1. Ex-ovo Cultura protocollo: <ol><li> Incubare uova di gallina fecondate <ol class="lroman"><li> Uova di gallina fecondate possono essere conservati a 13 ° C fino a 5 giorni prima di incubazione senza avviare lo sviluppo. Un raffreddamento di vino rosso in grado di mantenere questa temperatura. </li><li> Incubare le uova lato smussato in un incubatore di umidità del 60% costante con dondolo continuo a 37,5 ° C per 72 ore. </li></ol></li><li> Preparare amache (Figura 1a) <ol class="lroman"><li> Riempire ¾ di 9 bicchieri di plastica oz con acqua calda sterile. Un regolare 9 oz tazza ha un diametro superiore di 8 cm. Abbiamo scoperto che queste dimensioni era più adatto per l&#39;applicazione della cultura lungo tempo presentato qui. </li><li> Taglio di circa 20 cm pellicola trasparente. Circonferenza apporre sulla parte superiore della coppa mettendo un elastico intorno. Plastica dovrebbe essere ripartito in cima l&#39;acqua. Tagliare la plastica in eccesso intorno alla band. Abbiamo trovato pellicola trasparente per essere più appropriato per questa applicazione in quanto impedisce l&#39;adesione del tuorlo, con conseguente suddivisione tuorlo durante il trasporto. </li><li> Pulire la plastica con Kimwipes (Kimberly-Clark, Inc.) bagnate con il 70% di etanolo. </li></ol></li><li> Rimuovere le uova da incubatrice dopo 72 ore di incubazione. </li><li> Sterilizzare le uova strofinando la superficie con etanolo al 70% Kimwipes bagnata. </li><li> Deporre le uova in orizzontale per 1-2 minuti per il corretto posizionamento degli embrioni (Figura 1b). Una scatola di uova può essere utilizzato per deporre le uova in orizzontale. Gli embrioni ruoterà verso l&#39;alto dopo 1-2 minuti. </li><li> Trasferimento degli embrioni su amache in modo asettico in una cappa a flusso laminare. <ol class="lroman"><li> Usando un bordo tagliente (come il bordo di un secchio di metallo o di un bicchiere di vetro), toccare delicatamente l&#39;uovo fino a quando vi è una piccola ammaccatura sul lato inferiore delle uova (Figura 1c). Ricordate, l&#39;embrione è posizionato sulla superficie superiore. </li><li> Mettete i pollici in lati opposti della ammaccatura e tirare i gusci a parte in orizzontale (Figura 1d). </li><li> Mettere il tuorlo con embrione così come l&#39;albumina sulla amaca (figura 1e). Albumina fornirà un ambiente di smorzamento intorno al tuorlo di assorbire gli shock indotto spostando il sistema così come fornisce nutrimento all&#39;embrione per le pratiche della cultura lungo. Acqua sotto l&#39;amaca garantirà l&#39;embrione rimane parallelo al terreno durante il trasporto e fungere da conduttore di calore durante l&#39;imaging se un riscaldatore a circolazione d&#39;acqua è usato. </li><li> L&#39;embrione deve essere già posizionato sulla parte superiore se il trasferimento viene effettuato correttamente. In caso contrario, utilizzare un contrasto, un oggetto sterile (es. Blunt punta di forbici curve chiuse) e direzionale accarezzare il tuorlo in modo che l&#39;embrione ruota verso l&#39;alto. </li><li> Posizionare un diametro di 10 centimetri piatto Petri in cima alla coppa per sigillare l&#39;embrione. </li></ol></li><li> Luogo cultura tazza in incubatore a 37,5 ° C. Se un incubatore portatile è usato, ponendo 01-02 settembre tazze oz riempita con acqua distillata mantiene l&#39;umidità a ~ 60% (Fig. 1f). </li><li> Se gli embrioni al di là di coltura embrionale giorni 7, pezzi di guscio d&#39;uovo tritato devono essere sparsi per la periferia dell&#39;embrione come fonte di calcio per lo sviluppo e la maturazione delle ossa. </li></ol> Equipaggiamento accessorio per esterno incubatore di imaging / manipolazione: Un bagno d&#39;acqua in circolo è stato costruito in casa per mantenere la temperatura di incubazione, mentre imaging o manipolazione (Figura 3). Un bagno d&#39;acqua che può andare bene la coppa è collegato tramite tubazione dimensioni adeguate ad un serbatoio d&#39;acqua. Un riscaldatore resistenza riscalda l&#39;acqua nel serbatoio, che viene regolata manualmente seguendo la temperatura dell&#39;acqua. Una pompa acqua circola l&#39;acqua. Rappresentazione pittorica raffigura la facilità di imaging tramite ultrasongraphy / microscopia florescent (Figura 3). <img src="/files/ftp_upload/2154/2125fig1.jpg" alt="Figura 1" /> Figura 1. Preparazione di amaca e il trasferimento di embrioni di pollo al amache. <img src="/files/ftp_upload/2154/2125fig2.jpg" alt="Figura 2" /> Figura 2. Risultati rappresentativi -. Diversi embrioni di pollo fase cresciuta attraverso la cultura tecnica ex ovo embrioni da SS 17 in poi possono essere coltivate attraverso questa tecnica. <img src="/files/ftp_upload/2154/2125fig3.jpg" alt="Figura 3" /> Figura 3. Immagini schematiche e rappresentante del sistema di circolazione dell&#39;acqua utilizzata in congiunzione con l&#39;ex-ovo sistema di coltura per mantenere gli embrioni a temperatura ideale di sviluppo. 