Nós agradecemos o Sr. Hajime Ichijo conselhos para a gravação de vídeo e útil para editar o vídeo. Agradecemos também Drs.Yuji Tsunekawa e Kaichi Yoshizaki para assistente de tipo para gravação de vídeo. Este trabalho é apoiado por KAKENHI em B Jovem Cientista ea prioridade Ciência Cérebro Áreas-Molecular de MEXT do Japão. Agradecemos o apoio da Global COE Programa &quot;Centro de Investigação Básica e translacional para a Ciência do Cérebro Global&quot; a partir MEXT do Japão e de Pesquisa Núcleo de Ciência e Tecnologia Evolutiva (CREST) ​​de Ciência japonês e Corporação de Tecnologia de Ciência do Japão e Agência de Tecnologia (JST) .

1. Configurando o sistema WEC <ol><li> Conjunta de uma membrana M 0,22 filtro para o tubo de silicone de entrada no sistema um WEC). </li><li> Abrir a válvula de um cilindro de gás contendo O 2 (95%) e CO 2 (5%). Fluxo da mistura de gases no cilindro através da garrafa borbulhando contendo água esterilizada. </li><li> Ajustar o volume do fluxo de mistura gasosa a 50 cc. </li><li> Insira o tubo de silicone para deixar em uma garrafa de água e verificar o fluxo de mistura gasosa. </li><li> Girar o tambor com a velocidade de 20 rpm / min. </li></ol> 2. Preparação de meio de cultura <ol><li> Rat-degelo imediatamente centrifugado (IC) de soro em 37,0 ° C b). Adicionar D-glicose (2 mg / ml) em soro descongeladas em um copo ml autoclavado 100. Meio de cultura deve ser preparado no nível de cultura primária (por exemplo, usando um capuz). </li><li> Adicionar antibióticos antimicótico líquido (100x) para o soro com 1: 400 de diluição. </li><li> Vaporize isoflurano restantes (devido à anestesia de ratos durante a coleta de sangue) do soro por 20 minutos em temperatura ambiente (RT) c). </li><li> Esterilizar o meio de cultura usando um filtro de membrana 0,45 mM. </li><li> Despeje 3,0 ml do meio em cada frasco de cultura e selar a parte superior de cada frasco com uma ficha de silício, coberta com folha de alumínio (ver ref. 14) d). </li><li> Prepare três ou quatro pratos disposição de Petri contendo solução salina e de Tyrode). </li></ol> 3. Anestesia do rato e do isolamento do útero <ol><li> Anestesiar um rato timed-grávidas profundamente com anestésicos inalatórios ou parenteral. </li><li> Limpar a área abdominal do rato anestesiado grávida de etanol 70%. </li><li> Pick up a pele com uma pinça e exteriorizar a parede abdominal, cortando a pele com uma tesoura grande f). </li><li> Pick up da parede abdominal com um par de fórceps e fazer em forma de U grandes incisões longitudinal na parede abdominal com uma tesoura grande até o nível torácico g). </li><li> Arribar do intestino para o lado esquerdo com um par de fórceps e expor uma extremidade do útero ligado ao ovário. </li><li> Pegue o final do útero com uma pinça e corte a posição entre o útero e ovário com uma tesoura grande. </li><li> Tirar o útero de ratos fêmeas grávidas, cortando a gordura ao redor do útero até a outra extremidade do útero. </li><li> Transferência do útero para uma placa de Petri contendo solução salina de Tyrode. </li><li> Euthanize o rato por excesso de administração de anestésicos ou deslocamento cervical após a remoção do útero. </li></ol> 4. Remoção dos embriões do útero <ol><li> Brevemente lavar o útero em solução salina Tyrode e transferi-lo para a segunda placa de Petri com um par de pinças finas h). </li><li> Sob um microscópio binocular, pegar a parede uterina com um par de pinças finas e cortar a parede uterina no lado oposto ao mesométrio conectar-se com os vasos sanguíneos usando uma tesoura oftálmica em linha reta. </li><li> Insira a ponta de uma tesoura oftálmica em linha reta no espaço entre a parede uterina e decídua. Cortar a parede uterina longitudinalmente ao longo do lado antimesometrial. </li><li> Moita a decidua no lado mesometrial e conceptos separado do útero através de dois pares de pinças finas i). </li><li> Transferência de conceptos à placa de Petri terceiro com uma pipeta de vidro esterilizados, com um diâmetro adequado. </li></ol> 5. Dissecação de embriões de ratos <ol><li> Inserir uma ponta do micropinças na decídua e fazer uma incisão transversal na decídua em torno do concepto com dois pares de fórceps. </li><li> Inserir as pontas dos pinça na decídua no lado placentário e fazer duas incisões longitudinais na decídua até o topo. </li><li> Remova a decídua no lado placentário rasgando que com dois pares de fórceps. Remover o decidua restantes com dois pares de fórceps. </li><li> Pick up membrana de Reichert, faça uma incisão horizontal sobre o mesmo, e separar a membrana do concepto no lado anti-placentária. </li><li> Faça um pequeno furo no saco vitelino perto da cabeça do embrião com os dois pares de