1. Protocolo Cultura Ex-ovo: <ol><li> Incubar ovos de galinha fertilizados <ol class="lroman"><li> Ovos de galinha fertilizados podem ser armazenadas a 13 ° C até 5 dias antes da incubação sem iniciar o desenvolvimento. Um refrigerador de vinho tinto pode manter esta temperatura. </li><li> Incubar ovos lado sem corte em um 60% de umidade constante incubadora com balanço contínuo a 37,5 ° C por 72 horas. </li></ol></li><li> Prepare redes (Figura 1a) <ol class="lroman"><li> Preencha ¾ de 9 oz copos de plástico com água estéril morna. A regularidade 9 oz copo tem um diâmetro superior de 8 cm. Descobrimos que este tamanho era o mais adequado para aplicação de longa data a cultura aqui apresentado. </li><li> Cortar cerca de 20 cm filme plástico. Apor circunferencialmente em cima do copo, colocando uma faixa de borracha ao redor. Plástico deve ser espalhado em cima da água. Corte o excesso de plástico em torno da banda. Encontramos filme plástico a ser mais adequado para esta aplicação, uma vez que previne a adesão da gema, resultando na separação da gema durante o transporte. </li><li> Limpe o plástico com kimwipes (Kimberly-Clark, Inc.) umedecido com álcool 70%. </li></ol></li><li> Retire os ovos da incubadora após 72 horas de incubação. </li><li> Esterilizar os ovos, limpando a superfície com álcool 70% kimwipes molhada. </li><li> Colocar os ovos na horizontal por 1-2 minutos para o posicionamento adequado do embrião (Figura 1b). Uma caixa de ovos pode ser usado para colocar ovos na horizontal. Embriões irá girar ao topo após 1-2 minutos. </li><li> Transferência de embriões em redes assepticamente em uma capela de fluxo laminar. <ol class="lroman"><li> Usando uma borda afiada (como a borda de um balde de metal ou um copo de vidro), toque o ovo delicadamente até que haja um pequeno dente na parte inferior do ovo (Figura 1c). Lembre-se, o embrião é posicionado no upperside. </li><li> Colocar os polegares em lados opostos do dente e puxe as cascas para além horizontalmente (Figura 1d). </li><li> Coloque a gema com o embrião, bem como a albumina para a rede (figura 1e). Albumina irá proporcionar um ambiente de amortecimento em torno da gema para absorver os choques induzidos movendo o sistema, bem como proporciona nutrição para o embrião para as práticas da cultura de comprimento. Água por baixo da rede vai garantir o embrião vai ficar paralelos ao chão durante o transporte e servir como um condutor de calor durante o exame, se um aquecedor de circulação de água é utilizada. </li><li> O embrião já deve ser colocada em cima se a transferência for realizada corretamente. Se não, use um unsharp, objeto estéril (ou seja, de ponta Blunt tesoura curva fechada) e acariciar direcionalmente a gema de tal forma que o embrião gira ao topo. </li><li> Lugar a 10 cm de diâmetro placa de Petri em cima do copo para selar o embrião. </li></ol></li><li> Colocar a cultura copo no set incubadora a 37,5 ° C. Se uma incubadora portátil é utilizado, colocando setembro 1-2 copos oz preenchido com água destilada mantém a umidade em ~ 60% (Figura 1f). </li><li> Se os embriões cultura além do dia embrionárias 7, pedaços de casca de ovo esmagada devem ser espalhadas em torno da periferia do embrião como fonte de cálcio para o desenvolvimento ósseo e maturação. </li></ol> Equipamento acessório para a incubadora fora imagem / manipulação: Um banho de água circulante foi construída em casa para manter a temperatura de incubação, enquanto imagens ou manipulação (Figura 3). Um banho de água que pode caber o cálice é ligado através de tubos de tamanho apropriado para um reservatório de água. Um aquecedor de resistência aquece a água no reservatório, que é regulada manualmente, seguindo a temperatura da água. Uma bomba de água circula a água. Representação pictórica mostra a facilidade de imagens via ultrasongraphy / microscopia fluorescente (Figura 3). <img src="/files/ftp_upload/2154/2125fig1.jpg" alt="Figura 1" /> Figura 1. Preparação de rede e transferência de embriões de galinha para redes. <img src="/files/ftp_upload/2154/2125fig2.jpg" alt="Figura 2" /> Figura 2. Resultados representativos -. Embriões de galinha cresceu estágio diferente através de técnica de cultura de embriões ex ovo HH 17 em diante podem ser cultivadas através desta técnica. <img src="/files/ftp_upload/2154/2125fig3.jpg" alt="Figura 3" /> Figura 3. Imagens esquemáticas e representante do sistema de circulação de água utilizada em conjunto com o sistema de cultura ex-ovo para manter os embriões à temperatura ideal de desenvolvimento. 