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

Mevcut protokolün geliştirilmesi için finansman özellikle Ulusal Tayvan Üniversitesi, Tayvan (hibe ID-104R350144) yanı sıra, Tayvan Bilim ve Teknoloji Bakanlığı (hibe kimliği &quot;Top Üniversitesi Planı Amaç&quot; tarafından desteklenmektedir: EN 104 -2313-B-002-039-My3). Biz özellikle mevcut çalışma için bir hayvan odası ve laboratuar alanı sağlamak için Ulusal Tayvan Üniversitesi Biyoteknoloji Merkezi teşekkür ederiz.

Not:.. Bu prosedür ve Bauer ve ark, 2013 ve Nakazawa ve arkadaşları, 2011 tarafından geliştirilen 2,9 tavuk modeli Kültür Protokolü bir modifikasyonudur 1. Sağlıklı Embriyonik Gün 5 embriyolar Japon Bıldırcınlarında gelen hazırlayın <ol><li> Aynı kafeste birlikte 1 erkek ve 3 kadın cinsel olgun Japon bıldırcın yerleştirin. Besleme yem ve su ad libitum verilmiştir. Işığın 14 saat ve karanlığın 10 saat hayvan odasında ışık ayarlayabilirsiniz. </li><li> öğleden sonra her gün plastik torbalarda döllenmiş yumurta toplamak ve embriyoların büyüme hızını yavaşlatmak için bir 16 ° C buzdolabında saklayın. </li><li> İki haftalık bir süre içinde yumurta yeterli sayıda topladıktan sonra, beş gün boyunca iyi bir havalandırma ile 37 ° C inkübatör döllenmiş yumurta kuluçkaya yatmaktadır. </li></ol> 2. Bazal Orta ve Yıkama Çözümleri hazırlayın <ol><li> Büyüme ortamı olarak (pH 7.2 a ayarlanmıştır), DMEM / F-12 ortamı kullanın, ve ek% 10 yeni doğmuş buzağı serumu (NBCs) ve% 1 Pen-Strep Ampho ile. Çözelti (PSA, penisilin, 10 5 birim / L streptomisin, 100 mg / l; amfoterisin B, 0.25 mg / L). </li><li> depolama için 10x konsantrasyonda fosfat tamponlu tuz (PBS) çözeltisi hazırlayın; yıkamak için (pH 7.2 ayarlamak, KCI, 2,667 mM; KH 2 PO 4, 1.471 mM;; Na 2 HPO 4 -7 2 O, 8.06 mM NaCl, 137,93 mM içeren) ve yolk kesesi membran süre nemlendirin 1x PBS çözümü kullanın yumurta sarısı ve albümin gelen endoderm ayıran. </li><li> endoderm pelet yeniden askıya (pH 7.2 a ayarlanmıştır) DMEM ortamı kullanın. </li></ol> Japon Bıldırcın YSMs Primary EECS 3. Koleksiyonu <ol><li> 5 gün (adım 1.3) için toplanan yumurta kuluçkaya yatmaktadır. normal embriyonik gelişmeyi teyit etmek için bir yumurta Candler tarafından yumurta inceleyin. NOT: embriyonik 5. günde, YSM mezodermin sınır çok net ve 3 embriyonik tabakalar daha az birbirine sıkıca bağlı olan, bu nedenle ob kolaydırtain Endoderm. Sadece YSM koleksiyonları normal kapiller dolaşımı gelişim gösteren yumurta kullanın. </li><li> dikkatle taze su ve% 75 etanol kullanarak kabuğu temizleyin. nazikçe, bir makas ile hava hücre konumundan yumurta kabuğu açın dışarı soyun ve 37 ° C 1x PBS ile dolu bir 10 cm kültür çanak forseps ve yerine göre bütün YSM toplamak. </li><li> bir makas embriyo, yumurta sarısı ve yumurta beyazı kaldırmak ve nazikçe 1x PBS ile YSM yıkayın üç kez ve 10 cm kültür çanak PBS çözüm YSM yerleştirin kullanma. </li><li> YSM gelen ektoderm kaldırmak için forseps ve bir makas kullanın. 2 forseps kullanabilir, bir sıkıca endodermal hücre tabakası tutmak için, ve diğer kılcal mezoderm tutun. </li><li> bir mikroskop altında ayrı çekin ve embriyo doğru yönde mezodermin kenarından endoderm ayırın. 