. 1 Ex-ovo Kultur-Protokoll: <ol><li> Inkubieren befruchtete Hühnereier <ol class="lroman"><li> Befruchtete Hühnereier können bei 13 ° C gelagert werden bis zu 5 Tage vor der Inkubation ohne Einleitung Entwicklung. Ein Rotwein Kühler kann diese Temperatur. </li><li> Inkubieren Eier stumpfe Seite nach oben in eine 60% ige konstante Luftfeuchtigkeit Inkubator mit kontinuierlichem Schütteln bei 37,5 ° C für 72 Stunden. </li></ol></li><li> Bereiten Hängematten (Abbildung 1a) <ol class="lroman"><li> Füllen ¾ 9 Unzen Plastikbecher mit warmem sterilem Wasser. Eine regelmäßige 9 Unzen Tasse hat einen oberen Durchmesser von 8 cm. Wir fanden, dass dieser Größe am besten geeignet für lange Kultur-Anwendung präsentiert hier war. </li><li> Cut ca. 20 cm Frischhaltefolie. Befestigen Sie den Umfang auf der Tasse, indem ein Gummiband herum. Plastic sollte auf der Wasseroberfläche verteilt werden. Schneiden Sie die überschüssige Kunststoff rund um die Band. Wir fanden Frischhaltefolie am geeignetsten für diese Anwendung, da sie die Haftung der Dotter verhindert, was in den Dotter Spaltung während des Transports. </li><li> Wischen Sie die Plastik mit Kimwipes (Kimberly-Clark, Inc.) mit 70% Ethanol benetzt. </li></ol></li><li> Entfernen Sie Eier aus Inkubator nach 72 Stunden Inkubationszeit. </li><li> Sterilisieren Eier durch Abwischen mit 70% Ethanol benetzt Kimwipes. </li><li> Legen Sie die Eier horizontal für 1-2 Minuten für die richtige Positionierung des Embryos (Abb. 1b). Ein Eierkarton kann Eier legen horizontal verwendet werden. Embryonen nach oben nach 1-2 Minuten zu drehen. </li><li> Transfer-Embryonen auf Hängematten aseptisch in einer Sterilbank. <ol class="lroman"><li> Mit einer scharfen Kante (wie die Kante eines Metall-Eimer oder ein Becherglas), tippen Sie auf das Ei vorsichtig, bis es eine kleine Delle an der Unterseite des Eies (Abbildung 1c). Denken Sie daran, den Embryo auf der Oberseite positioniert. </li><li> Legen Sie den Daumen in den gegenüberliegenden Seiten des dent und ziehen die Schalen horizontal auseinander (Abbildung 1d). </li><li> Legen Sie das Eigelb mit Embryo sowie Albumin auf die Hängematte (Abbildung 1e). Albumin wird einen dämpfenden Umgebung rund um das Eigelb geben, Schocks, indem Sie das System sowie die Ernährung bietet, den Embryo für lange Kultur Praktiken induzierte absorbieren. Wasser unter der Hängematte wird dafür sorgen, den Embryo wird parallel bleiben, um den Boden beim Transport und dienen als Wärmeleiter während der Bildgebung, wenn ein Wasserkreislauf Heizung verwendet wird. </li><li> Der Embryo ist bereits oben positioniert werden, wenn die Übertragung korrekt durchgeführt wird. Wenn nicht, verwenden Sie eine unscharfe, sterile Objekt (zB Blunt Spitze geschlossen gebogene Schere) und direktional streicheln das Eigelb, dass der Embryo nach oben dreht. </li><li> Legen Sie eine 10 cm Durchmesser Petrischale auf dem Becher, den Embryo zu versiegeln. </li></ol></li><li> Legen Tasse Kultur in den Inkubator gesetzt bei 37,5 ° C. Wenn ein tragbarer Inkubator verwendet wird, indem 1-2 9 Unzen Becher mit destilliertem Wasser gefüllt hält die Feuchtigkeit auf ~ 60% (Abbildung 1f). </li><li> Wenn Kultivierung Embryonen außerhalb embryonalen Tag 7 sollten zerdrückt Eierschale Stücke rund um die Peripherie des Embryos als Kalzium-Quelle für die Entwicklung der Knochen und die Reifung verstreut werden. </li></ol> Zusatzausrüstung für außerhalb Inkubator Imaging / Manipulation: Ein zirkulierendes Wasserbad wurde im Haus gebaut, um Inkubationstemperaturen zu halten, während Bildgebung oder Manipulation (Abbildung 3). Ein Wasserbad, dass die Tasse passen kann, ist über entsprechende Größe Schlauch an ein Wasser-Reservoir verbunden. A Widerstandsheizung erwärmt das Wasser in das Reservoir, das manuell, indem Sie die Wassertemperatur geregelt. Eine Wasserpumpe sorgt für die Wasserzirkulation. Bildliche Darstellung zeigt Bildgebung bequem über ultrasongraphy / florescent Mikroskopie (Abbildung 3). <img src="/files/ftp_upload/2154/2125fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Vorbereitung der Hängematte und die Übertragung von Hühnerembryonen zu Hängematten. <img src="/files/ftp_upload/2154/2125fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Repräsentative Ergebnisse -. Unterschiedlichen Stadium Hühnerembryonen via ex ovo Kultur Technik gewachsen Embryonen aus HH 17 seit über diese Technik angebaut werden. <img src="/files/ftp_upload/2154/2125fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Schematische und repräsentative Bilder der Wasserkreislauf in Verbindung mit dem ex-ovo Kultur System verwendet, um Embryonen zu idealen Entwicklung Temperatur zu halten. 