O financiamento para o desenvolvimento de protocolo atual é particularmente apoiada por &quot;Aim para o Plano Universidade Top&quot;, da Universidade Nacional de Taiwan, Taiwan (concessão ID-104R350144), bem como o Ministério da Ciência e Tecnologia de Taiwan (ID concessão: a maioria dos 104 -2313-B-002-039-MY3). Agradecemos especialmente o Centro de Biotecnologia da Universidade Nacional de Taiwan para fornecer uma sala de animais e espaço de laboratório para o estudo atual.

NOTA: Este procedimento é uma modificação de um protocolo de cultura modelo de galinha desenvolvido por Bauer et al 2013 e Nakazawa et al, 2011. 2,9.. 1. Prepare Healthy Embryonic Dia 5 embriões de codorniz japonesa <ol><li> Coloque 1 do sexo masculino e 3 do sexo feminino sexualmente maduros codornas japonesas juntos na mesma gaiola. Alimentação de alimentação e água ad libitum. Ajustar a luz na sala de animal para 14 horas de luz e 10 horas de escuridão. </li><li> Recolher os ovos fertilizados em sacos de plástico a cada dia no período da tarde e mantê-los em um 16 ° C geladeira para retardar a taxa de crescimento dos embriões. </li><li> Após recolha de um número suficiente de ovos dentro de um período de duas semanas, incubar os ovos fertilizados de uma incubadora a 37 ° C com bom arejamento durante cinco dias. </li></ol> 2. Prepare Basal Médio e soluções de lavagem <ol><li> Use DMEM / F-12 (ajustado a pH 7,2) como meio de crescimento, e completá-locom o novo 10% de soro vitelo recém-nascido (NBCS) e 1% de Pen-Strep Ampho. Solução (PSA; penicilina, 10 5 unidades / L; estreptomicina, 100 mg / l; anfotericina B, 0,25 mg / L). </li><li> Preparar a solução salina tamponada com fosfato (PBS) a uma concentração 10x para o armazenamento; usar a solução 1x PBS (contendo 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; acertar a pH 7,2) para lavar e umedecer o tempo membrana saco vitelino separação da endoderme de gema de ovo e albumina. </li><li> Use meio DMEM (ajustado para pH 7,2) para re-suspender as pelotas endoderme. </li></ol> 3. Recolha de EECS primária de japoneses codorniz YSMs <ol><li> Incubar os ovos recolhidos durante 5 dias (passo 1.3). Examine os ovos por um candler ovo para confirmar o desenvolvimento embrionário normal. NOTA: No dia embrionário 5, o limite da YSM mesoderme é muito clara e as 3 camadas embrionárias são menos bem-ligados entre si, por isso é fácil de obendoderme Tain. Utilize apenas os ovos que apresentem o desenvolvimento normal circulação capilar para coleções YSM. </li><li> Limpar a casca de ovo com cuidado usando água doce e 75% de etanol. Abra a casca de ovo a partir da posição das células de ar por um par de tesouras, descascar delicadamente fora e recolher toda a YSM por uma pinça e coloque em um prato de cultura 10 centímetros preenchido com 37 ° C 1x PBS. </li><li> Utilizando um par de tesouras remover o embrião, gema e clara de ovo, e lavar cuidadosamente o YSM com 1x PBS três vezes e colocar YSM em solução de PBS numa placa de cultura de 10 cm. </li><li> Use uma pinça e um par de tesouras para remover o ectoderme de YSM. Use 2 fórceps, um para segurar firmemente a camada de células endoderme, e a outra para segurar o mesoderme capilar. </li><li> Sob um microscópio de dissecação, separar e separar a endoderme a partir da borda da mesoderme em direcção ao embrião. Recolhe-se o endoderme amarelo claro em 50 ml de tubos de centrífuga usando um conta-gotas e o lugar em 15 ml de meio de crescimento numa Wat 37 ° Cbanho er até que todas as coleções de tecidos estão concluídas. </li></ol> 4. A digestão do Endoderma fatias por digestão de colagenase <ol><li> Recentemente dissolver 6,5 unidades de colagenase tipo IV em 10 ml de DMEM. </li><li> Centrifugar os tubos de 50 ml (3,4) a 130 xg durante 3 minutos à temperatura ambiente. Aspirar o meio e colocar toda a endoderme em uma placa de cultura de 6 centímetros, usando uma pipeta. Adicionar solução de colagenase 1 ml a esta placa de cultura. </li><li> Cortar a endoderme recolhidos a partir de 2 embriões de codorniz com um par de tesouras curvas até que o tamanho de cada fatia é de aproximadamente 2 a 3 mm. Usar um conta-gotas, recolher todas as fatias para um tubo de centrífuga de 50 ml, juntamente com 9 ml de solução de colagenase fresco. </li><li> Digerir o tecido com colagenase durante 30 minutos num banho de água com agitação a 37 ° C a 175 rpm. Esta digestão parcial com colagenase é utilizado para melhorar a proliferação de células dos explantes. </li><li> Centrifugar a 130 xg durante 3 minutos à temperatura ambiente e suavemente remover a fracção do sobrenadante utilizando uma pipeta. Lava-se a pelete por suspensão em 20 ml de DMEM e remover a fracção do sobrenadante após a centrifugação de 130 x g durante 3 min. </li><li> Re-suspender o sedimento com 12 ml de meio de crescimento (DMEM / F12 com 10% de NBCS e 1% de PSA) e sementes suavemente e igualmente numa placa de 24 poços (cada poço uma solução de células com 500 ul). </li><li> Incubar explantes de tecido a 37 ° C em 5% de CO 2 em ar durante 2 dias para permitir que as células a proliferar para fora dos tecidos. </li><li> Incubar as células por mais 2 dias e caracterizar quanto à viabilidade celular por um protocolo de ensaio de viabilidade celular. 15 NOTA: Living células manter um ambiente de redução dentro do citoplasma. Resazurina, o ingrediente activo, é, de um composto não-tóxico permeável célula que é azul em cor e virtualmente não fluorescente. Quando adicionado a culturas de células, a Resazurina é reduzida a resorufina no citosol por enzimas mitocondriais. Resorufina é de cor vermelha e altamente fluorescente. As células viáveis ​​converter continuamente resazurina a resorufina, increasing a fluorescência global e cor do meio de cultura de células circundante. </li><li> Realizar endoderme célula epitelial de isolamento de ARN total, a transcrição e quantitativa PCR em tempo real de expressão de ARNm de gene marcador reversa como descrito 16,17. <ol><li> Use de PCR de transcriptase reversa para detectar SOAT1 ARNm (sentido directo: 5&#39;-GAAGGGGCCTATCTGGAACG-3 &#39;; anti-sentido: 5&#39;-ATCTGCACGTGACATGACCA-3&#39;; produto de PCR = 168 pb) como um marcador específico para as células CEE. Use β-actina de ARNm (sentido directo: 5&#39;-TGGTGAAGCTGTAGCCTCTC-3 &#39;; anti-sentido: 5&#39;-GTGATGGACTCTGGTGATGG-3&#39;; produto de PCR = 151 pb), como um gene de manutenção e o controlo interno. </li><li> Use electroforese em gel para detectar o tamanho do produto de RT-qPCR. 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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.

