Diese Arbeit wurde von der Margaret M. Alkek Stiftung RHF unterstützt.

Teil 1: Bench einrichten <ol><li> Eine feuchte Kammer, indem Kimwipe / ddH 2 O in Kunststoff-Kammer vorbereitet. </li><li> Eine Falcon-Röhrchen an Albumin, Geschirr für Kultur, Ringe, Uhrglas und Entsorgung sammeln auf der Bank platziert. </li><li> Pyrex Schüssel wird mit 1,4 l Kochsalzlösung gefüllt (siehe Erläuterungen [a]). </li></ol> Teil 2: Embryo ist in Kochsalzlösung explantierten <ol><li> Die Eier werden aus dem Inkubator nach 16 Stunden (Stufe 4) entfernt. Das Ei ist, offener, indem Sie die Schale mit einer Pinzette. Shell Stück entfernt werden. </li><li> Die dünne Albumin ist in Falcon-Röhrchen gesammelt. Die dicken Albumin ist mit einer Pinzette entfernt. </li><li> Der Embryo wird in einer Plastikschale in der Saline Schale gelegt. Die verbleibenden Albumin ist mit einer Pinzette entfernt. </li></ol> Teil 3: Embryo ist am Ring zentriert <ol><li> Der Dottersack wird mit einer Pinzette so geneigt, dass Embryos nach oben zeigt. </li><li> Der Dottersack ist auf der Ebene des Äquators oder unten abgeschnitten. </li><li> Mit feinen Pinzetten, ist die Dotterhaut schnell geschält. Die Dotterhaut ist so, dass seine körnigen Seite (nicht shinny) nach oben zeigt orientiert. </li><li> Mit feinen Pinzetten, ist die Dotterhaut auf dem Uhrglas gelegt. </li><li> Mit feinen Pinzette, ein Glas Ring an der Oberseite der Dotterhaut aufgetragen und der Embryo ist zentriert. </li><li> Die Dotterhaut ist an den Kanten des Glases Ring gehoben. Die Montage wird von der Saline Gericht entfernt. </li></ol> Teil 4: Die Kultur wird unter dem Mikroskop eingestellt <ol><li> Die Dotterhaut wird über den Glasring mit feinen Pinzetten unter dem Mikroskop angehoben. </li><li> Mit Pasteurpipette wird die Kochsalzlösung aus dem äußeren Rand des Ringes entfernt. </li><li> Mit einer feinen Schere wird überschüssige Dotterhaut von der Innenkante des Glases Ring entfernt. Care ist nicht zu durchdringen die Membran mit Pipette oder einer Zange genommen. </li><li> Der Embryo wird vorsichtig mit Kochsalzlösung gespült, um lose Membranen und Dotter Zellen zu entfernen. </li><li> 200 ul Kochsalzlösung zu den äußeren Rand des Ringes (dies wird später Transfer zu erleichtern) aufgenommen. </li><li> Die Montage erfolgt mit einem umgekehrten Plastikschale bedeckt. </li></ol> Teil 5: Die Kultur ist Inkubator überführt <ol><li> Eine Falcon Kulturschale gekennzeichnet ist. </li><li> 2,5 ml dünne Albumin ist an der Unterseite des Falcon Gericht aufgenommen. </li><li> 200 ul von dünnen Albumin ist an der inneren Kante des Deckels der Falcon Gericht aufgenommen. </li><li> Die invertierte Gericht ist aus dem Verbund lösen. </li><li> Mit feinen Pinzette wird das Glas Ring entlang der Kante des Uhrglas geschoben, und an den Falcon Gericht. </li><li> Alle übrigen Kochsalzlösung ist von der Innenfläche des Ringes entfernt. </li><li> Die Falcon Gericht ist mit Deckel abgedeckt und versiegelt. </li><li> Die Montage erfolgt in feuchten Kammer und dann in den Brutschrank gestellt. </li><li> Die Embryonen werden für bis zu 24 Stunden bei 38 ° C kultiviert </li></ol> Teil 6: Im Anschluss an Kultur, ist der Embryo fixiert; Kultur ist Inkubator überführt <ol><li> Eine Fixierung Gericht wird mit eiskaltem PBS gefüllt (oder DEPC-PBS, wenn Embryonen für die in-situ-Hybridisierung verarbeitet werden sollen). </li><li> Die Kultur ist aus dem Brutschrank entnommen und sofort auf Eis gelegt. Die Kulturschale wird sofort mit eiskaltem PBS / DEPC-PBS