37 ° C wat büyüme ortamına, 15 ml bir damlalık ve yer kullanarak 50 ml santrifüj tüplerine açık sarı endoderm toplamaker banyosu tüm doku koleksiyonları tamamlanana kadar. </li></ol> Kollajenaz Sindirim tarafından Endoderm Dilimleri 4. Sindirim <ol><li> Taze DMEM, 10 ml tip IV kollajenaz 6.5 birim çözülür. </li><li> Oda sıcaklığında 3 dakika boyunca 130 x g&#39;de 50 ml tüpler (3.4) santrifüjleyin. orta aspire ve bir pipet kullanarak 6 cm kültür çanak tüm endoderm yerleştirin. Bu kültür kabı 1 ml kolajenaz çözeltisi ekleyin. </li><li> Her dilimin büyüklüğüne kadar kavisli bir makasla 2 bıldırcın embriyolardan alınan endoderm dilimleyin yaklaşık 2 ila 3 mm. Bir damlalık kullanarak taze kolajenaz çözeltisi 9 ml ile birlikte bir 50 ml santrifüj tüpüne tüm dilimleri toplar. </li><li> 175 rpm&#39;de 37 ° C&#39;de ve çalkalama su banyosunda 30 dakika boyunca kolajenaz ile doku Digest. kollajenaz ile Bu kısmi sindirim eksplant hücre çoğalmasını iyileştirmek için kullanılır. </li><li> Oda sıcaklığında 3 dakika boyunca 130 x g&#39;de santrifüj ve yavaşça bir pipet kullanılarak süpernatan fraksiyonu kaldırma. 20 ml DMEM içinde süspansiyon pelet yıkayın ve 3 dakika boyunca 130 x g santrifüj sonra süpernatan fraksiyonu çıkarın. </li><li> Yeniden askıya büyüme ortamında (% 10 NBCs ve% 1 PSA DMEM / F12) 12 ml pelet ve 24 oyuklu bir plakaya yavaşça ve eşit (her bir 500 ul hücre çözelti) tohum. </li><li> Hücreler doku üzerinden çoğalmaya izin 2 gün boyunca havada% 5, 37 ° C&#39;de CO2 doku eksplantlarında inkübe edin. </li><li> Başka bir 2 gün boyunca inkübe hücreleri ve hücre canlılığı tahlil protokolü tarafından hücre canlılığı için karakterize eder. 15 NOT: Yaşayan hücreler sitoplazmada içinde bir indirgeyici bir ortam sağlamak. Resazurin, aktif terkip maddesi, mavi renkte ve hemen hemen floresan olmayan bir toksik-olmayan, hücre geçirgen bir bileşiktir. hücre kültürlerine eklendiğinde, resazurin mitokondriyal enzimlerin sitosolde resorufine azaltılır. Resorufm kırmızı renkte ve oldukça floresan olduğunu. Canlı hücreler sürekli resazurin Resorufm dönüştürmek, artışlarlahücreleri çevreleyen kültür ortamının genel floresans ve renk asing. </li><li> Açıklandığı gibi transkripsiyon ve PCR marker geni mRNA kantitatif gerçek zamanlı ters endoderm epitel hücresi total RNA izolasyonu gerçekleştirin. 16,17 <ol><li> SOAT1 mRNA tespit etmek için ters transkriptaz PCR kullanarak (sens: 5&#39;-GAAGGGGCCTATCTGGAACG-3 &#39;; antisens: 5&#39;-ATCTGCACGTGACATGACCA-3&#39; PCR ürünü = 168 bp) EEC hücreleri için bir özgün bir işaret olarak. Bir temizlik geni ve iç kontrol olarak β-Aktin mRNA (PCR ürünü = 151 bp: &#39;;; 5&#39;-GTGATGGACTCTGGTGATGG-3 anlamsız&#39; 5&#39;TGGTGAAGCTGTAGCCTCTC-3 duygusu) kullanın. </li><li> RT-qPCR ürün boyutunu tespit etmek için jel elektroforezi kullanın. % 1 agaroz jeli hazırlayın ve 2 ul yükleme boyası 10 ul qPCR karıştırın. Yük 10 ul de% 1 agaroz jeli içinde her birine Karışım 30 dakika süre ile 100V ile jel üzerinde çalıştırıldı. </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.