fórceps, e cortar o saco vitelino com uma tesoura oftálmica curvas. É importante para não danificar os vasos sanguíneos. </li><li> Pegue o âmnio no local em torno da cabeça do embrião com os dois pares de pinças finas, e retire com cuidado o embrião a partir do saco vitelino, rasgando o âmnio h). O corpo do embrião deve ser puxado para fora do saco vitelino para aumentar a oferta de oxigênio para o embrião no estágio midgestation. </li></ol> 6. Cultura de embriões todo <ol><li> Verificar danos na placenta, saco vitelino, e da condição do coração batendo e circulação sanguínea. </li><li> Estar ciente de não esticar o cordão umbilical, a transferênciao embrião de um frasco de cultura com uma pipeta de vidro esterilizado. Reporte de solução salina Tyrode para garrafas de cultura deve ser o mínimo possível. </li><li> Remover um plug de silicone não-buraco do tambor rotador. Conectar o frasco de cultura com uma ficha com um buraco no tambor rotator do sistema WEC perpendicularmente. Tenha cuidado para não colocar força no tambor em direções inadequadas desde o drum rotador é um equipamento muito bem. Os frascos contendo o embrião dissecados são transferidos para a cultura, um por um; abertura da porta da frente da incubadora devem ser minimizados para evitar que a temperatura da incubadora é diminuída j). </li><li> Após 10 h de cultura, aumentar o volume de fluxo da mistura de gás de até 75 cc e até 100 cc após 24 h (Tabela 1). </li><li> O meio de cultura deve ser mudado em torno de 24 horas através da transferência dos embriões cultivados em uma garrafa nova cultura com o meio recém preparado. Aumentar o volume de fluxo de até 125 cc, depois de 34 horas e até 150 cc após 48 h (Tabela 1) </li><li> Ocasionalmente verificar a condição dos embriões cultivados contando taxa de batimentos cardíacos (ie, 120-150/min é ótima) e observando a circulação sanguínea. Se necessário contar o número de somitos para julgar o crescimento de embriões em solução salina de Tyrode. No desenvolvimento normal, um somito é adicionado em duas horas. </li><li> WEC quando estiver terminada, pare de alimentação de mistura de gás e desconectar o tubo de entrada eo filtro de membrana para evitar o refluxo para dentro do tubo de entrada da garrafa borbulhando. </li></ol> 7. Notas <ol class="lalpha"><li> A temperatura dentro do sistema WEC é constantemente mantido a 37,0-37,5 ° C. </li><li> 100% rat IC soro é utilizado rotineiramente em nosso WEC de embriões de rato e do rato, e mantidos a -20 ° C antes do uso. </li><li> Nós compramos rato IC soro, que é coletado de ratos que estão anestesiados com isoflurano. </li><li> Garrafas descartáveis ​​também estão disponíveis a partir de Ikemoto Rika. Garrafas e plugs de silicone devem ser autoclavados. </li><li> Dissecação de embriões devem ser realizados à temperatura ambiente porque a manutenção de embriões em temperatura baixa ou alta durante a dissecção afetam o desenvolvimento posterior no WEC. </li><li> Ferramentas para dissecção são esterilizados por calor seco. </li><li> A pele e parede abdominal devem ser cortados separadamente para evitar a contaminação dos cabelos em um prato possível. Mudamos um par de tesouras e pinças grandes com um novo par deles quando cortamos a pele e isolar o útero, respectivamente. </li><li> Utilizando uma pinça, cujas pontas são muito bem é muito importante para dissecar tecidos com certeza. Nós manualmente afiar as pontas de uma pinça com uma pedra e óleo de máquina. </li><li> Usamos um lado da pinça como uma faca para evitar excesso de pressão de embriões. </li><li> Quando a temperatura dentro do sistema WEC diminui, abrindo a porta repetidamente, continuamos a acender a luz para manter a temperatura dentro do sistema. </li></ol> 8. Resultados representativos A Figura 1 mostra os procedimentos de dissecação do embrião de rato e embriões de ratos cultivadas. <img src="/files/ftp_upload/2170/2170fig1.jpg" alt="Figura 1" /> Figura 1. Cultura de embriões Whole do embrião de rato E12.5. (A) A concepto dissecados do útero de ratos fêmeas grávidas. (B) Remoção da decídua no lado placenta. (C) remoção da membrana de Reichert. (D) Abertura do saco vitelino. (E) O cultivo de embriões de rato na garrafa durante 6 horas após o início do WEC. (F) O embrião de rato cultivadas por 42 hr. d, decidua; R; membrana de Reichert; p, placenta; ys, saco vitelino, e, embrião. <table border="1"><tbody><tr><td></td><td> 0 hr </td><td> 12 hr </td><td> 24 hr </td><td> 36 hr </td><td> 48 horas </td></tr><tr><td> E12.5 embrião de rato </td><td> 95% 50 cc </td><td> 95% 75 cc (10 h) </td><td> 95% 100 cc </td><td> 95% 125 cc (34 hr) </td><td> 95% a 150 cc </td></tr></tbody></table> Tabela 1. Condições de oxigênio Optimal.