2. Applicazioni selezionato: I. Microiniezione Questa cultura embrione è adatto per applicazioni microiniezione in cui le soluzioni devono essere iniettati per l&#39;imaging vascolare di contrasto a base (es. microscopia a fluorescenza, micro-topografia computerizzata, ecc) (Figura 4). Di seguito è riportato il protocollo microiniezione: <ol><li> Preparare il apparec iniezionetus <ol class="lroman"><li> Moda tirato 0,75 tubi di vetro capillari ID in microaghi punta smussata con un microforge. Dimensioni della punta richiesta dipende vena obiettivo diametro / portata desiderata. </li><li> Collegare la siringa a 0,03 pollici tubo in silicone ID. </li><li> Caricare il tubo con la soluzione da iniettare e collegare il microago al tubo. </li><li> Posizionare il microago sul micromanipolatore e siringa titolare siringa o pompa a siringa. </li></ol></li><li> Aggiungere acqua sterile in tazza in modo da accrescere l&#39;embrione. Abbiamo sperimentato che per la penetrazione corretta dell&#39;ago in un vaso sanguigno (es. vasi vitellina), la penetrazione orizzontale è necessario. Ciò richiede l&#39;embrione di essere sullo stesso piano, come i bordi della tazza. <ol class="lroman"><li> Estrarre l&#39;elastico tenendo la plastica sotto l&#39;embrione. </li><li> Sollevare un lato della plastica delicatamente e aggiungere un po &#39;di acqua sterile per la coppa. </li><li> Fissare l&#39;elastico sulla plastica di nuovo. </li></ol></li><li> Iniettare la soluzione di un vaso embrionale. Nella nostra esperienza, quando la soluzione viene iniettata a una nave vitellina, microaghi doveva essere di dimensioni ° circa 1 / 10 del diametro del vaso per mantenere la perfusione efficace senza causare sanguinamento. <ol class="lroman"><li> Pompa la soluzione per il microago garantendo l&#39;assenza di formazione di bolle d&#39;aria. </li><li> Allineare la punta del microago sulla nave selezionata. Per le navi vitellina, selezionando un&#39;area forchetta darà superficie massima di penetrare la nave. </li><li> Spingere l&#39;ago verso la nave. La nave per prima ritrattare. Una volta che l&#39;ago penetra, avviare perfusione. Il colore del sangue che scorre dovrebbe cambiare con perfusione. Se il cambiamento di colore non si vede, l&#39;ago potrebbe essere penetrato dalla nave. In questo caso, ritirare lentamente il microago allo stesso tempo la soluzione di pompaggio fino a cambiare colore nel vaso si vede. Una volta che la perfusione è completo, ritirare il microago fuori dal vaso. </li></ol></li></ol><img src="/files/ftp_upload/2154/2125fig4.jpg" alt="Figura 4" /> Figura 4: microiniezione di un colorante fluorescente nel sistema vascolare di un ex-ovo-embrione di pollo in coltura. II. Microchirurgico Procedura - Sinistra Legatura atriale Questo ex-ovo configurazione cultura è l&#39;ideale per l&#39;esecuzione di applicazioni chirurgiche di embrioni di pollo in quanto fornisce la piena accessibilità per l&#39;embrione. Di seguito è riportato il protocollo per una tecnica, ad esempio, la legatura atriale sinistro (LAL) (Figura 5). La procedura viene eseguita circa 24 ore nel periodo di coltura (HH24): <ol><li> Preparare overhand nodi ~ 0,5 mm di diametro con 10-0 sutura chirurgica nylon. </li><li> Con una pinza sottile, localmente rimuovere le membrane coriali e allantoide oltre l&#39;embrione e sollevare l&#39;embrione dal retro per ruotare verticalmente in modo tale che il lato sinistro dell&#39;embrione è visibile. </li><li> Utilizzando una pinza sottile, rimuovere il pericardio sopra l&#39;atrio sinistro. </li><li> Allineare il nodo sopra l&#39;atrio sinistro e stringerlo. Il nodo dovrebbe essere legata in modo da vietare ~ 75% del flusso di sangue originale sul lato sinistro. Tagliare i bordi del nodo con microscissors e rimuovere con cautela e scartare sutura in eccesso. </li><li> Ruotare l&#39;embrione al suo orientamento originale (lato destro in alto). </li><li> Controlli Sham coinvolgerà la stessa procedura, ma la sutura sarà solo attraversato e non legato. Efficace trattamento LAL (75 costrizione%) sarà confermato a 24 ore dopo la chirurgia, e gli embrioni squalificato saranno esclusi. </li></ol><img src="/files/ftp_upload/2154/2154fig5.jpg" alt="Figura 5" /> Figura 5:. Un controllo e una sinistra embrione atriale pulcino legatura Arrow mostra gli atri sinistra, che è circa il 75% più piccolo nell&#39;embrione legato.