Önceki kültürü sistemi, sadece sınırlı bir başarı olduğundan, daha iyi bir kültür sistemi gereklidir. Japon bıldırcın YSM endoderm gibi ex-vivo eksplant daha iyi bir büyüme performansı elde etmek için hücre-hücre kavşakları gevşetmek için kollajenaz gibi proteolitik enzimlerle tedavi gerektirir. Verilerimiz kısmi sindirimi tedavi hücre sayısı 2 gün sonra (Şekil 1) için tohumlama sonra sindirilmemiş doku kültüründen daha fazla olduğunu göstermektedir. Bu nedenle, kı...

Kuş embriyo önemli beslenme kaynağı% 33 lipit,% 17 protein oluşan sarısı, ve% 1 kül. 1 embriyo gelişimi sırasında, sarısı kese zarının (YSM) embriyonik karın boşluğu içinde yetişen ve yavaş yavaş sarısı kapsar yüzey. Embriyonik gün 2 başlayarak, lipit metabolizması ve anjiyojenez ile ilişkili gen ekspresyonu kademeli YSM artar ve YSM yavaş tüy benzeri çıkıntılar geliştirir. 8,9 Bu çıkıntılar embriyo gelişimini desteklemek için sarısı besin emilimini artırır. YSM üç germ katmanları, endoderm, mezoderm ve ektoderm içeren bir extraembryonic dokudur. 14 yolk kesesi ektoderm nazikçe sarısı kesesi kapsayacak şekilde vitellin membran ile albümin ve bağlantılar ile karşı karşıyadır. Endodermal epitel hücreleri Kadm bölünebilir yumurta sarısı doğru doğrudan karşı karşıya ve besin kullanım portalları olarak hizmet vermektedir. 6 YSM genişledikçe, Endodermal epitel hücre (EECS) vardırİki grup, bölge vitelline ve alan vaskulosa içine e ve işlevsellik. 7 Konum Vitellin endodermal hücrelerden oluşan ve embriyo uzak olmaktadır; Konum vaskulosa mezodermal hücrelerden oluşan ve kan damarları ve bağ dokuları ile farklılaştırılmış EECS kapsar. embriyonik gün 5 ile, yumurta sarısı tamamen YSM ve ektoderm ve endoderm ile kaplıdır ve vasküler alan hızla büyüdü. YSM embriyonik dolaşım sistemine (sarısı türetilen çok düşük yoğunluklu lipoprotein) ve proteinler. 9, 2 Bu nedenle, lipit mekanizmaları incelemek için, birincil Japon bıldırcın embriyonik endodermal epitel hücre kültür sistemi tesis yeniden oluşturur ve serbest bırakır lipidler, emer kuş embriyonik gelişim sırasında YSM içinde kullanımı. Böyle triaçilgliserol, lesitin, fosfolipid ve kolesterol esteri (CE) olarak lipidler kuş embriyolar için birincil enerji kaynaklarıdır. gelişiminin erken aşamalarında, yumurta sarısı lipidler c vardırsadece% 1.3 CE omposed ve kuş embriyonik gelişim 3 orta vadede% 10-15 yükselir, 11. Kolesterol ester kuş embriyo YSM sterol O-asiltransferaz 1 (SOAT1) tarafından kolesterolden sentezlenir. 4 Kolesterol depo formu CE, CE lipoproteinler taşınır ve lipoproteinler dokulara dolaşımı ile taşınmaktadır. 13 Bir hafta ambar önce kuş embriyoların hızlı bir büyüme var. Yaklaşık sarısı kalan lipit içeriğinin% 68, bu aşamada emilir. 10 lipidler bir AET araştırma modeli ile açıklık kazanabilir kullanılmaktadır sarısı hangi mekanizma. Bir tavuk EEC kültürü protokolü bu araştırma hedefe ulaşmak için kurulmuştur. 2, 9 Ancak, gelişmiş AET hücre kültürü prosedürü ile besin kullanımını aracılık belirli enzimler ve proteinlerin fonksiyonlarını incelemek için gerekli doku eksplant düşük başarı oranına gelişimi sırasında kuş embriyolar.