Uma vez que o sistema de cultura anterior tem apenas um sucesso limitado, é necessário um sistema de cultura melhor. A endoderme codornizes YSM japonesa requer tratamento com enzimas proteolíticas tais como a colagenase para soltar as junções célula-célula para alcançar um melhor desempenho do crescimento nos explantes ex-vivo. Os nossos dados mostram que o número de células de tratamento digestão parcial foi maior do que a partir da cultura de tecido não digerido após a sementeira, durante 2 dias (...

O recurso principal nutricional do embrião aviário é gema, composto de 33% de lípidos, proteínas 17%, e 1% de cinzas. 1 durante o desenvolvimento embrionário, a membrana vitelina (YSM) cresce a partir de dentro da cavidade abdominal embrionário e cobre gradualmente a gema superfície. Começando no dia embrionário 2, a expressão de genes associados com o metabolismo dos lípidos e a angiogénese é gradualmente aumentada em YSM, e o YSM desenvolve lentamente projecções das vilosidades semelhantes. Estas projecções 8,9 aumentar a absorção de nutrientes gema para suportar o desenvolvimento embrionário. O YSM é um tecido extra-embrionário que contém três camadas germinativas, endoderme, mesoderme e ectoderme. 14 do ectoderma saco vitelino enfrenta a clara e links com a membrana vitelina para cobrir suavemente o saco vitelino. As células epiteliais da endoderme são confrontados diretamente para a gema de ovo e servir como portais de utilização de nutrientes. 6 À medida que a ADM se expande, células epiteliais endodérmico (EECS) pode ser dividido por shape e funcionalidade em dois grupos, a área vitelínicas e vasculosa área. 7 vitelina área é composta por células da endoderme e está distante do embrião; vasculosa área é composta por células mesodérmicas e cobre EECS diferenciadas com vasos sanguíneos e tecidos conjuntivos. Por dia embrionário 5, a gema é totalmente coberta por ectoderma e endoderma de YSM ea área vascular tem crescido rapidamente. O YSM absorve, recompõe e liberta lípidos (como-gema derivado lipoproteína de muito baixa densidade) e proteínas no sistema circulatório embrionário. 9, 2 Assim, foi estabelecido um embrionário sistema de codorniz japonesa primária endodérmica epitelial de cultura de células, para estudar os mecanismos de lípido utilização em YSM durante o desenvolvimento embrionário aviária. Os lípidos tais como triglicerídeos, lecitina, de fosfolípidos e ésteres de colesterol (CE) são as fontes de energia primárias para embriões de aves. Nas fases iniciais de desenvolvimento, lípidos da gema são Composed de apenas 1,3% do CE e ele sobe para 10-15% a meio do período de desenvolvimento embrionário aviária 3, 11. éster de colesterol é sintetizado a partir do colesterol, esteróis O-aciltransferase 1 (SOAT1) em YSM embrião aviária. 4 A forma de armazenamento de colesterol é CE, CE é realizada nas lipoproteínas e lipoproteínas são transportados pela circulação para os tecidos. 13 Uma semana antes escotilha há um crescimento rápido dos embriões de aves. Aproximadamente 68% do conteúdo de lípidos restantes na gema são absorvidos durante esta fase. 10 O mecanismo pelo qual a gema de lípidos são utilizados pode ser clarificada por um modelo de pesquisa CEE. Um protocolo de cultura de frango CEE foi estabelecido para atingir este objectivo de pesquisa. 2, 9 No entanto, devido à baixa taxa de sucesso de explantes de tecidos, um processo de cultura de células CEE melhorada é necessária para estudar a função de certas enzimas e proteínas na mediação de a utilização de nutrientes pelos embriões de aves durante o desenvolvimento.