2. Ausgewählte Anwendungen: I. Mikroinjektion Dieser Embryo Kultur ist geeignet für die Mikroinjektion Anwendungen, bei denen Lösungen müssen injiziert werden, um für den Kontrast für die Bildverarbeitung (zB Fluoreszenz-Mikroskopie, Mikro-Computertomographie etc.) (Abbildung 4) Gefäßsystem werden. Unten ist die Mikroinjektion Protokoll: <ol><li> Bereiten Sie die Injektion apparatus <ol class="lroman"><li> Fashion gezogen 0,75 ID Glaskapillaren in abgeschrägte Spitze Mikronadeln mit einem microforge. Größe der Spitze hängt von Ziel Venendurchmesser / Durchfluss gewünscht. </li><li> Verbinden Sie die Spritze auf 0,03 Zoll ID Silikonschlauch. </li><li> Legen Sie den Schlauch mit der Lösung injiziert werden und verbinden Sie die Mikronadel, um den Schlauch. </li><li> Legen Sie die Mikronadel auf den Mikromanipulator und Spritze zu Spritze Inhaber oder Spritzenpumpe. </li></ol></li><li> Add sterilem Wasser in die Tasse damit erhöhen den Embryo. Wir erfahren, dass für eine ordnungsgemäße Eindringen der Nadel in ein Blutgefäß (dh vitelline Schiff), horizontale Durchdringung notwendig ist. Dies erfordert den Embryo auf der gleichen Ebene wie die Ränder des Bechers sein. <ol class="lroman"><li> Nehmen Sie das Gummiband hält der Kunststoff unter der Embryo. </li><li> Heben Sie die eine Seite der Kunststoff leicht und fügen Sie einige sterilem Wasser in die Tasse. </li><li> Sichern Sie das Gummiband auf den Kunststoff wieder. </li></ol></li><li> Injizieren Sie die Lösung in einen embryonalen Gefäß. In unserer Erfahrung, wenn Lösung für ein vitelline Gefäß injiziert wird, Mikronadeln benötigt, um einer Größe von ungefähr 1 / 10 th der Gefäßdurchmesser, um eine effiziente Perfusion ohne Blutungen zu halten. <ol class="lroman"><li> Pump die Lösung für die Sicherstellung Mikronadel keine Bildung von Luftblasen. </li><li> Richten Sie die Spitze des Mikronadel auf das ausgewählte Schiff. Für vitelline Schiffe wird durch die Auswahl einer Gabel Bereich geben maximale Fläche für das Schiff eindringt. </li><li> Drücken Sie die Nadel in Richtung des Schiffes. Das Schiff wird zuerst eingezogen. Sobald die Nadel eindringt, beginnen Perfusion. Die Farbe des strömenden Blutes sollte mit Perfusion ändern. Wenn Farbwechsel nicht gesehen wird, kann die Nadel aus dem Schiff eingedrungen sind. In diesem Fall langsam einfahren Mikronadel gleichzeitig Pumpenlösung bis Farbwechsel in das Schiff zu sehen ist. Sobald die Durchblutung abgeschlossen ist, zurückziehen Mikronadel aus dem Behälter. </li></ol></li></ol><img src="/files/ftp_upload/2154/2125fig4.jpg" alt="Abbildung 4" /> Abbildung 4: Mikroinjektion von einem fluoreszierenden Farbstoff in das Gefäßsystem eines Ex-ovo kultivierten Hühnerembryo. II. Mikrochirurgische Verfahren - linksatrialen Ligation Diese ex-ovo Kultur Setup ist ideal für die Durchführung von chirurgischen Anwendungen zum Hühnerembryonen, da sie volle Zugänglichkeit bietet, um den Embryo. Unten ist das Protokoll für ein Beispiel Technik, linksatrialen Ligation (LAL) (Abbildung 5). Das Verfahren ist etwa 24 Stunden in die Kultur Periode (HH24) durchgeführt: <ol><li> Bereiten overhand Knoten ~ 0,5 mm Durchmesser mit 10-0 Nylon chirurgisches Nahtmaterial. </li><li> Mit feinen Pinzetten, lokal entfernen Sie die Chorion-Allantois-und Membranen über den Embryo und heben den Embryo von der Rückseite zu drehen vertikal, so dass die linke Seite des Embryo sichtbar ist. </li><li> Mit feinen Pinzette entfernen Herzbeutel über den linken Vorhof. </li><li> Richten Sie den Knoten in den linken Vorhof und anziehen. Der Knoten sollte so gebunden werden, da ~ 75% des ursprünglichen Blutflusses auf die linke Seite zu verbieten. Schneiden Sie die Kanten des Knotens mit Mikroschere und vorsichtig entfernen und entsorgen Überschuss Naht. </li><li> Drehen Sie den Embryo wieder in seine ursprüngliche Orientierung (rechts oben). </li><li> Sham Kontrollen beinhalten das gleiche Verfahren, aber der Faden wird nur durchfahren werden, und nicht gebunden. Effektive LAL Behandlung (75% Einschnürung) wird nach 24 Stunden nach der Operation bestätigt werden, und disqualifiziert Embryonen ausgeschlossen wird. </li></ol><img src="/files/ftp_upload/2154/2154fig5.jpg" alt="Abbildung 5" /> Abbildung 5:. A-Steuerung und einen linken Vorhof ligiert Hühnerembryo Pfeil zeigt die linken Vorhof, die etwa 75% kleiner in der ligierten Embryo ist.