<ol>
	<li>Zamir, E. A., Czirok, A., Cui, C., Little, C. D., Rongish, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesodermal+cell+displacements+during+avian+gastrulation+are+due+to+both+individual+cell-autonomous+and+convective+tissue+movements.">Mesodermal cell displacements during avian gastrulation are due to both individual cell-autonomous and convective tissue movements.</a> Proc Natl Acad Sci U S A. 103, 19806-19811 (1980).</li><li>Ehrman, L. A., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+regulation+in+the+heart+forming+region+of+avian+embryos.">Lack of regulation in the heart forming region of avian embryos.</a> Dev Biol. 207, 163-175 (1999).</li><li>Clark, E. B., Hu, N., Rosenquist, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+conotruncal+constriction+on+aortic-mitral+valve+continuity+in+the+stage+18,+21+and+24+chick+embryo.">Effect of conotruncal constriction on aortic-mitral valve continuity in the stage 18, 21 and 24 chick embryo.</a> Am J Cardiol. 53, 324-327 (1984).</li><li>deAlmeida, A., McQuinn, T., Sedmera, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+ventricular+preload+is+compensated+by+myocyte+proliferation+in+normal+and+hypoplastic+fetal+chick+left+ventricle.">Increased ventricular preload is compensated by myocyte proliferation in normal and hypoplastic fetal chick left ventricle.</a> Circ Res. 100, 1363-1370 (2007).</li><li>Nakamura, H., Funahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+DNA+into+chick+embryos+by+in+ovo+electroporation.">Introduction of DNA into chick embryos by in ovo electroporation.</a> Methods. 24, 43-48 (2001).</li><li>Butcher, J. T., McQuinn, T. C., Sedmera, D., Turner, D., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transitions+in+early+embryonic+atrioventricular+valvular+function+correspond+with+changes+in+cushion+biomechanics+that+are+predictable+by+tissue+composition.">Transitions in early embryonic atrioventricular valvular function correspond with changes in cushion biomechanics that are predictable by tissue composition.</a> Circ Res. 100, 1503-1511 (2007).</li><li>McQuinn, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+ultrasonographic+imaging+of+avian+cardiovascular+development.">High-frequency ultrasonographic imaging of avian cardiovascular development.</a> Dev. Dyn. 236, 3503-3513 (2007).</li><li>Korn, M. J., Cramer, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Windowing+chicken+eggs+for+developmental+studies.">Windowing chicken eggs for developmental studies.</a> J Vis Exp. , (2007).</li><li>Auerbach, R., Kubai, L., Knighton, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+procedure+for+the+long-term+cultivation+of+chicken+embryos.">A simple procedure for the long-term cultivation of chicken embryos.</a> Dev Biol. 41, 391-394 (1974).</li></ol>