<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

Nós estabelecemos um modelo de cultura de células epiteliais endodérmico (CEE) para estudar a função de certas enzimas e proteínas na mediação de utilização de nutrientes por embriões de aves durante o desenvolvimento. Fertilizados ovos de codorniz Japonesa foram incubadas a 37 ° C durante 5 dias e, em seguida, yolk sac membranas (YSM) foram recolhidas para se estabelecer o sistema de cultura de CEE. Isolamos a camada endoderma embrionário de YSM, e cortado da membrana em 2 - 3 mm pedaços e parcialmente digerido com colagenase antes de semear em placas de cultura de 24 poços. O EECS proliferam para fora do tecido e estão prontos para os estudos de cultura de células. Descobrimos que o EECS tinha características típicas de YSM in vivo, por exemplo, acúmulo de gotículas lipídicas, expressão de esteróis O-aciltransferase e lipoproteína lipase. O tratamento a digestão parcial aumentou significativamente a taxa de sucesso da cultura CEE. Utilizando o EECS, foi demonstrado que a expressão de SOAT1 foi regulado pelo AMPc dproteína quinase ependent Um caminho relacionado. Este sistema de cultura de codorna japonesa CEE primária é uma ferramenta útil para estudar o transporte de lipídios embrionário e para esclarecer o papel de genes envolvidos na mediação de utilização de nutrientes em YSM durante o desenvolvimento embrionário aviária.

A fim de alcançar o objetivo de estabelecer um modelo de celular consistente e útil, nós precisamos de alargar e estabilizar a taxa de proliferação e desempenho dos EECS aviária. Comparamos a incubação directa da endoderme sem digestão com enzima endoderme parcialmente digerido com enzimas proteolíticas, tais como colagenase ou colagenase mais 0,6 L de dispase. Dispase é um amino-endopeptidase que hidrolisa as ligações peptidicas N-terminais de resíduos de aminoácidos não...

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.

Primária endodérmico epitelial Cultura de Células da gema Sac Membrana de japoneses codorniz Embriões

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

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8.8K visualizações.  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.

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

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

Um Sanduíche papel de filtro submerso por longo prazo<em&gt; Ex Ovo</em&gt; Imagem Time-lapse de galinha precoce embriões

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

Biologia do Desenvolvimento

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.

Método para a Cultura de Embriões início pintainho ex vivo (Nova Cultura)

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

Biologia

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.

Análise de migração da crista neural e Diferenciação por Cross-espécies de Transplantes

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

Neurociência

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.

Abordagem em duas fases para explorar e tarde-estágios iniciais de formação do órgão no modelo aviária: O Timo e glândulas paratireoides organogênese paradigma

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

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.

Cultura de organotypic método para estudar o desenvolvimento de tecidos embrionários de galinha

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.

Membrana corioalantóica de codorna - Uma ferramenta para diagnóstico fotodinâmico e terapia

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

Pesquisa do câncer

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.

Isolamento de tecidos embrionários e formação de órgãos quiméricoes codorna-frango, usando o exemplo do Timo

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 Cultura das Células Epitelias de diferentes regiões anatômicas da membrana amniótica humana

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

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.

Isolamento e Cultura da Avian embrionárias Células Progenitoras Valvular

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.

Preparação e Análise Morfológica das Culturas celulares da crista neural do pintinho

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