2. Os pedidos seleccionados: Microinjeção I. Esta cultura de embriões é adequado para aplicações microinjeção onde as soluções precisam ser injetado para vasculatura para geração de imagens baseado em contraste (microscopia de fluorescência ou seja, micro-topografia computadorizada, etc) (Figura 4). Abaixo está o protocolo de microinjeção: <ol><li> Prepare comparti injeçãotus <ol class="lroman"><li> Moda puxado 0,75 tubos ID capilar de vidro em ponta chanfrada microagulhas usando um microforge. Tamanho da ponta necessária depende taxa alvo veia diâmetro / fluxo desejado. </li><li> Conectar a seringa ao tubo 0,03 polegadas ID silicone. </li><li> Carregar o tubo com a solução a ser injetado e conecte o microagulhas para a tubulação. </li><li> Coloque o microagulhas e micromanipulador para a seringa para titular seringa ou bomba de seringa. </li></ol></li><li> Adicionar água esterilizada no copo, assim, elevar o embrião. Nós experimentamos que para a penetração adequada da agulha em um vaso sanguíneo (navio vitelínico ou seja), a penetração horizontal é necessária. Isso requer o embrião estar no mesmo plano que as bordas do copo. <ol class="lroman"><li> Retire a banda de borracha que segura o plástico sob o embrião. </li><li> Levantar um dos lados do plástico com cuidado e acrescente um pouco de água estéril para o copo. </li><li> Segura o elástico para o plástico de novo. </li></ol></li><li> Injetar a solução para um navio embrionárias. Em nossa experiência, quando a solução é injetada a um navio vitelínico, microagulhas precisava ser ª cerca de 1 / 10 de tamanho do diâmetro do vaso para manter a perfusão eficiente sem causar sangramento. <ol class="lroman"><li> Bombear a solução para o microneedle garantindo que não haja formação de bolhas de ar. </li><li> Alinhe a ponta do microagulhas para o navio selecionado. Para os navios vitelínico, a seleção de uma área de garfo dará área máxima para penetrar na embarcação. </li><li> Empurre a agulha em direção ao navio. O navio irá primeiro retrair. Uma vez que a agulha penetra, iniciar a perfusão. A cor do sangue que flui deve mudar com a perfusão. Se mudar de cor não é visto, a agulha pode ter penetrado para fora da embarcação. Nesse caso, lentamente retrair o microagulhas, ao mesmo tempo de bombeamento solução até mudar de cor no vaso é visto. Uma vez que a perfusão está completo, recolher o microagulhas para fora da embarcação. </li></ol></li></ol><img src="/files/ftp_upload/2154/2125fig4.jpg" alt="Figura 4" /> Figura 4: microinjeção de um corante fluorescente na vasculatura de um embrião de galinha ex-ovo cultivadas. II. Procedimento de microcirurgia - Ligadura Atrial Esquerda Esta configuração cultura ex-ovo é ideal para a realização de aplicações cirúrgicas de embriões de galinha, uma vez que oferece acessibilidade total para o embrião. Abaixo está o protocolo de uma técnica de exemplo, deixou ligadura atrial (LAL) (Figura 5). O procedimento é realizado em aproximadamente 24 horas para o período de cultura (HH24): <ol><li> Prepare overhand nós ~ 0,5 mm de diâmetro com fio de náilon 10-0 cirúrgico. </li><li> Com uma pinça fina, localmente remover as membranas coriônica e alantóide sobre o embrião e levante o embrião de trás para girá-lo na vertical de tal forma que o lado esquerdo do embrião é visível. </li><li> Utilizando uma pinça fina, retire o pericárdio sobre o átrio esquerdo. </li><li> Alinhar o nó sobre o átrio esquerdo e aperte-a. O nó deve ser amarrado de modo a proibir ~ 75% do fluxo sangüíneo original para o lado esquerdo. Cortar as bordas do nó com microtesoura e remova cuidadosamente e descarte de sutura em excesso. </li><li> Gire o embrião de volta à sua orientação original (lado direito em cima). </li><li> Controles Sham envolverá o mesmo procedimento, mas a sutura só vai ser transmitido através de e não vinculados. Tratamento eficaz LAL (75% constrição) será confirmado com 24 horas pós-cirurgia, e os embriões desqualificado serão excluídas. </li></ol><img src="/files/ftp_upload/2154/2154fig5.jpg" alt="Figura 5" /> Figura 5:. Um controle e um embrião de galinha atrial esquerda ligada seta mostra o átrio esquerdo, que é cerca de 75% menor no embrião ligado.