<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

Biz gelişim sırasında kuş embriyolar tarafından besin kullanımı aracılık belirli enzimler ve proteinlerin fonksiyonlarını incelemek için bir Endodermal epitel hücre kültürü modeli (AET) kuruldu. Döllenmiş Japon bıldırcın yumurta 5 gün boyunca 37 ° C&#39;de inkübe edildi ve daha sonra SAC membranları (YSM) EEC kültür sistemi kurmak için toplandı sarısı edildi. Biz YSM embriyonik endoderm katman izole edilir ve 2&#39;ye membran dilimlenmiş - 3 mm parçalar kısmen 24 oyuklu kültür plakaları içinde, tohumlamadan önce kollajenaz ile sindirilir. EECS dokusu içinde çoğalan ve hücre kültür çalışmaları için hazırdır. Bu EECS, örneğin, in vivo olarak YSM tipik özelliklerini, lipid damlaları birikimine, sterol O-asiltransferaz ve lipoprotein lipaz ekspresyonunu olduğu bulundu. Kısmi sindirim tedavisi önemli ölçüde AET kültürünün başarılı oranı arttı. EECS kullanarak, biz SOAT1 sentezlenmesi cAMP d düzenlenir olduğunu göstermiştirependent protein A ile ilgili yol kinaz. Bu birincil Japon bıldırcın EEC kültür sistemi embriyonik lipid ulaşım incelemek ve kuş embriyonik gelişim sırasında YSM besin kullanımını aracılık yer alan genlerin rolü açıklığa kavuşturmak için bir araçtır.

tutarlı ve yararlı bir hücre modeli oluşturulması amaca ulaşmak için, biz uzanan ve çoğalma hızı ve avyan EECS performansını stabilize etmek gerekir. Bu kısmen, kolajenaz ya da kollajenaz artı dispase 0.6 U gibi proteolitik enzimler ile sindirilmiş endoderm ile bir enzimi sindirimi ile endoderm doğrudan inkübasyon karşılaştırıldı. Dispase polar olmayan amino asit kalıntılarının N-terminal peptid bağlarını hidrolize bir amino-endopeptidazdır. Proteaz sindir...

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.

Japon Bıldırcın Embriyolar Yolk Sac Membran Primary Endodermal Epitel Hücre Kültürü

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

Research

JoVE Journal

Developmental Biology

8.8K Görüntüleme.  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.

Video: 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.

Erken Civciv Embriyolar ex vivo (Kültür) Kültür Yöntemi

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

Biyoloji

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.

Çapraz türler Nakli Sinir Crest Göç ve Farklılaşma Analiz

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

Nörobilim

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.

İlk - ve geç-aşamalarında Organ oluşumu kuş modelindeki keşfetmek için iki adımlı yaklaşım: timus ve paratiroid bezleri Organogenesis paradigma

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

Gelişim Biyolojisi

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.

Embriyonik tavuk doku gelişimi çalışmaya Organotypic kültür yöntemi

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.

Bıldırcın Korioallantoik Membran - Fotodinamik Tanı ve Tedavi için Bir Araç

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

Kanser Araştırmaları

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.

Yalıtım embriyonik doku ve timus örnek kullanarak bıldırcın-tavuk Chimeric organ oluşumu

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.

İnsan Amniyotik Membranının Farklı Anatomik Bölgelerinden Epitel Hücrelerin In Vitro Kültürü

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.

Bir Eski-ovo</emGörüntüleme ve Mikrocerrahi Uygulamaları için uygundur> Tavuk Embriyo Kültürü Sistemi

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.

Kuş Embriyonik Kapak Progenitör Hücreler izolasyonu ve Kültür

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.

Civciv Kraniyal Nöral Kret Hücre Kültürlerinin Hazırlanması ve Morfolojik Analizi

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