<ol>
	<li>New, D. A. T., Copp, A. J., Cockroft, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intoroduction.+In+mammalian+postimplantation+embryos.">Intoroduction. In mammalian postimplantation embryos.</a> In Mammalian Postimplantation Embryos A Practical Approach. , 1-14 (1990).</li><li>Cockroft, D. L., Copp, A. J., Cockroft, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+and+culture+of+post+implantation+embryos.">Dissection and culture of post implantation embryos.</a> In Mammalian Postimplantation Embryos A Practical Approach. , 15-40 (1990).</li><li>Morris-Kay, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postimplantation+mammalian+embryos.">Postimplantation mammalian embryos.</a> Essential Developmental Biology A Practical Approach. , 55-66 (1993).</li><li>Fujinaga, M., Tuan, R. S., Lo, C. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rotating+bottle+culture+method+with+continuous+replacement+of+the+gas+phase.">A rotating bottle culture method with continuous replacement of the gas phase.</a> Experientia. 35, 138-140 (1979).</li><li>Osumi-Yamashita, N., Ninomiya, Y., Eto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+craniofacial+embryology+in+vitro.">Mammalian craniofacial embryology in vitro.</a> Int. J. Dev. Biol. 41, 187-194 (1997).</li><li>Inoue, T., Nakamura, S., Osumi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+mapping+of+the+mouse+prosencephalic+neural+plate.">Fate mapping of the mouse prosencephalic neural plate.</a> Dev. 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Existem dois passos críticos no WEC roedores para o sucesso. Primeiro, o procedimento de dissecção deve ser preciso para não danificar embriões, especialmente os vasos sanguíneos. Segundo, o procedimento deve ser o mais rápido possível, porque o oxigênio e nutrientes não são mais fornecidos através da placenta após o isolamento do útero. Isto é crítico para os embriões mais velhos. No caso do WEC de ratos após E12.5, devemos transferir embriões dissecado em garrafas de cultura em 30 minutos. <p cl...

