Name: primary endodermal epithelial cell culture from the yolk sac membrane of japanese quail embryos
Uploaded: 2016-03-10T13:00:00.000+00:00
Duration: 11 min 53 s
Description: Watch this Scientific Journal Video about Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos at JoVE.com

Die Finanzierung für die Entwicklung des aktuellen Protokolls ist vor allem durch &quot;Ziel für die Top-Universität Plan&quot; von der National Taiwan University, Taiwan (Zuschuss ID-104R350144) sowie das Ministerium für Wissenschaft und Technologie von Taiwan (Erteilungs ID unterstützt: MOST 104 -2313-B-002-039-MY3). Wir danken vor allem das Zentrum für Biotechnologie der National Taiwan University zur Bereitstellung eines Tierraum und Laborflächen für die aktuelle Studie.

Hinweis: Dieses Verfahren ist eine Modifikation eines Huhns Modellkulturprotokoll von Bauer entwickelte et al 2013 und Nakazawa et al, 2011. 2,9.. 1. Bereiten Sie gesunde embryonale Tag 5 Embryonen aus der japanischen Wachtel <ol><li> Platz 1 männliche und 3 weibliche geschlechtsreif japanischen Wachtel zusammen im selben Käfig. Versorgung Futter und Wasser ad libitum. Stellen Sie die Licht im Tierraum bis 14 h Licht und 10 h Dunkelheit. </li><li> Sammeln Sie befruchtete Eier in Plastiktüten jeden Tag am Nachmittag und halten sie in einem 16 ° C Kühl die Wachstumsrate von Embryonen zu verzögern. </li><li> eine ausreichende Anzahl von Eiern in einem Zeitraum von zwei Wochen, brüten die befruchteten Eier in einem 37 ° C Inkubator mit guter Belüftung für fünf Tage Nach dem Sammeln. </li></ol> 2. Bereiten Sie Basal Medium und Waschlösungen <ol><li> Verwenden DMEM / F-12-Medium (eingestellt auf pH 7,2) als Wachstumsmedium und ergänzen esmit 10% Neugeborenen Kälberserum (NBCS) und 1% Pen-Strep Ampho. Lösung (PSA; Penicillin, 10 5 Einheiten / L; Streptomycin, 100 mg / L; Amphotericin B, 0,25 mg / L). </li><li> Bereiten phosphatgepufferter Salzlösung (PBS) Lösung bei einer 10-fach-Konzentration für die Lagerung; verwenden 1x PBS - Lösung (enthaltend NaCl, 137,93 mM; KCl, 2,667 mM; KH 2 PO 4, 1,471 mM; Na 2 HPO 4 · 7H 2 O, 8,06 mM; einstellen auf pH 7,2) , um den Dottersack Membran , während zu waschen und zu befeuchten Trennen des Endoderm aus Eigelb und Albumin. </li><li> Verwenden DMEM-Medium (eingestellt auf pH 7,2) auf Endoderm Pellets resuspendieren. </li></ol> 3. Erhebung von Primär EECs aus der japanischen Wachtel YSMs <ol><li> Inkubieren Sie die gesammelten Eier für 5 Tage (Schritt 1.3). Untersuchen Sie die Eier von einem Ei candler zur normalen embryonalen Entwicklung bestätigen. HINWEIS: Bei den embryonalen Tag 5, ist die Grenze von YSM Mesoderm sehr klar, und die drei embryonalen Schichten sind weniger eng miteinander verbunden sind, so ist es leicht zu OBTain endoderm. Verwenden Sie nur die Eier normalen Kapillarzirkulation Entwicklung für YSM Sammlungen zeigen. </li><li> Reinigen Sie die Eischale sorgfältig frisches Wasser und 75% Ethanol. Öffnen Sie die Eischale aus der Luftzelle Position durch eine Schere, sanft schälen heraus und das gesamte YSM mit einer Pinzette und in eine 10 cm Kulturschale gefüllt mit 37 ° C 1x PBS. </li><li> Mit Schere den Embryo, Eigelb und Eiweiß, zu entfernen und vorsichtig die YSM mit 1x PBS dreimal waschen und legen YSM in PBS-Lösung in einer 10 cm Kulturschale. </li><li> Verwenden einer Pinzette und eine Schere das Ektoderm von YSM zu entfernen. Verwenden Sie zwei Zangen, ein fest halten Sie