Nessun conflitto di interessi dichiarati.

Accesso ottico e sperimentazione negli embrioni aviaria è una sfida a causa dei vincoli del guscio d&#39;uovo. Windowing limita in modo significativo il numero di accesso per gli approcci di iniezione e microchirurgia 8 microvasi. Di conseguenza solo gli embrioni precoci possono essere manipolati e costante osservazione non è possibile. Primi ex-ovo culture utilizzando piastre di Petri erano di uso limitato a causa di un inadeguato controllo della tensione superficiale sull&#39;embrione impedito a ...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Fertile white Leghorn chicken eggs</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Model GB1, Avery Incubators, Hugo CO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Saran Wrap</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Kimwipes</td>
<td>&nbsp;</td>
<td>Kimberly-Clark, Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Rubber bands</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Warm sterile water</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>9 oz plastic cup</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<tr>
<td>100 mm diameter Petri dish</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>1602N thermal air</td>
<td>&nbsp;</td>
<td>GQF Manufacturing CO, Savannah GA</td>
<td>&nbsp;</td>
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<td>Fluorescein-conjugated dextran (2 MDa, 1% w/v in phosphate buffered saline)</td>
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<td>Sigma Aldrich Inc.</td>
<td>&nbsp;</td>
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<td>Microforge</td>
<td>&nbsp;</td>
<td>Glassworx, Inc, St Louis MO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Glass capillary tubes (0.75 mm ID)</td>
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<td>&nbsp;</td>
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<td>Micromanipulator</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Sarasota FL</td>
<td>Model M3301L</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent microscopy</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>Z20</td>
<td>&nbsp;</td>
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<td>Fine 55-forceps</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Sarasota FL</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>10-0 nylon surgical suture</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Tubing</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>&nbsp;</td>
<td>1mm OD</td>
</tr>
<tr>
<td>3 mL syringe</td>
<td>&nbsp;</td>
<td>BD</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>200 &mu;L pipetter and pipette tips</td>
<td>&nbsp;</td>
<td>VWR</td>
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an ex-ovo chicken embryo culture system suitable for imaging and microsurgery applications

Comprendere le relazioni tra fattori genetici e microambientali che guidano normali e malformate sviluppo embrionale è fondamentale per scoprire nuove strategie terapeutiche. I progressi nella tecnologia di imaging hanno permesso indagine quantitativa dell&#39;organizzazione e della maturazione del piano di corpo, ma una fase successiva morfogenesi embrionale è meno chiara. Embrioni di pollo sono un interessante modello animale vertebrato sistema per questa applicazione a causa della sua facilità di cultura e di manipolazione chirurgica. Embrioni precoci possono essere coltivate per un breve periodo su anelli carta da filtro, che permette l&#39;accesso completo ottici per patterning cellulare e il destino 1,2 studi. Lo studio dei processi avanzati dello sviluppo come morfogenesi cardiaca sono tradizionalmente svolte attraverso una finestra del guscio 3-5, ma questa tecnica limita l&#39;accesso ottico a causa delle dimensioni della finestra. Abbiamo già messo a punto un metodo semplice per la cultura embrioni interi franco-ovo su esagonale pesare imbarcazioni fino a 10 giorni, che ha permesso di imaging ad alta risoluzione tramite ecografia 6,7. Queste culture sono state difficili da trasportare, limitando i tipi di strumenti di imaging disponibili per esperimenti dal vivo. Noi qui presenti una migliore senza guscio sistema di coltura con un costo-efficace, camera portatile ambientale. Uova erano incrinate su un amaca creato da una membrana poliuretanica (pellicola trasparente) apposto la circonferenza un bicchiere di plastica parzialmente piena d&#39;acqua sterile. Le dimensioni della circonferenza e la profondità della amaca erano entrambi critici per mantenere la tensione superficiale, mentre la meccanica del amaca e acqua sotto aiutato smorzare le vibrazioni indotte dal trasporto. Un piccolo ingombro circolante bagno d&#39;acqua è stato anche sviluppato per consentire un controllo continuo della temperatura durante la sperimentazione. Dimostriamo la capacità di embrioni cultura in questo modo per almeno 14 giorni senza difetto morfogenici o ritardi ed impiegare questo sistema in microchirurgia diversi e applicazioni di imaging.

Watch this Scientific Journal Video about An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications at JoVE.com

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In questo articolo presentiamo una metodologia semplice per permettere a lungo termine<em&gt; Ex-ovo</em&gt; Cultura embrione aviaria. Questa tecnica è ideale per la sperimentazione longitudinale che richiedono una completa accessibilità ottici e / o il trasporto sterile negli embrioni aviaria.

