Name: mouse fetal whole intestine culture system for ex vivo manipulation of signaling pathways and three-dimensional live imaging of villus development
Uploaded: 2014-09-04T14:00:00.000+00:00
Duration: 6 min 46 s
Description: Watch this Scientific Journal Video about Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development at JoVE.com

We gratefully acknowledge Dr. Deborah L. Gumucio as our advisor and for her invaluable support in defining the culture and imaging methods. We also thank Dr. Jim Brodie, Dr. Hong-Xiang Lu, Dr. Charlotte Mistretta, and Dr. Ann Grosse for their contributions to the development of the whole intestine organ culture system. Helpful discussions on imaging provided excellent advice from Dr. Chip Montrose, Michael Czerwinski and Sasha Meshinchi. All imaging was performed in the Microscopy and Image Analysis Laboratory at the University of Michigan. Funding support was provided by NIH R01 DK065850.

NOTA: Todos os camundongos foram tratados com humanidade usando protocolos aprovados pela Universidade de Michigan Medical School Unidade de Medicina Laboratorial Animal e de acordo com as orientações do Comitê da Universidade de uso e cuidados dos animais. 1. Whole sistemas de órgãos Cultura <ol><li> Preparação de meios de comunicação e placas de cultura <ol><li> Em uma cultura capuz tecido, remover 5 ml de meio BGJb da garrafa estoque e adicionar 5 ml de Pen / Strep. Prepare um estoque de trabalho de mídia em um tubo de 50 ml, adicionando 1 ml de 5 mg / ml de ácido ascórbico a 49 ml de meio BGJb (com Pen / Strep). </li><li> Preparar uma placa de cultura de 6 poços por adição de 1 ml por baixo de cada um dos transwells. NOTA: Se todos os 6 transwells não são utilizados, mova transwells extras em uma nova estéril placa de 6 poços para uso posterior. Adicione 3 mL de meios de trabalho BGJb para cada uma de três placas de Petri de 10 centímetros estéreis. </li></ol></li><li> Preparação para dissecção <ol><li> Mergulhe ferramentas de dissecação em 70% ethanol e não se esqueça de manter as ferramentas limpas, enquanto trabalhava. </li><li> Configure a área de dissecação com um grande balde de gelo e coloque a placa de cultura de 6 poços e 10 centímetros de Petri com meio BGJb no gelo. Trabalhar no gelo, tanto quanto possível, enquanto dissecar e preparar o intestino para a cultura. </li><li> Anestesiar ratos fêmea adulta em uma câmara com isofluorano (100 l) antes de eutanásia por deslocamento cervical para a exploração dos intestinos fetais. </li><li> Após a colheita os fetos de sua mãe, encenar cada feto cuidadosamente de acordo com o gráfico 13 Theiler encenação. Não confie nos dias pós-coito para determinar a idade de cada feto como variações de até 24 horas, em fase de desenvolvimento são rotineiramente observado em uma única ninhada. </li></ol></li><li> Dissecção da intestinos fetais <ol><li> Eutanásia fetos por decapitação antes da remoção do intestino. </li><li> Dissecar cada intestino em 1x de Dulbecco Phosphate-Buffered Saline (DPBS) e place-lo em uma placa de petri 10 centímetros com o trabalho de mídia BGJb. </li><li> Cuidadosamente remover o baço do estômago e do pâncreas do estômago e duodeno superior. </li><li> Suavemente separar o omento tendo o cuidado de deixar o omento ligado, tanto quanto possível, e que separa as ligações apenas o suficiente para permitir que o intestino para ser esticado. NOTA: Para o intestino mais velhos do que os que são ou E14.5 cultivadas mais do que 24 horas, cuidadosamente separar o intestino em três segmentos iguais para permitir conteúdo luminal para fluir através do intestino e ser expelido das extremidades cortadas. No entanto, para as culturas iniciadas antes E13.5, esperar até que após 24 horas de cultura antes de se separar os três segmentos do intestino para permitir que a serosa para crescer adequadamente ao longo do comprimento do intestino. </li><li> Usar uma pipeta de boca para transferir as peças intestinais para os transwells da placa de cultura (1.1.2). </li><li> Gentilmente reposicionar os intestinos como necessário para que eles fiquem em frente a Transwell. NOTA: Uma vez que os intestinos começam a se mover conteúdo luminal através do tubo, todas as torções no intestino fará uma cópia de segurança e danos ao intestino. </li></ol></li><li> Cultura e tratamento <ol><li> Remova a mídia sob o transwell, adicionar 700 mL de mídia (media de tratamento que trabalham com proteínas recombinantes adicionados ou inibidores farmacológicos), sob o transwell. </li><li> Adicionar 300 mL de meio de tratamento gota a gota, em cima dos intestinos e deixe de molho por meio da transwell. Reposicionar os intestinos, se necessário. </li><li> Mudar meio de tratamento, pelo menos, uma vez por dia ou mais frequentemente, dependendo da meia-vida do reagente de tratamento. </li></ol></li><li> Preparação de esferas de agarose de proteína embebido por uma fonte localizada de tratamento reagente <ol><li> Ressuspender as esferas de agarose em seu recipiente de armazenagem e transferência de 100 ul de esferas de agarose para um tubo de microcentrífuga de 1,5 ml. </li><li> Encher o volume restante do tubo estéril com 1x Phosphate-Buffered Saline (PBS) e misturar as contas para lavá-los. Colocar o tubo de microcentrífuga num bastidor durante alguns minutos para permitir que os grânulos se depositem no fundo e, em seguida, para fora da pipeta a partir de PBS no topo dos grânulos. Tornar a encher o tubo de microcentrífuga