die endodermaler Zellschicht, und die andere die Kapillare Mesoderm zu halten. </li><li> Unter einem Binokular, auseinanderziehen und die endoderm vom Rand des Mesoderms in Richtung auf den Embryo zu trennen. Sammeln Sie die hellgelbe endoderm in 50 ml Zentrifugenröhrchen eine Pipette und in 15 ml Wachstumsmedium in einem 37 ° C wat miter Bad, bis alle Gewebesammlungen sind abgeschlossen. </li></ol> 4. Die Verdauung des Endoderm Scheiben von Collagenaseverdau <ol><li> auflösen frisch 6,5 Einheiten vom Typ IV-Kollagenase in 10 ml DMEM. </li><li> Zentrifugieren der 50-ml-Röhrchen (3.4) bei 130 × g für 3 min bei RT. Saugen Sie das Medium und legen alle endoderm in einer 6 cm Kulturschale mit einer Pipette. 1 ml Kollagenase-Lösung dieses Kulturschale. </li><li> Schneiden Sie sammelte die endoderm von 2 Wachteln Embryonen mit einem Paar von gebogenen Schere, bis die Größe jeder Scheibe beträgt ca. 2 bis 3 mm. Verwenden Sie eine Pipette, sammeln Sie alle Scheiben in ein 50 ml Zentrifugenröhrchen mit 9 ml frischem Kollagenase-Lösung. </li><li> Digest des Gewebes mit Kollagenase für 30 min in einem 37 ° C Schüttelwasserbad bei 175 Umdrehungen pro Minute. Diese partielle Digestion mit Kollagenase wird verwendet, um das Explantat-Zellproliferation zu verbessern. </li><li> Zentrifuge bei 130 × g für 3 min bei RT und entfernen leicht die Überstandsfraktion mit einer Pipette. Waschen Sie das Pellet durch Suspension in 20 ml DMEM und entfernen Sie die überstehenden Fraktion nach 130 xg Zentrifugation für 3 min. </li><li> Re-suspend das Pellet mit 12 ml Wachstumsmedium (DMEM / F12 mit 10% NBCS und 1% PSA) und sanft und gleichmäßig in einer 24-Well-Platte (jeweils gut mit 500 ul Zelllösung) Samen. </li><li> Inkubieren Gewebeexplantaten bei 37 ° C in 5% CO 2 in Luft für 2 Tage Zellen zu ermöglichen aus dem Gewebe zu vermehren. </li><li> Inkubiere Zellen für weitere 2 Tage und Charakterisierung von für die Lebensfähigkeit der Zellen durch eine Assayprotokoll die Zellebensfähigkeit. 15 HINWEIS: Lebende Zellen pflegen eine reduzierende Umgebung im Cytosol. Resazurin, der Wirkstoff, ein nicht-toxisches, zellpermeablen Verbindung, die in der Farbe blau ist und praktisch nicht fluoreszierend. Wenn zu Zellkulturen gegeben, ist Resazurin reduzierte durch mitochondriale Enzyme im Cytosol zu Resorufin. Resorufin ist in der Farbe rot und stark fluoreszierend. Lebensfähige Zellen kontinuierlich konvertieren Resazurin zu Resorufin, increAsing die Gesamtfluoreszenz und die Farbe des Kulturmediums umgebenden Zellen. </li><li> Führen Sie endoderm Epithelzelle Gesamt - RNA Isolierung, reverse Transkription und quantitative Echtzeit - PCR von Marker - Gen - mRNA - Expression , wie beschrieben. 16,17 <ol><li> Verwendung der PCR-Reverse-Transkriptase-SOAT1 mRNA zu detektieren (sense: 5&#39;-GAAGGGGCCTATCTGGAACG-3 &#39;; Antisense: 5&#39;-ATCTGCACGTGACATGACCA-3&#39;; PCR product = 168 bp) als ein spezifischer Marker für den EWG-Zellen. Verwenden β-Actin-mRNA (Sense: 5 &#39;TGGTGAAGCTGTAGCCTCTC-3&#39;; Antisense: 5 &#39;GTGATGGACTCTGGTGATGG-3&#39;; PCR-Produkt = 151 bp) als Housekeeping-Gen und die interne Kontrolle. </li><li> Verwenden Sie Gel-Elektrophorese die Produktgröße von RT-qPCR zu erkennen. Bereiten Sie 1% Agarose-Gel und mischen 10 ul qPCR-Produkt mit 2 &amp; mgr; l Lade Farbstoff. Last 10 ul Gemisch in jede Vertiefung in der 1% Agarose-Gel, und führen Sie das Gel durch 100V für 30 min. </li></ol></li></ol>