gefüllt. </li><li> Der Embryo ist von der Dotterhaut abgelöst. Der Embryo wird die Fixierung Schale überführt, mit dem stumpfen Ende einer Pasteurpipette. </li><li> Der Embryo wird nach unten mit stumpfem Ende feinen Pinzette merken. Die PBS für die Fixierung Schale wird entfernt. Fixativ gegeben wird (siehe Anhang [b]). </li><li> Je nach Anwendung wird der Embryo für 6-8 Stunden bei 4 ° C fixiert (Kryostat), O / N bei 4 ° C (in situs), oder 1 Stunde bei RT für ganze Berg Immunhistochemie. </li><li> Der Fixateur wird entfernt und mit eiskaltem PBS / DEPC PBS ersetzt. </li><li> Für Downstream-Anwendungen wie in-situ-Hybridisierung oder immantibody Färbung: das Nervensystem und das Herz sind perforiert mit einem stumpfen Ende Mikrokapillare Nadel oder ein Mikrodissektion Messer, das wird verhindern, Abfangen von Sonde / Antikörper in späteren Schritten. Anmerkungen: [a] Saline besteht aus: Lösung A (für 1 l): 121,0 g NaCl, 15,5 g KCl, 10,4 g CaCl 2 .2 H 2 O, 12,7 g MgCl 2 .6 H 2 O; Lösung B (für 1 l) : 2,4 g Na 2 HPO 4 · 2H 2 O, 0,2 g NaH 2 PO 4 · 2H 2 O; Autoclave; Vor dem Gebrauch mischen 120 ml A mit 2700 ml H 2 O; Add 180 ml ​​B. Mix [1]; [b] Fixativ ist unmittelbar vor Gebrauch vorbereitet (4% PFA in PBS oder DEPC-PBS für in situs). </li></ol> Teil 7: Repräsentative Ergebnisse Vor Kultur, ist der Embryo in Primitivstreifens Stufe (HH4). Am Ende der Kultur Zeit hat der Embryo HH10 (Länge 2-3 mm) entwickelt und wird sichtbar in der Mitte der Kulturschale. Es ist möglich, eine Gruppe von Zellen mit fluoreszierenden Carbocyanin DiI Labelkurz vor dem Kultur (0h) und folgen ihrer Bewegung während der Kulturzeit. In diesem Fall wurden die Zellen unter Hensen den Knoten (HN) mit DiI markiert. Diese Zellen sind nachweislich auf eine stufenweise Entwicklung Somiten (som) und Chorda (n) beitragen. <img src="/files/ftp_upload/old/903_2008_10_20_2/903_Figure-01.png" alt="" width="780" height="228" /> Teil 8: Fehlersuche <table style="width: 698px; height: 241px;" border="1" cellspacing="0" cellpadding="0" width="698"><tbody><tr><th> Problem </th><th> Ursache </th><th> Abhilfe </th></tr><tr><td> Embryo bleibt mit Eigelb statt kommenden off mit Membran (Schritt 2) </td><td> Schlechte Qualität von Eiern </td><td> Shop Eier bei 13 ° C nach Erhalt; Anfrage frisch produzierten Eier. Inkubieren Eier am selben Tag wie Anreise. </td></tr><tr><td> Dotterhaut Rutsche vom Uhrmacher Glas folgende Platzierung der Ring (Schritt 3) </td><td> Albumin Reste </td><td> In Schritt 2, um sicherzustellen, dass alle Albumin, indem Sie sie weg von Membran mit gekippten Pinzette entfernt </td></tr><tr><td> Embryo nicht zugänglich ist: liegt unterhalb der Membran (Schritt 4) </td><td> Falschen Seite der Membran wird nach oben </td><td> In Schritt 3 sicher, dass die Seite der Membran mit Dotterschollen nach oben zeigt. Die glänzende, polierte Seite sollte nach unten zeigen. </td></tr><tr><td> Embryo in Kochsalzlösung / Albumin folgenden Kultur eingetaucht (Schritt 6) </td><td> Saline / Albumin linken inneren Ring vor Kultur, Loch in der Membran </td><td> In Schritt 5 Stellen Sie sicher, dass alle Albumin / Kochsalzlösung aus dem Inneren des Ringes wird entfernt; In Schritt 4, sicherzustellen, dass Sie nicht gewaltsam öffnen Membran mit einer Pinzette (Einsatz mit stumpfen Enden Zangen) </td></tr><tr><td> Embryo zerfiel nach Kultur </td><td> Bakterielle Infektion </td><td> Sterilisieren Sie alle Werkzeuge und Glaswaren; Verwenden Antibiotikum / Antimykotikum </td></tr></tbody></table>