Un Ex-ovo Sistema di embrioni di pollo Cultura Adatto per applicazioni di imaging e Microchirurgia

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

an-ex-ovo-chicken-embryo-culture-system-suitable-for-imaging

Research

JoVE Journal

Biology

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

22.9K Views.  Cornell University. In questo articolo presentiamo una metodologia semplice per permettere a lungo termine Ex-ovo Cultura embrione aviaria. Questa tecnica è ideale per la sperimentazione longitudinale che richiedono una completa accessibilità ottici e / o il trasporto sterile negli embrioni aviaria.

Video: An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform routine activities, but prosthetic suspensions using traditional socket technology can prove to be cumbersome and uncomfortable for a person with limb loss.  Moreover, for those with high proximal amputations, limited residual limb length may prevent exoprosthesis attachment all together. Osseointegrated implant technology is a novel operative procedure which allows firm skeletal attachment between the host bone and an implant. Preliminary results in European amputees with osseointegrated implants have shown improved clinical outcomes by allowing direct transfer of loads to the bone-implant interface.  Despite the apparent advantages of osseointegration over socket technology, the current rehabilitation procedures require long periods of restrictive load bearing prior which may be reduced with expedited skeletal attachment via electrical stimulation.  The goal of the osseointegrated intelligent implant design (OIID) system is to make the implant part of an electrical system to accelerate skeletal attachment and help prevent periprosthetic infection. To determine optimal electrode size and placement, we initiated proof of concept with computational modeling of the electric fields and current densities that arise during electrical stimulation of amputee residual limbs.  In order to provide insure patient safety, subjects with retrospective computed tomography scans were selected and three dimensional reconstructions were created using customized software programs to ensure anatomical accuracy (Seg3D and SCIRun) in an IRB and HIPAA approved study.  These software packages supported the development of patient specific models and allowed for interactive manipulation of electrode position and size. Preliminary results indicate that electric fields and current densities can be generated at the implant interface to achieve the homogenous electric field distributions   required to induce osteoblast migration, enhance skeletal fixation and may help prevent periprosthetic infections.  Based on the electrode configurations experimented with in the model, an external two band configuration will be advocated in the future.

There is a need to develop alternative prosthesis attachment due to limb loss attributed to vascular occlusive diseases and trauma. The goal of the work is to introduce an osseointegrated intelligent implant design system to increase skeletal fixation and reduce periprosthetic infection rates for patients needing osseointegrated technology.

Analisi di un Sistema di bioelettrica Osteointegrata Intelligent Design impianto per amputati

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform ...

Ricerca

Biologia

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

Pulcino Ex ovo Cultura e Ex ovo CAM saggio: Come funziona veramente

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

Lo xenotrapianto di cellule staminali umane in embrioni di pollo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

Il metodo di coltura degli embrioni roditori intero utilizzando il Rotator tipo di sistema cultura Bottiglia

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice. HYPERStack vessels allow closed system scale up from the current stacked plate products and delivers >2.5X more cells in the same volumetric footprint. The HYPERStack vessels function via gas permeable material which allows gas exchange to occur, therefore eliminating the need for internal headspace within a vessel. The elimination of headspace allows the compartment where cell growth occurs to be minimized to reduce space, allowing more layers of cell growth surface area within the same volumetric footprint.
For many applications such as cell therapy or vaccine production, a closed system is required for cell growth and harvesting. The HYPERStack vessel allows cell and reagent addition and removal via tubing from media bags or other methods.
This protocol will explain the technology behind the gas permeable material used in the HYPERStack vessels, gas diffusion results to meet the metabolic needs of cells, closed system cell growth protocols, and various harvesting methods.

An Introduction into the technology, protocol and handling of the Corning HYPERStack Vessels and accessories used for high yield adherent cell culture. The protocol will show how to use the closed system vessels for increasing cell harvesting over current stacked plate products.

Chiuso Sistema Cultura protocollo cellulare con imbarcazioni HyperStack con la tecnologia dei materiali gas permeabili

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice.   ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

Il trasferimento genico in embrioni di pollo da anziani<em&gt; Ex ovo</em&gt; Elettroporazione

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

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 ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

Un<em&gt; Ex vivo</em&gt; Cultura sistema per studiare lo sviluppo della tiroide

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Preparazione di Rat Siero Adatto per mammiferi tutta la cultura Embrione

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Istituzione di un pH extracellulare acido sistema cultura

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