<ol>
	<li>Zamir, E. A., Czirok, A., Cui, C., Little, C. D., Rongish, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesodermal+cell+displacements+during+avian+gastrulation+are+due+to+both+individual+cell-autonomous+and+convective+tissue+movements.">Mesodermal cell displacements during avian gastrulation are due to both individual cell-autonomous and convective tissue movements.</a> Proc Natl Acad Sci U S A. 103, 19806-19811 (1980).</li><li>Ehrman, L. A., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lack+of+regulation+in+the+heart+forming+region+of+avian+embryos.">Lack of regulation in the heart forming region of avian embryos.</a> Dev Biol. 207, 163-175 (1999).</li><li>Clark, E. B., Hu, N., Rosenquist, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+conotruncal+constriction+on+aortic-mitral+valve+continuity+in+the+stage+18,+21+and+24+chick+embryo.">Effect of conotruncal constriction on aortic-mitral valve continuity in the stage 18, 21 and 24 chick embryo.</a> Am J Cardiol. 53, 324-327 (1984).</li><li>deAlmeida, A., McQuinn, T., Sedmera, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+ventricular+preload+is+compensated+by+myocyte+proliferation+in+normal+and+hypoplastic+fetal+chick+left+ventricle.">Increased ventricular preload is compensated by myocyte proliferation in normal and hypoplastic fetal chick left ventricle.</a> Circ Res. 100, 1363-1370 (2007).</li><li>Nakamura, H., Funahashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+DNA+into+chick+embryos+by+in+ovo+electroporation.">Introduction of DNA into chick embryos by in ovo electroporation.</a> Methods. 24, 43-48 (2001).</li><li>Butcher, J. T., McQuinn, T. C., Sedmera, D., Turner, D., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transitions+in+early+embryonic+atrioventricular+valvular+function+correspond+with+changes+in+cushion+biomechanics+that+are+predictable+by+tissue+composition.">Transitions in early embryonic atrioventricular valvular function correspond with changes in cushion biomechanics that are predictable by tissue composition.</a> Circ Res. 100, 1503-1511 (2007).</li><li>McQuinn, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-frequency+ultrasonographic+imaging+of+avian+cardiovascular+development.">High-frequency ultrasonographic imaging of avian cardiovascular development.</a> Dev. Dyn. 236, 3503-3513 (2007).</li><li>Korn, M. J., Cramer, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Windowing+chicken+eggs+for+developmental+studies.">Windowing chicken eggs for developmental studies.</a> J Vis Exp. , (2007).</li><li>Auerbach, R., Kubai, L., Knighton, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+procedure+for+the+long-term+cultivation+of+chicken+embryos.">A simple procedure for the long-term cultivation of chicken embryos.</a> Dev Biol. 41, 391-394 (1974).</li></ol>