<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>

Optischer Zugang und Experimentieren in Vogelembryonen ist eine Herausforderung aufgrund von Einschränkungen der Eischale. Windowing deutlich begrenzt die Anzahl der Mikrogefäßen zugänglich für Injektionen und mikrochirurgische Ansätze 8. Als Ergebnis nur frühe Embryonen können manipuliert werden und eine kontinuierliche Beobachtung ist nicht möglich. Frühe ex-ovo Kulturen mit Petrischalen wurden von begrenztem Nutzen, weil unzureichender Kontrolle der Oberflächenspannung auf den Embryo ve...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
<td>&nbsp;</td>
<tr>
<td>100 mm diameter Petri dish</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1602N thermal air</td>
<td>&nbsp;</td>
<td>GQF Manufacturing CO, Savannah GA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescein-conjugated dextran (2 MDa, 1% w/v in phosphate buffered saline)</td>
<td>&nbsp;</td>
<td>Sigma Aldrich Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microforge</td>
<td>&nbsp;</td>
<td>Glassworx, Inc, St Louis MO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glass capillary tubes (0.75 mm ID)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<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>
<tr>
<td>Fine 55-forceps</td>
<td>&nbsp;</td>
<td>World Precision Instruments, Sarasota FL</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>10-0 nylon surgical suture</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<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>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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an ex-ovo chicken embryo culture system suitable for imaging and microsurgery applications