<ol>
	<li>Pannett, P. A., Compton, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cultivation+of+tissues+in+saline.">The cultivation of tissues in saline.</a> Lancet. 206, 381-384 (1924).</li><li>New, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+for+the+cultivation+of+the+chick+embryo+in+vitro.">A new technique for the cultivation of the chick embryo in vitro.</a> J. Embryol. Exp. Morph. 3, 326-331 (1955).</li><li>Alvarez, I. S., Araujo, M., Nieto, 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=Neural+induction+in+whole+chick+embryo+cultures+by+FGF.">Neural induction in whole chick embryo cultures by FGF.</a> Dev. Biol. 199, 42-54 (1998).</li><li>Psychoyos, D., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+the+organizer+after+radical+ablation+of+Hensen's+node+and+the+anterior+primitive+streak+in+the+chick+embryo.">Restoration of the organizer after radical ablation of Hensen's node and the anterior primitive streak in the chick embryo.</a> Development. 122, 3263-3273 (1996).</li><li>Psychoyos, D., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+and+migratory+routes+of+primitive+streak+cells+in+the+chick+embryo.">Fates and migratory routes of primitive streak cells in the chick embryo.</a> Development. 122, 1523-1534 (1996).</li><li>Voiculescu, O., Papanayotou, C., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatially+and+temporally+controlled+electroporation+of+early+chick+embryos.">Spatially and temporally controlled electroporation of early chick embryos.</a> Nature Protoc. 3, 419-426 (2008).</li></ol>