Não há conflitos de interesse declarados.

Acesso óptico e experimentação em embriões de aves é um desafio devido às limitações da casca do ovo. Windowing limita significativamente o número de microvasos acessível para injeção e abordagens microcirúrgicas 8. Como resultado, apenas embriões podem ser manipulados e observação contínua não é possível. Início ex-ovo culturas usando placas de Petri eram de uso limitado por causa do controle inadequado da tensão superficial sobre o embrião impediu a cultura a longo prazo <sup...

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		<th>Catalogue Number</th>
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<tr>
<td>Fertile white Leghorn chicken eggs</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Model GB1, Avery Incubators, Hugo CO</td>
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<td>Saran Wrap</td>
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<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Kimwipes</td>
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<td>Kimberly-Clark, Inc.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Rubber bands</td>
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<td>Warm sterile water</td>
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<td>9 oz plastic cup</td>
<td>&nbsp;</td>
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<td>100 mm diameter Petri dish</td>
<td>&nbsp;</td>
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<td>1602N thermal air</td>
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<td>GQF Manufacturing CO, Savannah GA</td>
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<td>Fluorescein-conjugated dextran (2 MDa, 1% w/v in phosphate buffered saline)</td>
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<td>Sigma Aldrich Inc.</td>
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<td>Microforge</td>
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<td>Glassworx, Inc, St Louis MO</td>
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<td>Glass capillary tubes (0.75 mm ID)</td>
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<td>Micromanipulator</td>
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<td>World Precision Instruments, Sarasota FL</td>
<td>Model M3301L</td>
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<td>Fluorescent microscopy</td>
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<td>Zeiss</td>
<td>Z20</td>
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<td>Fine 55-forceps</td>
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<td>World Precision Instruments, Sarasota FL</td>
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<td>10-0 nylon surgical suture</td>
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<td>Ethicon</td>
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<td>Tubing</td>
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<td>VWR</td>
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<td>1mm OD</td>
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<td>3 mL syringe</td>
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<td>BD</td>
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<td>200 &mu;L pipetter and pipette tips</td>
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<td>VWR</td>
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an ex-ovo chicken embryo culture system suitable for imaging and microsurgery applications

Compreender as relações entre fatores genéticos e microambientais que a unidade normal e malformados desenvolvimento embrionário é fundamental para descobrir novas estratégias terapêuticas. Avanços na tecnologia de imagem têm permitido investigação quantitativa da organização e amadurecimento do plano do corpo, mas numa fase posterior morfogênese embrionária é menos clara. Embriões de galinha são um atrativo vertebrados sistema modelo animal para esta aplicação devido à sua facilidade de cultura e manipulação cirúrgica. Embriões podem ser cultivadas por um curto período em anéis de papel de filtro, que permite acesso óptico completo para padronização celular e 1,2 estudos destino. Estudar processos avançados de desenvolvimento, como a morfogênese cardíaca são tradicionalmente realizada através de uma janela da casca do ovo 3-5, mas esta técnica limita o acesso óptico devido ao tamanho da janela. Nós já desenvolveu um método simples para a cultura de todo o embrião ex-ovo no hexagonal pesar barcos para até 10 dias, o que permitiu imagens de alta resolução através de ultra-sonografia 6,7. Essas culturas foram difíceis de transportar, limitando os tipos de ferramentas de imagem disponíveis para experimentos ao vivo. Nós aqui apresentar um sistema de cultura melhorou shell-less com um custo-benefício, a câmara portátil ambiental. Os ovos estavam rachados em uma rede criada por uma membrana de poliuretano (filme plástico) aposta circunferencialmente para um copo de plástico parcialmente cheio com água estéril. As dimensões da circunferência ea profundidade da rede eram fundamentais para manter a tensão superficial, enquanto os mecânicos da rede e água abaixo ajudou amortecem as vibrações induzidas por transporte. A pegada pequena circulação banho de água também foi desenvolvido para permitir o controle de temperatura contínua durante a experimentação. Nós demonstramos a capacidade de cultura de embriões, desta forma, pelo menos, 14 dias sem defeito ou atraso morfogênicos e empregar este sistema em vários de microcirurgia e aplicações de imagem.

