore. Utilizzando questo sistema, cuori interi da anziani embrioni di topo (E14.5-E16.5) possono essere mantenute in coltura per un massimo di quattro giorni. L&#39;intero plesso coronario è mantenuto, a differenza delle culture fetta, quindi eventuali spunti di segnalazione che si verificano da diverse regioni del cuore rimangono presenti. Inoltre, coloranti fluorescenti live-cellulari possono permeare la Matrigel per permettere la visualizzazione del cuore in diretta, e perle di proteine ​​coniugate possono essere collocati in prossimità del cuore di fornire una fonte di segnalazione localizzata. Insieme, questi vantaggi fanno di questa tecnica un metodo ideale per lo studio dei processi di sviluppo in embryonic cuore del mouse.

<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

Studi di sviluppo nel topo sono ostacolati dalla inaccessibilità dell&#39;embrione durante la gestazione. Così, protocolli di isolare e cultura singoli organi di interesse sono essenziali per fornire un metodo di entrambe visualizzando cambiamenti nello sviluppo e consentendo nuove strategie terapeutiche. Per promuovere la cultura a lungo termine del cuore embrionale in fase avanzata di gestazione, abbiamo sviluppato un protocollo in cui il cuore asportato è colto in un semi-solido, Matrigel diluito. Questo substrato fornisce supporto sufficiente per mantenere la struttura tridimensionale, ma è sufficientemente flessibile per consentire una continua contrazione. In breve, i cuori sono tagliate dal embrione e collocate in una miscela di Matrigel freddo diluito 1:1 con terreno di crescita. Dopo la Matrigel diluito solidifica, è aggiunto terreno di crescita al piatto cultura. Cuori asportati il ​​più tardi giorno embrionale 16,5 erano vitali per quattro giorni dopo la dissezione. Analisi del plesso coronarico dimostra che questo metodo noninterrompere lo sviluppo vascolare coronarico. Così, presentiamo un nuovo metodo per coltura a lungo termine di cuori embrionali.

Usando questa tecnica, il cuore mantiene la sua morfologia tridimensionale e rimane vitale, come indicato dalle continue contrazioni (Film 1). Queste contrazioni sono sempre più prominente negli atri che nei ventricoli. Seguendo cultura, cuori possono essere fissati e trattati per immunoistochimica sia o istologia per esaminare l&#39;espressione marker specifico o strutture. Figura 1A mostra la base dei ventricoli e grandi arterie di un cuore embrionale di topo che è stato coltivato p...

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

Studi di sviluppo nel topo sono ostacolati dalla inaccessibilità dell&#39;embrione durante la gestazione. Per promuovere la cultura a lungo termine del cuore embrionale in fase avanzata di gestazione, abbiamo sviluppato un protocollo in cui il cuore asportato è colto in un semi-solido, Matrigel diluito.

A Novel<em&gt; Ex vivo</em&gt; Metodo di Cultura per il mouse embrionale del cuore

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 Views.  University of North Carolina at Chapel Hill. Studi di sviluppo nel topo sono ostacolati dalla inaccessibilità dell'embrione durante la gestazione. Per promuovere la cultura a lungo termine del cuore embrionale in fase avanzata di gestazione, abbiamo sviluppato un protocollo in cui il cuore asportato è colto in un semi-solido, Matrigel diluito.

Video: 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.

Metodo per la Cultura di Chick primi embrioni ex vivo (Nuova Cultura)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Ricerca

Biologia

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 elettroporazione in vivo ed ex utero per l&#39;espressione genica in cellule gangliari della retina del mouse

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.

Embrionali di topo Cultura Lung, un sistema per valutare i meccanismi molecolari della 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 sistema per<em&gt; Ex vivo</emColtura&gt; del pancreas embrionale

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.

Un nuovo metodo per la Cultura e polarizzata stimolazione umani espianti mucosa intestinale

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.

Isolamento della embrionali ventricolari cellule endoteliali

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.

Embrionali di topo di sviluppo in un embrione di tutta la cultura di sistema privo di siero

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 Cultura embrionali di topo della pelle e live-imaging delle Migrazioni Melanoblast

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.

Proteine ​​Isolamento dal Sviluppare embrionali di topo Cuore Valve Regione

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.

La proteina C-FOS immunoistologiche Detection: uno strumento utile come indicatore di percorsi centrali coinvolti nelle risposte fisiologiche specifiche<em&gt; In Vivo</em&gt; e<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, ...