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There is no competing financial interests or other conflicts of interest. We did not get any funding from any private company.

Da die vorherige Kultursystem nur begrenzten Erfolg hat, wird eine bessere Kultursystem erforderlich. Die japanische Wachtel YSM endoderm erfordert eine Behandlung mit proteolytischen Enzymen wie Kollagenase, die Zell-Zell-Verbindungen zu lösen bessere Wachstumsleistung in den ex-vivo-Explantate zu erreichen. Unsere Daten zeigen , dass die Zellzahlen von partiellen Verdau Behandlung waren größer als der unverdauten Gewebekultur nach 2 Tagen nach der Aussaat (Abbildung 1). Daher ist die partielle prot...

Der Hauptnahrungsressource des Vogelembryo yolk ist, bestehend aus 33% Lipiden, 17% Protein und 1% Asche. 1 Während der embryonalen Entwicklung, der Dottersack - Membran (YSM) wächst aus der embryonalen Bauchhöhle und erstreckt sich allmählich das Eigelb Oberfläche. Beginnend am embryonalen Tag 2, die Genexpression , die mit Lipid - Metabolismus und die Angiogenese wird in YSM allmählich erhöht, und die YSM entwickelt sich langsam villus artige Vorsprünge. 8,9 Diese Vorsprünge erhöhen die Absorption von Nährstoffen yolk embryonalen Entwicklung zu unterstützen. Die YSM ist ein extraembryonic Gewebe , das drei Keimblätter, Endoderm, Mesoderm und Ektoderm enthält. 14 Der Dottersack ectoderm steht das Eiweiss und Verbindungen mit der Dotterhaut vorsichtig auf die Dottersack abzudecken. Die endodermaler Epithelzellen direkt auf das Eigelb und dienen als Nährstoffverwertung Portale konfrontiert. 6 Wie der YSM erweitert, endodermaler Epithelzelle (EECs) durch shap unterteilt werdene und Funktionalität in zwei Gruppen, Bereich Dotter und Bereich vasculosa. 7 Gebiet vitelline besteht aus Entodermzellen und ist weit entfernt von dem Embryo; Bereich vasculosa aus mesodermalen Zellen zusammengesetzt und umfasst differenzierte EECs mit Blutgefäßen und Bindegewebe. Durch die embryonale Tag 5 wird das Eigelb vollständig von Ektoderm und Entoderm von YSM bedeckt und das Gefäßbereich ist schnell gewachsen. Die YSM absorbiert, neu zusammensetzt und gibt Lipiden (wie Eigelb abgeleitetes Lipoprotein sehr niedriger Dichte) und Proteine ​​in die embryonalen Kreislaufsystem. 9, 2 Daher wir eine primäre japanischen Wachteln embryonalen endodermalen epithelialen Zellkultur - System, die Mechanismen von Lipid zu studieren Nutzung in YSM während aviären embryonalen Entwicklung. Lipids wie Triacylglycerol, Lecithin, Phospholipid und Cholesterinester (CE) sind die primären Energiequellen für Vogelembryonen. In den frühen Stadien der Entwicklung, yolk Lipide sind Cin Vogelembryo YSM omposed von nur 1,3% CE und es steigt auf 10-15% bei Halbzeit der aviären Embryonalentwicklung 3, 11. Cholesterinester wird aus Cholesterin durch Sterin O-Acyltransferase 1 (SOAT1) synthetisiert. 4 Die Speicherform von Cholesterin ist CE, CE in Lipoproteinen getragen wird, und Lipoproteine ​​durch Zirkulation zu den Geweben transportiert werden. 13 Eine Woche vor Luke dort schnelle Wachstum von Vogelembryonen ist. Etwa 68% der verbleibenden Lipidgehalte in Eigelb werden in dieser Phase absorbiert. 10 Der Mechanismus , mit dem Eigelb Lipide verwendet werden , können durch eine EWG - Forschungsmodell geklärt werden. Ein Huhn EWG Kulturprotokoll wurde festgestellt , diese Forschungsziel zu erreichen. 2, 9 jedoch aufgrund der geringen Erfolgsrate von Gewebeexplantaten wird eine verbesserte EWG Zellkulturverfahren benötigt , um die Funktion bestimmter Enzyme und Proteine ​​, die in der Vermittlung Nährstoffausnutzung zu untersuchen , indem Vogelembryos während der Entwicklung.