Watch this Scientific Journal Video about An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications at JoVE.com

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

Neste artigo, apresentamos uma metodologia simples para ativar a longo prazo<em&gt; Ex-ovo</em&gt; Cultura de embriões de aves. Esta técnica é ideal para a experimentação longitudinal exigindo acessibilidade óptico completo e / ou transporte estéril em embriões de aves.

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 ...

an-ex-ovo-chicken-embryo-culture-system-suitable-for-imaging

Research

JoVE Journal

Biology

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

22.9K visualizações.  Cornell University. Neste artigo, apresentamos uma metodologia simples para ativar a longo prazo Ex-ovo Cultura de embriões de aves. Esta técnica é ideal para a experimentação longitudinal exigindo acessibilidade óptico completo e / ou transporte estéril em embriões de aves.

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

Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform routine activities, but prosthetic suspensions using traditional socket technology can prove to be cumbersome and uncomfortable for a person with limb loss.  Moreover, for those with high proximal amputations, limited residual limb length may prevent exoprosthesis attachment all together. Osseointegrated implant technology is a novel operative procedure which allows firm skeletal attachment between the host bone and an implant. Preliminary results in European amputees with osseointegrated implants have shown improved clinical outcomes by allowing direct transfer of loads to the bone-implant interface.  Despite the apparent advantages of osseointegration over socket technology, the current rehabilitation procedures require long periods of restrictive load bearing prior which may be reduced with expedited skeletal attachment via electrical stimulation.  The goal of the osseointegrated intelligent implant design (OIID) system is to make the implant part of an electrical system to accelerate skeletal attachment and help prevent periprosthetic infection. To determine optimal electrode size and placement, we initiated proof of concept with computational modeling of the electric fields and current densities that arise during electrical stimulation of amputee residual limbs.  In order to provide insure patient safety, subjects with retrospective computed tomography scans were selected and three dimensional reconstructions were created using customized software programs to ensure anatomical accuracy (Seg3D and SCIRun) in an IRB and HIPAA approved study.  These software packages supported the development of patient specific models and allowed for interactive manipulation of electrode position and size. Preliminary results indicate that electric fields and current densities can be generated at the implant interface to achieve the homogenous electric field distributions   required to induce osteoblast migration, enhance skeletal fixation and may help prevent periprosthetic infections.  Based on the electrode configurations experimented with in the model, an external two band configuration will be advocated in the future.

There is a need to develop alternative prosthesis attachment due to limb loss attributed to vascular occlusive diseases and trauma. The goal of the work is to introduce an osseointegrated intelligent implant design system to increase skeletal fixation and reduce periprosthetic infection rates for patients needing osseointegrated technology.

Análises bioelétrica de um Sistema de Design Inteligente osseointegrados Implant para Amputados

The projected number of American amputees is expected to rise to 3.6 million by 2050.  Many of these individuals depend on artificial limbs to perform ...

Biologia

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

Pintinho Ex ovo Cultura e Ex ovo CAM Ensaio: como ele realmente funciona

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

Xenotransplante de células-tronco humanas no embrião de galinha

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

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

Whole embryo culture (WEC) technique has been developed in 1950's by New and his colleagues, and applied for developmental biology 1. Although development and growth of mammalian embryos are critically dependent on the function of the placenta, WEC technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages during embryonic day (E) 6.5-E12.5 in the mouse or E8.5-E14.5 in the rat 2, 3, 4. In WEC, we can directly target desired areas of embryos using fine glass capillaries because embryos can be manipulated under the microscope. Therefore, rodent WEC is very useful technique when we want to study dynamic developmental processes of postimplanted mammalian embryos. Up to date, several types of WEC systems have been developed 1. Among those, the rotator-type bottle culture system is most popular and suitable for long-term culture of embryos at midgestation, i.e., after E9.5 and E11.5 in the mouse and rat, respectively 1. In this video protocol, we demonstrate our standard procedures of rat WEC after E12.5 using a refined model of the original rotator system, which was designed by New and Cockroft 5, 6, and introduce various applications of WEC technique for studies in mammalian developmental biology.