<table border="1"><tbody><tr><td> Nome </td><td> Tipo </td><td> Companhia </td><td> Número de catálogo </td><td> Comentário </td></tr><tr><td colspan="5"> Criação WEC </td></tr><tr><td> WEC sistema (10-0310) </td><td> Ferramenta </td><td> Ikemoto Rika </td><td> 010-0310 </td><td> Um outro modelo pequeno também está disponível. </td></tr><tr><td> Silicon plug sem um buraco </td><td> Ferramenta </td><td> Ikemoto Rika </td><td> 010-032-08 </td><td></td></tr><tr><td> Cilindro de mistura de gás </td><td> Ferramenta </td><td> Nikko Sanso </td><td> - </td><td> Contendo 95% de oxigênio e dióxido de carbono de 5%. Ordem feita sob encomenda. </td></tr><tr><td> Regulador de gás </td><td> Ferramenta </td><td> Ono Seisakusho </td><td> WR-11 </td><td></td></tr><tr><td> 0,22 mM filtro Millex GS </td><td> Ferramenta </td><td> Millipore </td><td> SLGS033SS </td><td></td></tr><tr><td colspan="5"> Anestesia e isolamento do útero </td></tr><tr><td> Tesouras grandes </td><td> Ferramenta </td><td> Napox </td><td> B-7H </td><td></td></tr><tr><td> Fórceps </td><td> Ferramenta </td><td> Napox </td><td> A-3-2 </td><td></td></tr><tr><td> Prato disposição Petri (90 mm x 15 mm) </td><td> Ferramenta </td><td> Iwaki </td><td> SH90-15 </td><td> Deep-tipo de prato é o melhor para dissecção. </td></tr><tr><td> Isoflurano </td><td> Reagente </td><td> Abbott </td><td> B506 </td><td> Para anestesia </td></tr><tr><td> Pentobarbital sódico </td><td> Reagente </td><td> Schering-Plough Saúde Animal </td><td> - </td><td> Para anestesia </td></tr><tr><td> Tyrode de solução salina </td><td> Reagente </td><td> - </td><td> - </td><td> De acordo como o protocolo na Ref. 2. Salvar a 4 ° C. </td></tr><tr><td> Cronometrado grávidas de ratos Sprague-Dawley </td><td> Animal </td><td> Charles Rivers Laboratories Japão </td><td> - </td><td></td></tr><tr><td colspan="5"> Preparação de meio de cultura </td></tr><tr><td> Garrafa de vidro cultura </td><td> Ferramenta </td><td> Ikemoto Rika </td><td> 010-032-05 </td><td></td></tr><tr><td> Frasco de cultura disposição </td><td> Ferramenta </td><td> Ikemoto Rika </td><td> 010-032-06 </td><td></td></tr><tr><td> Silicon ficha com buraco </td><td> Ferramenta </td><td> Ikemoto Rika </td><td> 010-032-07 </td><td></td></tr><tr><td> Folha de alumínio </td><td> Ferramenta </td><td> Qualquer empresa </td><td> - </td><td></td></tr><tr><td> Saco de autoclave </td><td> Ferramenta </td><td> Hogy </td><td> HM-26 </td><td> Para garrafas de cultura </td></tr><tr><td> Saco de autoclave </td><td> Ferramenta </td><td> Hogy </td><td> HM-14A </td><td> Para plugues de silicone </td></tr><tr><td> 0,45 mM filtro Millex HA </td><td> Ferramenta </td><td> Millipore </td><td> SLHA033SS </td><td></td></tr><tr><td> 50 tubo cônico ml </td><td> Ferramenta </td><td> Becton Dickinson </td><td> 352070 </td><td></td></tr><tr><td> 100 ml copo </td><td> Ferramenta </td><td> Iwaki </td><td> TE-32 </td><td></td></tr><tr><td> Rat IC soro </td><td> Reagente </td><td> Charles Rivers Laboratories Japão </td><td> - </td><td> Referem-se a: Sr. Kunihiko Morisaki, TEL: +81- (0) 45-474-9336; FAX: +81- (0) 45-474-9340 </td></tr><tr><td> D (+)-Glicose </td><td> Reagente </td><td> Wako </td><td> 041-00595 </td><td></td></tr><tr><td> Antibióticos antimyotic líquido </td><td> Reagente </td><td> Gibco </td><td> 15240 </td><td></td></tr><tr><td colspan="5"> Dissecação de embriões </td></tr><tr><td> Tesoura oftálmica reta </td><td> Ferramenta </td><td> Napox </td><td> MB50-7 </td><td> Para o corte da parede uterina </td></tr><tr><td> Tesoura oftálmica curvas </td><td> Ferramenta </td><td> Napox </td><td> MB54-2 </td><td> Para cortar o saco vitelino </td></tr><tr><td> Fórceps # 5 </td&gt; <td> Ferramenta </td><td> Vigor </td><td> T6715 </td><td></td></tr><tr><td> Fórceps # 5F </td><td> Ferramenta </td><td> Regine </td><td> T6819 </td><td></td></tr><tr><td> Pipeta de vidro </td><td> Ferramenta </td><td> - </td><td> - </td><td> Feito à mão usando pipeta Pasteur. </td></tr><tr><td> Saco de autoclave </td><td> Ferramenta </td><td> Hogy </td><td> HM-4 </td><td> Para pipetas de vidro </td></tr><tr><td> Microscópio binocular </td><td> Ferramenta </td><td> Leica </td><td> Mz7s </td><td></td></tr><tr><td> Luz </td><td> Ferramenta </td><td> Leica </td><td> CLS 150XD </td><td></td></tr><tr><td> Óleo de pedra </td><td> Ferramenta </td><td> Yoko Uchida </td><td> 833-2000 </td><td> Para afiar de fórceps </td></tr><tr><td> Óleo de máquina </td><td> Ferramenta </td><td> Yoko Uchida </td><td> 835-0000 </td><td> Para afiar de fórceps </td></tr></tbody></table>