Die neue Kultur-Methode 2 kann für eine Vielzahl von Anwendungen, von der Transplantate Wachstumsfaktor mit Perlen 3, um whole mount in situ Hybridisierung und Immunhistochemie ganze Berg 4 verwendet werden. Kultur über einen 24-Stunden-Periode ermöglicht die kontinuierliche Überwachung der embryonalen Entwicklung in Anwendungen wie Zeitraffer Zellbewegung Analyse 5 oder Überwachung von GFP mit elektroporiert Konstrukte 6.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Eggs</td>
<td>Animal</td>
<td>Charles River Laboratories</td>
<td>Premium Fertile</td>
<td>&nbsp;</td>
<tr>
<td>Stereomicroscope</td>
<td>Microscope</td>
<td>Leica Microsystems</td>
<td>MZ9.5 or similar</td>
<td>&nbsp;</td>
<tr>
<td>Marsh Automatic Incubator</td>
<td>Tool</td>
<td>Lyon</td>
<td>RX</td>
<td>&nbsp;</td>
<tr>
<td>Hybridization Incubator</td>
<td>Tool</td>
<td>Robbins Scientific</td>
<td>M1000</td>
<td>&nbsp;</td>
<tr>
<td>Pyrex dish (2)</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Watchmaker&#x2019;s glass 50mm</td>
<td>Tool</td>
<td>VWR</td>
<td>66112-060</td>
<td>&nbsp;</td>
<tr>
<td>Glass rings</td>
<td>Tool</td>
<td>Physical Plant facility</td>
<td>&nbsp;</td>
<td>cut 4 mm thick sections of glass tubing (27 mm outer diam, 25 mm inner diam). Do not fine polish.</td>
</tr>
<tr>
<td>Curved Forceps (1)</td>
<td>Surgery</td>
<td>Electron Microscopy Sciences</td>
<td>72991-4C</td>
<td>&nbsp;</td>
<tr>
<td>Forceps (2)</td>
<td>Surgery</td>
<td>Fine Science Tools</td>
<td>11002-13</td>
<td>blunt ended using sharpening Stone and 100ul mineral oil</td>
</tr>
<tr>
<td>Sharpening Stone Dan&#x2019;s Black Arkansas</td>
<td>Surgery</td>
<td>Electron Microscopy Sciences</td>
<td>62082-00</td>
<td>&nbsp;</td>
<tr>
<td>Fine scissors</td>
<td>Surgery</td>
<td>Fine Science Tools</td>
<td>14161-10</td>
<td>&nbsp;</td>
<tr>
<td>Plastic dishes</td>
<td>Tool</td>
<td>Falcon</td>
<td>353001</td>
<td>&nbsp;</td>
<tr>
<td>Rubber Bulb</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>70980</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur Capillary Pipette</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>70950-12</td>
<td>round edge under flame</td>
</tr>
<tr>
<td>Microcapillary tube</td>
<td>Surgery</td>
<td>Sigma</td>
<td>P1049-1PAK</td>
<td>Pull using vertical micropipette puller; blunt end with fine forceps</td>
</tr>
<tr>
<td>Microdissecting knife</td>
<td>Surgery</td>
<td>Fine Science Tools</td>
<td>10056-12</td>
<td>Use to puncture cavities prior to in situ hybridization</td>
</tr>
<tr>
<td>Minuten pins 0.2mm diam</td>
<td>Surgery</td>
<td>Fine Science Tools</td>
<td>26002-20</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184 Silicon Elastomer Curing Agent and Base</td>
<td>Reagent</td>
<td>Dow Corning</td>
<td>0001986475</td>
<td>Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C</td>
</tr>
<tr>
<td>Diethylpyrocarbonate (depc)</td>
<td>Reagent</td>
<td>Acros Organics</td>
<td>10025025</td>
<td>Add 1ml depc to 1l PBS; shake; autoclave</td>
</tr>		</tbody>
		</table>
		</div>

method for culture of early chick embryos ex vivo (new culture)

Der Hühnerembryo ist ein wertvolles Werkzeug in der Studie der frühen Embryonalentwicklung. Seine Transparenz, Zugänglichkeit und einfache Handhabung machen ihn zu einem idealen Werkzeug zur Untersuchung der Bildung und Strukturierung des Gehirns, Neuralrohr, Somiten und Herz Primordien. Anwendungen von Hühnerembryonen Kultur gehören die Elektroporation von DNA-oder RNA-Konstrukte, um Genfunktionen, Transplantate growth factor Kügelchen wie FGFs und BMPs sowie whole mount in situ Hybridisierung und Immunhistochemie analysiert. Dieses Video zeigt die verschiedenen Schritte in Hühnerembryo Kultur; Erste, der Embryo in Kochsalzlösung explantiert. Dann wird der Embryo auf einem Glasring zentriert. Die Membranen um den Embryo sind die Wände entlang des Rings aufgehoben. Der Ring wird dann in einer Kulturschale mit einem Pool von Albumin platziert. Die Kulturschale wird verschlossen und in einer feuchten Kammer, wo der Embryo bis zu 24 Stunden kultiviert. Schließlich ist der Embryo vom Ring entfernt, fixiert und für weitere Anwendungen. Ein Leitfaden für die Fehlersuche wird ebenfalls vorgestellt.