Whole embryo culture technique allows us to culture mouse and rat embryos ex vivo condition during limited periods corresponding to midgestation stages. In this video protocol, we demonstrate our standard procedures of rat whole embryo culture after E12.5 using the rotator-type bottle culture system.

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 ...

Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice. HYPERStack vessels allow closed system scale up from the current stacked plate products and delivers >2.5X more cells in the same volumetric footprint. The HYPERStack vessels function via gas permeable material which allows gas exchange to occur, therefore eliminating the need for internal headspace within a vessel. The elimination of headspace allows the compartment where cell growth occurs to be minimized to reduce space, allowing more layers of cell growth surface area within the same volumetric footprint.
For many applications such as cell therapy or vaccine production, a closed system is required for cell growth and harvesting. The HYPERStack vessel allows cell and reagent addition and removal via tubing from media bags or other methods.
This protocol will explain the technology behind the gas permeable material used in the HYPERStack vessels, gas diffusion results to meet the metabolic needs of cells, closed system cell growth protocols, and various harvesting methods.

An Introduction into the technology, protocol and handling of the Corning HYPERStack Vessels and accessories used for high yield adherent cell culture. The protocol will show how to use the closed system vessels for increasing cell harvesting over current stacked plate products.

Sistema de Protocolo fechado Cultura de Células utilizando navios com HYPERStack Gas Technology material permeável

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice.   ...

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

The chicken embryo provides an excellent model system for studying gene function and regulation during embryonic development. In ovo electroporation is a powerful method to over-express exogenous genes or down-regulate endogenous genes in vivo in chicken embryos1. Different structures such as DNA plasmids encoding genes2-4, small interfering RNA (siRNA) plasmids5, small synthetic RNA oligos6, and morpholino antisense oligonucleotides7 can be easily transfected into chicken embryos by electroporation. However, the application of in ovo electroporation is limited to embryos at early incubation stages (younger than stage HH20 - according to Hamburg and Hamilton)8 and there are some disadvantages for its application in embryos at later stages (older than stage HH22 - approximately 3.5 days of development). For example, the vitelline membrane at later stages is usually stuck to the shall membrane and opening a window in the shell causes rupture of the vessels, resulting in death of the embryos; older embryos are covered by vitelline and allantoic vessels, where it is difficult to access and manipulate the embryos; older embryos move vigorously and is difficult to control the orientation through a relatively small window in the shell.
In this protocol we demonstrate an ex ovo electroporation method for gene transfer into chicken embryos at late stages (older than stage HH22). For ex ovo electroporation, embryos are cultured in Petri dishes9 and the vitelline and allantoic vessels are widely spread. Under these conditions, the older chicken embryos are easily accessed and manipulated. Therefore, this method overcomes the disadvantages of in ovo electroporation applied to the older chicken embryos. Using this method, plasmids can be easily transfected into different parts of the older chicken embryos10-12.

Gene Transfer into Older Chicken Embryos by ex ovo Electroporation

A method of gene transfer into chicken embryos at later incubation stages (older than Hamburger and Hamilton stage (HH) 22) is described. This method overcomes disadvantages of in ovo electroporation applied to older chicken embryos and is a useful technique to study gene function and regulation at older developmental stages.

Transferência de Embriões em Gene de frango por mais velhos<em&gt; Ovo ex</em&gt; Eletroporação

The chicken embryo provides an  excellent model system for studying gene function and regulation during  embryonic development. In ovo electroporation is ...

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 ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

Uma<em&gt; Ex vivo</em&gt; Sistema Cultura para estudar o desenvolvimento da tireóide

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

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 ...