the method of rodent whole embryo culture using the rotator-type bottle culture system

Todo embrião cultura técnica (WEC) foi desenvolvido em 1950 pela Nova e seus colegas, e aplicadas em biologia do desenvolvimento 1. Embora o desenvolvimento e crescimento de embriões de mamíferos dependem criticamente a função da placenta, WEC técnica nos permite rato cultura e embriões de ratos condição de ex vivo durante períodos limitados, correspondendo a fases midgestation durante o dia embrionário (E) 6,5 E12.5 na mouse ou E8.5-E14.5 no rato 2, 3, 4. No WEC, nós podemos diretamente alvo desejado áreas de embriões utilizando capilares de vidro fina porque os embriões podem ser manipulados sob o microscópio. Portanto, WEC roedores é uma técnica muito útil quando queremos estudar processos dinâmicos de desenvolvimento de embriões de mamíferos postimplanted. Até à data, vários tipos de sistemas têm sido desenvolvidos WEC 1. Entre estes, o rotator-tipo de sistema de cultura de garrafa é mais popular e adequada para longo prazo de cultura de embriões em midgestation, ou seja, após E9.5 e E11.5 em camundongos e ratos, respectivamente 1. Neste protocolo de vídeo, demonstramos nossos procedimentos padrão de WEC ratos após E12.5 usando um modelo refinado do sistema original rotador, que foi desenhado por New Cockroft e 5, 6, e introduzir várias aplicações da técnica do CME para estudos em desenvolvimento de mamíferos biologia.

Watch this Scientific Journal Video about The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System at JoVE.com

The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

Técnica de cultura todo embrião de rato permite-nos cultura e embriões de ratos<em&gt; Ex vivo</em&gt; Condição durante períodos limitados, correspondendo a fases midgestation. Neste protocolo de vídeo, demonstramos nossos procedimentos padrão de cultura de embriões de ratos após toda E12.5 usando o rotator-tipo de sistema de cultura de garrafa.

O método de cultura de embriões de roedores inteiro usando o Rotator do tipo Sistema de Cultura Garrafa

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development ...

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23.6K visualizações.  Tohoku University Graduate School of Medicine. Técnica de cultura todo embrião de rato permite-nos cultura e embriões de ratos Ex vivo Condição durante períodos limitados, correspondendo a fases midgestation. Neste protocolo de vídeo, demonstramos nossos procedimentos padrão de cultura de embriões de ratos após toda E12.5 usando o rotator-tipo de sistema de cultura de garrafa.

Vídeo: The Method of Rodent Whole Embryo Culture using the Rotator-type Bottle Culture System

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

Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.

Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system.

Cultura do mouse pulmão embrionário, um sistema para avaliar os mecanismos moleculares de Ramificação

Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of ...