Watch this Scientific Journal Video about Method for Culture of Early Chick Embryos ex vivo New Culture at JoVE.com

Method for Culture of Early Chick Embryos ex vivo New Culture

Dieses Video zeigt neue Kultur, eine Methode, durch die Hühnerembryonen außerhalb der Eizelle bis zu 24 h kultiviert. Diese Methode ermöglicht ein bis frühen Entwicklung (Primitivstreifens bis 14 som.), Ein Zeitraum entsprechend E7-9 in der Maus zu studieren. Anwendungen dieser Technik sind die Elektroporation, in situ Hybridisierung und Immunhistochemie.

Method for Culture of Early Hühnerembryonen ex vivo (New Culture)

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

method-for-culture-of-early-chick-embryos-ex-vivo-new-culture

Research

JoVE Journal

Biology

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

15.6K Aufrufe.  Institute of Biosciences and Technology - Texas A&M Health Science Center. Dieses Video zeigt neue Kultur, eine Methode, durch die Hühnerembryonen außerhalb der Eizelle bis zu 24 h kultiviert. Diese Methode ermöglicht ein bis frühen Entwicklung (Primitivstreifens bis 14 som.), Ein Zeitraum entsprechend E7-9 in der Maus zu studieren. Anwendungen dieser Technik sind die Elektroporation, in situ Hybridisierung und Immunhistochemie.

Serie: Method for Culture of Early Chick Embryos ex vivo New Culture

Double Whole Mount in situ Hybridization of Early Chick Embryos

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  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Doppel Whole Mount in situ Hybridisierung von Anfang Hühnerembryonen

Forschung

Biologie

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 ...

In vivo-like Organotypic Murine Retinal Wholemount Culture

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages.
Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used.
By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5.
In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.

In vivo-like Organotypic Murine Retinal Wholemount Culture

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

In vivo-ähnlichen Organotypische Murine Retinal Wholemount Kultur

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

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 ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Ex vivo-Kultur von primären humanen Eileiter Epithelzellen

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

Tracking Morphogenetic Tissue Verformungen in der frühen Hühnerembryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Scale-Up of Mammalian Cell Culture using a New Multilayered Flask

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale expansion, may become cumbersome, laborious and time-consuming when large numbers of cells are required. To address this need, the performance of a new multi-layered cell culture vessel to facilitate easy scale up of cells from single layered T-flasks will be discussed. The flasks tested are available in 3- and 5-layer format and enable culture and complete recovery of three and five times the number of cells respectively, compared to T-175 flasks. A key feature of the BD Multi-Flask is a mix/equilibration port that allows rapid in-vessel mixing as well as uniform distribution of cells and reagents within and 
between layers of each vessel and consistently produce cells that can be cultured in an environment that is congruent to T-175 flasks.
The design of these Multi-Flasks also allows for convenient pipette access for adding reagents and cells directly into the flasks as well as efficient recovery of valuable cells and reagents and reduces risk of contamination due to pouring. For applications where pouring is preferred over pipetting, the design allows for minimal residual liquid retention so as to reduce wastage of valuable cells and reagents.

Cells play an instrumental and increasing role in research, and the discovery and development of new therapeutics. With this increasing need for greater number of cells we need more efficient and effective ways for growing and harvesting attachment dependent cells. A Multilayered flask with the right features can serve this purpose.

Scale-Up von Mammalian Cell Culture mit einem New Mehrschichtige Flask

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

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

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

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

Die Slice-Kultur-Verfahren zur Nach Entwicklung der Zahnkeime In Explantation Kultur

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

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

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...