Das Verständnis der Zusammenhänge zwischen genetischen und Mikroumgebung Faktoren, die normale und fehlerhafte embryonale Entwicklung Laufwerk ist von grundlegender Bedeutung für die Entdeckung neuer therapeutischer Strategien. Fortschritte in der Imaging-Technologie haben die quantitative Untersuchung der Organisation und Reifung des Körpers Plan aktiviert, aber später embryonalen Morphogenese ist weniger klar. Hühnerembryonen sind eine attraktive Wirbeltier-Modellsystem für diese Anwendung wegen seiner Leichtigkeit von Kultur und chirurgische Manipulation. Frühe Embryonen können für eine kurze Zeit auf Filterpapier Ringe, die komplette optische Zugang für die Zell-Strukturierung und Schicksal Studien 1,2 ermöglicht kultiviert werden. Studieren fortgeschrittenen Entwicklungsprozessen, wie Herz-Morphogenese sind traditionell durch ein Fenster der Eischale 3-5 durchgeführt, aber diese Technik Grenzen optischer Zugang durch Fenstergröße. Wir zuvor entwickelte eine einfache Methode zur Kultur ganzen Embryonen ex-ovo auf sechseckigen wiegen Boote für bis zu 10 Tage, die hochauflösende Bildgebung mittels Ultraschall 6,7 aktiviert. Diese Kulturen wurden schwer zu transportieren, wodurch die Arten von Imaging-Tools für Live-Experimente. Wir stellen hier eine verbesserte Shell-less Kultur mit einem kostengünstigen, tragbaren Klimakammer. Die Eier wurden auf einer Hängematte mit einem Polyurethan-Membran (Frischhaltefolie) ringförmig an einem Plastikbecher teilweise mit sterilem Wasser gefüllt angebracht erstellt geknackt. Die Abmessungen des Umfangs und der Tiefe der Hängematte waren beide entscheidend für die Oberflächenspannung zu halten, während die Mechanik der Hängematte und Wasser unter geholfen dämpfen Vibrationen, die durch den Transport induziert. Eine kleine Stellfläche zirkulierenden Wasserbad wurde auch entwickelt, um kontinuierliche Temperaturkontrolle während der Experimente zu ermöglichen. Wir zeigen die Fähigkeit, Kultur Embryonen auf diese Weise für mindestens 14 Tage ohne morphogenic Defekt oder Verzögerung und beschäftigen dieses System in mehreren mikrochirurgischen und Imaging-Anwendungen.

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 diesem Artikel präsentieren wir Ihnen eine einfache Methode, um langfristig zu ermöglichen<em&gt; Ex-ovo</em&gt; Vogelembryo Kultur. Diese Technik ist ideal für die Längs-Experimente erfordern komplette optische Zugänglichkeit und / oder sterilen Transport in Vogelembryonen.

Ein Ex-ovo Chicken Embryo Culture-System Geeignet für Imaging und Mikrochirurgie Anwendungen

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

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Research

JoVE Journal

Biology

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

22.9K Aufrufe.  Cornell University. In diesem Artikel präsentieren wir Ihnen eine einfache Methode, um langfristig zu ermöglichen Ex-ovo Vogelembryo Kultur. Diese Technik ist ideal für die Längs-Experimente erfordern komplette optische Zugänglichkeit und / oder sterilen Transport in Vogelembryonen.

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

Windowing Chicken Eggs for Developmental Studies

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling.  In addition, it allows for molecular perturbations of several types, including placement of protein-coated beads and introduction of plasmid DNA using in ovo electroporation.  These experiments have yielded important data on the development of the central and peripheral nervous systems.  For many of these studies, it is necessary to open the eggshell and reclose it without perturbing the embryo's growth.  The embryo can be examined at successive developmental stages by re-opening the eggshell.  While there are several excellent methods for opening chicken eggs, in this article we demonstrate one method that has been optimized for long survival times.  In this method, the egg rests on its side and a small window is cut in the shell.  After the experimental procedure, the shell is used to cover the egg for the duration of its development.  Clear plastic tape overlying the eggshell protects the embryo and helps retain hydration during the remainder of the incubation period.  This method has been used beginning at two days of incubation and has allowed survival through mature embryonic ages.

The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling. In this article we demonstrate one egg preparation method that has been optimized for long survival times.

Windowing Chicken Eggs für Developmental Studies

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has ...

Forschung

Biologie

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.

Bioelectric Analysen eines osseointegrierte Intelligent Implant Design System für Amputierte

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 ...

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.

Küken Ex ovo Kultur und Ex ovo CAM-Assay: Wie es wirklich funktioniert

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.

Xenotransplantation von menschlichen Stammzellen in die Hühnerembryo

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.

Die Methode der Nager Whole Embryo Culture mit der Rotator-type Bottle Kultur-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 ...

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.

Closed System Cell Culture Protocol Mit HyperStack Schiffe mit gasdurchlässigen Material Technology

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.

Gentransfer in Hühnerembryonen durch Ältere<em&gt; Ex ovo</em&gt; Die Elektroporation

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.

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 ...

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.

Ein<em&gt; Ex-vivo-</em&gt; Kultur-System, um Schilddrüsenentwicklungsstudie

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.