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.

Um Ex-ovo Sistema de Cultura de embriões de frango Indicado para aplicações de imagem e Microcirurgia

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

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

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

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

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

A Novel<em&gt; Ex vivo</em&gt; Método de Cultura para o Rato coração embrionário

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

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Rato Desenvolvimento Embrionário em todo um sistema de cultura de embriões sem soro

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

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

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

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

O método de cultura fatia para acompanhar o desenvolvimento de germes dentários Em Explant Cultura

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

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

Preparação de Rat Serum Indicado para cultura de embriões de mamíferos Whole

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Establishment of an Extracellular Acidic pH Culture System

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the role of acidic extracellular pH in tumor aggressiveness and its underlying mechanism has not been extensively studied compared to hypoxic or nutrient starvation conditions. In addition, a well-defined culture method to mimic the acidic extracellular tumor microenvironment has not been fully reported.
Here we present a simple in vitro culture method to maintain acidic extracellular pH using reduced bicarbonate and increased lactate or HCl concentrations in the culture medium. The medium pH was sustained for at least 24 h and gradually decreased by 72 h following culture of PANC-1 and AsPC-1 pancreatic cancer cells. Three distinct acidic media conditions in this study highly upregulated pH-responsive genes such as MSMO1, INSIG1, and IDI1 compared to hypoxia or nutrient starvation. The upregulation of these genes can be used as a marker of acidic pH. These simple techniques are beneficial to elucidate underlying mechanisms of tumor malignancy under acidic tumor microenvironment. Therefore, our extracellular acidic pH culture system enables discovery of cellular acidic pH responses not only in cancer cells but also in primary cells, such as renal tubular cells, in relation to the other acidic disorders including, diabetic ketoacidosis, lactic acidosis, renal tubular acidosis, and respiratory acidosis.

The acidic tumor microenvironment plays critical roles in tumor progression. To assess the effects of acidic extracellular pH on cancer cells in vitro, we established simple acidic culture systems.

Estabelecimento de um pH extracelular ácido sistema de cultura

Conditions of the tumor microenvironment, such as hypoxia or nutrient starvation, play critical roles in cancer progression and malignancy. However, the ...

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

Avaliando as propriedades angiogenéticas de células-tronco do câncer de ovário usando o Sistema de Cocultura Tridimensional, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...

Process Development for the Production and Purification of Adeno-Associated Virus (AAV)2 Vector using Baculovirus-Insect Cell Culture System

Adeno-associated viruses (AAV) are promising vectors for gene therapy applications. Here, the AAV2 vector is produced by co-culture of Spodoptera frugiperda (Sf9) cells with Sf9 cells infected with baculovirus (BV)-AAV2-GFP (or therapeutic gene) and BV-AAV2-rep-cap in serum-free suspension culture. Cells are cultured in a flask in an orbital shaker or Wave bioreactor. To release the AAV particles, producer cells are lysed in buffer containing detergent, which is subsequently clarified by low-speed centrifugation and filtration. AAV particles are purified from the cell lysate using AVB Sepharose column chromatography, which binds AAV particles. Bound particles are washed with PBS to remove contaminants and eluted from the resin using sodium citrate buffer at pH 3.0. The acidic eluate is neutralized with alkaline Tris-HCl buffer (pH 8.0), diluted with phosphate-buffered saline (PBS), and further concentrated with tangential flow filtration (TFF). The protocol describes small-scale pre-clinical vector production compatible with scale-up to large-scale clinical-grade AAV manufacturing for human gene therapy applications.

Process Development for the Production and Purification of Adeno-Associated Virus AAV2 Vector using Baculovirus-Insect Cell Culture System

In this protocol, AAV2 vector is produced by co-culturing Spodoptera frugiperda (Sf9) insect cells with baculovirus (BV)-AAV2-green fluorescent protein (GFP) or therapeutic gene and BV-AAV2-rep-cap infected Sf9 cells in suspension culture. AAV particles are released from the cells using detergent, clarified, purified by affinity column chromatography, and concentrated by tangential flow filtration.

Desenvolvimento de processos para a produção e purificação do vírus associado ao Adeno (AAV)2 Vetor usando o sistema de cultura de células de baculovírus-inseto