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Gibco by Life technologies</td><td>12800-017 10 X 1 L</td><td>For wash the EECs pellets</td></tr><tr><td>D-MEM/F-12</td><td>Gibco by Life technologies</td><td>12400-024 10 X 1 L</td><td>As the basal medium in culturing EECs</td></tr><tr><td>NBCS</td><td>Gibco by Life technologies</td><td>16010-159</td><td>As the supplyment serum in culturing EECs</td></tr><tr><td>Pen-Strep Ampho. Solution</td><td>BI (Biological Industries)</td><td>03-033-1B 100 ml</td><td>For attenuating the possible infection&amp;nbsp;</td></tr><tr><td>Collagenase Type IV</td><td>Gibco by Life technologies</td><td>17104-019 1 g</td><td>&amp;nbsp;Collagenase is a protease with specificity for the bond between a neutral amino acid (X) and glycine in the sequence Pro-XGly-Pro. As the protease for dissociation of cells from primary tissue.</td></tr><tr><td>24-well plate</td><td>FALCON&lt;sup&gt;&amp;reg;&lt;/sup&gt;</td><td>REF-353047</td><td>For EECs to attach and extension</td></tr><tr><td>50 ml PP centrifuge tubes</td><td>Corning&lt;sup&gt;&amp;reg;&lt;/sup&gt; CentriStar&lt;sup&gt;TM&lt;/sup&gt;</td><td>430829</td><td>For transportion of membranes and enzyme digestion</td></tr><tr><td>50 ml Conical bottomed Tube with Cap</td><td>PRO TECH</td><td>CT-50-PL-TW</td><td>For transportion of membranes and enzyme digestion</td></tr><tr><td>Reciprocal shaking bath</td><td>DEAGLE</td><td>SB302</td><td>For better enzymatic digestion on membranes</td></tr></tbody></table>

primary endodermal epithelial cell culture from the yolk sac membrane of japanese quail embryos

Wir gründeten eine endodermaler epithelialen Zellkulturmodell (EWG) für die Funktion bestimmter Enzyme zu studieren und Proteine, die in Nährstoffverwertung von Vogelembryonen während der Entwicklung zu vermitteln. Befruchtete japanischen Wachteleier wurden für 5 Tage bei 37 ° C inkubiert und dann Dottersackmembranen (YSM) gesammelt wurden die EWG-Kultursystem zu etablieren. Wir isolierten die embryonale Endoderm Schicht aus YSM und in Scheiben geschnitten, die Membran in 2 - 3 mm Stücke und teilweise mit Kollagenase verdaut, bevor sie in 24-Well-Kulturplatten mit Animpfen. Die EECs proliferieren aus dem Gewebe und sind für die Zellkultur-Studien bereit. Wir fanden , dass die EECs typischen Eigenschaften YSM in vivo hatten, beispielsweise Akkumulation von Lipidtröpfchen, die Expression von Sterol O-Acyltransferase und Lipoprotein - Lipase. Die partielle Verdauung Behandlung signifikant erhöht die erfolgreiche Rate der EWG Kultur. Verwendung der EECs haben wir gezeigt, dass die Expression von SOAT1 durch die cAMP d reguliert wurdeependent Protein-A-verwandten Weg Kinase. Diese primäre japanische Wachtel EWG Kultursystem ist ein nützliches Werkzeug embryonale Lipidtransport zu untersuchen und die Rolle der Gene bei der Vermittlung der Nährstoffverwertung in YSM während avian Embryonalentwicklung beteiligt zu klären.

Um das Ziel der Schaffung eines konsistenten und nützliche Zellmodell zu erreichen, müssen wir die Proliferationsrate und Leistung der aviären EECs zu erweitern und zu stabilisieren. Wir verglichen direkte Inkubation der Endoderm ohne Enzymverdauung mit Endoderm teilweise mit proteolytischen Enzymen wie Kollagenase oder Kollagenase und 0,6 U von Dispase verdaut. Dispase ist ein amino-Endopeptidase, die die N-terminalen Peptidbindungen von nicht-polaren Aminosäureresten hydrolysiert. ...

Watch this Scientific Journal Video about Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos at JoVE.com

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

Primäre Endodermal Epithelial Cell Culture aus dem Dottersack-Membran der japanischen Wachtel Embry

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient ...

primary-endodermal-epithelial-cell-culture-from-yolk-sac-membrane

Universidad San Sebastian

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JoVE Journal

Developmental Biology

8.8K Aufrufe.  National Taiwan University. To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

Serie: Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

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

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

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

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

Forschung

Biologie

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Die Analyse der Neuralleiste Migration und Differenzierung von Cross-Spezies Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

Neurowissenschaften

Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the formation of quail-chicken chimeras and the use of the chorioallantoic membrane (CAM) to sustain the development of ectopic tissues date back to the last century. Nowadays, the combination of these classical techniques with recent in vitro methodologies offers novel prospects to further explore organ formation.
Here we describe a two-step approach to study early- and late-stages of organogenesis. Briefly, the embryonic region containing the presumptive territory of the organ is isolated from quail embryos and grown in vitro in an organotypic system (up to 48 h). Cultured tissues are subsequently grafted onto the CAM of a chicken embryo. After 10 days of in ovo development, fully formed organs are obtained from grafted tissues. This method also allows the modulation of signaling pathways by the regular administration of pharmacological agents and tissue genetic manipulation throughout in vitro and in ovo developmental steps. Additionally, developing tissues can be collected at any time-window to analyze their gene-expression profile (using quantitative PCR (qPCR), microarrays, etc.) and morphology (assessed with conventional histology and immunochemistry).
The described experimental procedure can be used as a tool to follow organ formation outside the avian embryo, from the early stages of organogenesis to fully formed and functional organs.

This article provides an updated approach to the classical quail-chicken chimera system to study organ formation, by combining novel in vitro and in ovo experimental procedures.

Zweistufigen Ansatz - und Ende-Frühstadium der Orgel Formation im Vogelgrippe-Modell zu erkunden: die Thymusdrüse und Nebenschilddrüsen Organogenese Paradigma

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the ...

Entwicklungsbiologie

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

Organotypischen Kultur-Methode, die Entwicklung der embryonalen Gewebe zu studieren

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Quail Chorioallantoic Membrane - A Tool for Photodynamic Diagnosis and Therapy

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties make it an excellent in vivo experimental model to study angiogenesis, tumor growth, drug delivery systems, or photodynamic diagnosis (PDD) and photodynamic therapy (PDT). At the same time, this model addresses the requirement for the replacement of experimental animals with a suitable alternative. Ex ovo cultivated embryo allows easy substance application, access, monitoring, and documentation. The most frequently used is chick CAM; however, this article describes the advantages of the Japanese quail CAM as a low-cost and high-throughput model. Another advantage is the shorter embryonic development, which allows higher experimental turnover. The suitability of quail CAM for PDD and PDT of cancer and microbial infections is explored here. As an example, the use of the photosensitizer hypericin in combination with lipoproteins or nanoparticles as a delivery system is described. The damage score from images in white light and changes in fluorescence intensity of the CAM tissue under violet light (405 nm) was determined, together with analysis of histological sections. The quail CAM clearly showed the effect of PDT on the vasculature and tissue. Moreover, changes like capillary hemorrhage, thrombosis, lysis of small vessels, and bleeding of larger vessels could be observed. Japanese quail CAM is a promising in vivo model for photodynamic diagnosis and therapy research, with applications in studies of tumor angiogenesis, as well as antivascular and antimicrobial therapy.

The chorioallantoic membrane (CAM) of the avian embryo is a very useful and applicable tool for various areas of research. A special ex ovo model of Japanese quail CAM is suitable for photodynamic treatment investigation.

Wachtel-Chorioallanto-Membran - ein Werkzeug für die photodynamische Diagnose und Therapie

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties ...

Krebsforschung

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed contributions to unveiling key processes in developmental biology.
Herein is described an optimized method to isolate embryonic tissues from quail and chickens by microsurgery and enzymatic digestion while preserving its biological properties. After isolation, tissues from both species are associated in an in vitro organotypic assay for 48 h. Quail and chicken tissues can be discriminated by distinct nuclear features and molecular markers allowing the study of the cellular cross-talk between heterospecific association of tissues. This approach is, therefore, a useful tool for studying complex tissue interactions in developmental processes with highly dynamic spatial modifications, such as those occurring during pharyngeal morphogenesis and the formation of the foregut endoderm-derived organs. This experimental approach was first developed to study the epithelial-mesenchymal interactions during early-stages of thymus formation. In this, the endoderm-derived prospective thymic rudiment and mesoderm-derived mesenchyme, were isolated from quail and chicken embryos, respectively.
The capacity of the associated tissues to generate organs can be further tested by grafting them onto the chorioallantoic membrane (CAM) of a chicken embryo. The CAM provides nutrients and allows gas exchanges to the explanted tissues. After 10 days of in ovo development, the chimeric organs can be analyzed in the harvested explants by conventional morphological methods. This procedure also allows studying tissue-specific contributions during organ formation, from its initial development (in vitro development) to the final stages of organogenesis (in ovo development).
Finally, the improved isolation method also provides three-dimensionally (3D) preserved embryonic tissues, that can also be used for high-resolution topographical analysis of tissue-specific gene-expression patterns.

This article provides a method to isolate pure embryonic tissues from quail and chicken embryos that can be combined to form ex vivo chimeric organs.

Isolierung der embryonalen Gewebe und die Bildung von Wachteln-Huhn Chimären Organe Thymus am Beispiel

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed ...

In Vitro Culture of Epithelial Cells from Different Anatomical Regions of the Human Amniotic Membrane

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that the amniotic epithelium is a homogeneous layer. The human amnion can be divided into three anatomical regions: reflected, placental, and umbilical. Each region has different physiological roles, such as in pathological conditions. Here, we describe a protocol to dissect human amnion tissue in three sections and maintain it in vitro. In culture, cells derived from the reflected amnion displayed a cuboidal morphology, while cells from both placental and umbilical regions were squamous. Nonetheless, all the cells obtained have an epithelial phenotype, demonstrated by the immunodetection of E-cadherin. Thus, because the placental and reflected regions in situ differ in cellular components and molecular functions, it may be necessary for in vitro studies to consider these differences, because they could have physiological implications for the use of HAEC in biomedical research and the promising application of these cells in regenerative medicine.

This protocol describes the isolation of epithelial cells from different anatomical regions of the human amniotic membrane to determine their heterogeneity and functional properties for possible application in clinical and physiopathological models.

In Vitro Kultur von Epithelzellen aus verschiedenen anatomischen Regionen der menschlichen Amnionmembran

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that ...

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

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

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

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

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 ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Isolierung und Kultivierung von Avian Embryonale Valvular Progenitorzellen

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

This versatile protocol describes the isolation of premigratory neural crest cells (NCCs) through the excision of cranial neural folds from chick embryos. Upon plating and incubation, migratory NCCs emerge from neural fold explants, allowing for assessment of cell morphology and migration in a simplified 2D environment.

Präparation und morphologische Analyse von Küken-Schädel-Neuralleistenzellkulturen

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like ...