estéril com 1x PBS e repetir o processo de lavagem mais duas vezes. </li><li> Prepare a proteína de interesse em duas vezes a concentração desejada para o tratamento. </li><li> Misturar 5 mL de pérolas de agarose lavados com 5 l de estoque a proteína 2x e agitar suavemente as pérolas em proteína. Colocar o tubo em gelo durante 1 hora, misturando suavemente cada 10-15 min. </li><li> Lavar as contas brevemente em estéril 1x PBS, antes de colocar as contas no intestino. </li></ol></li><li> Colocação de pérolas de agarose <ol><li> Coloque suavemente as esferas de agarose no topo do intestino utilizando uma pipeta de boca com agulhas capilares puxados. Coloque as contas com extrema cautela, como manuseio descuidado pode danificar o intestino e prevenir o desenvolvimento de vilosidades no local da lesão. </li><li> Inserir o dobro de grânulos como são necessários para a análise uma vez que os intestinos irá sofrer movimentos peristálticos e cerca de metade das pérolas colocadas sairão dos intestinos mais o tempo de cultura. </li></ol></li><li> Fixação de intestinos cultivadas <ol><li> Retire cuidadosamente a mídia de tratamento abaixo do transwell e permitir que os intestinos para o ar seco por 3 min. </li><li> Adicionar 1 ml de fixador sob a transwell durante 2 min, em seguida cobrir a parte superior do intestino com 2 ml de fixador para o restante do tempo de fixação. Fixar com paraformaldeído a 4% O / N a 4 ° C ou durante 2 horas à temperatura ambiente. </li></ol></li></ol> 2 Imagem de intestinos fixas <ol><li> Imaging Wholemount <ol><li> Coloque os intestinos inteiros (com fluorescência endógena) em um slide com 100 ml de 1x DPBS. Use uma pinça para organizar cuidadosamente os intestinos. </li><li> Use um lenço de laboratório para remover cuidadosamente os DPBS e imediatamente adicione 100 ml de glicerol a 70% para fazer a mo tecidore transparente e possível aumentar a profundidade da imagem. NOTA: Se a penetração do intestino é desejado, em vez de montagem no intestino em 70% de glicerol, embeber o intestino em algumas gotas de solução de focagem claro para 10-30 min. Pipeta off a solução foco claro e montar o intestino com o Monte Clear. </li><li> Mergulhe cada um dos quatro cantos de uma lamela em massa de modelar e pegar uma pequena quantidade de argila para usar como espaçadores entre lâmina e lamela para manter a lamela de achatamento do intestino. </li><li> Selar a lamela sobre a lâmina com valap derretida aplicado com um cotonete. </li><li> Imagem do intestino de cada um microscópio confocal vertical ou invertida. Consulte a discussão para mais detalhes. </li></ol></li><li> Vibratome corte de intestino fixos (para corte transversal das estruturas intestinais) <ol><li> Intestinos inserir em 7% agarose em pequenos moldes de plástico (10 x 10 x 15 mm) e permitir que os blocos de agarose paraesfriar e solidificar. </li><li> Remova os blocos de agarose a partir dos moldes de plástico e cola blocos de agarose para a etapa de coleta vibratome com cola instantânea. </li><li> Preencha a etapa de coleta com o frio do gelo 1x DPBS. </li><li> Cortar secções com uma espessura entre 100-150 m. Para seções mais grossas usar soluções de compensação (descritos no ponto 2.1.2) para visualizar mais profundamente na seção transversal. </li><li> Despeje vibratome secções a partir da fase de recolha para um poço de uma placa de 6 cavidades, para armazenamento a 4 ° C. </li></ol></li><li> Coloração de imunofluorescência, tecidos seccionados vibratome fixos <ol><li> Colocar 5-12 secções vibratome por cavidade em uma placa de 24 poços com 1 x DPBS. </li><li> Remover 1x DPBS e adicione 500 ml de solução de permeabilização por poço. Agite delicadamente as amostras por 25 min em temperatura ambiente. </li><li> Depois de permeabilização, lavar as amostras 3 vezes com PBS 1x. </li><li> Bloquear as amostras de, pelo menos, 30 minutos em 500 ul de solução de bloqueio. </li><li> Após o bloqueio, exchange a solução de bloqueio com 500 ul de anticorpos primários diluídos em solução de bloqueio e a manter as amostras a 4 ° CO / N. NOTA: As diluições de anticorpos primários que funcionam bem em cortes congelados e parafina geralmente funcionam bem em vibratome seções também, no entanto os anticorpos que exige recuperação do antígeno nuclear geralmente não funcionam bem com este protocolo (por exemplo, BrdU). </li><li> No dia seguinte, as amostras de lavar três vezes durante 15 min de cada vez com solução de bloqueio. </li><li> Após a lavagem, aplicam-se 500 ul do anticorpo secundário diluído em solução de bloqueio. Envolva a placa de 24 poços com folha de alumínio para proteger os fluoróforos de luz e balançar as amostras durante 45 minutos a 2 horas à temperatura ambiente. </li><li> Lave as amostras 3 vezes com PBS 1x por 15 minutos cada e montar as seções vibratome em slides como descrito na Seção 2.4. </li></ol></li><li> Montagem vibratome seções <ol><li> Preparar lâminas para vibratome montagem cortando fatias finas de double-stick fita e colocá-los ao longo dos lados longos dos slides. </li><li> Com cuidado, coloque 5-10 seções vibratome entre a fita dupla-stick nos slides em uma gota de 100 l de PBS 1x. </li><li> Desdobrar todas as seções e espalhá-los em uma única camada em todo o slide. </li><li> Remova qualquer excesso de agarose ou pedaços irregulares que impediria uma lamela de sentar-se plana. </li><li> Com cuidado, use um tecido de laboratório para absorver o excesso de PBS 1x de todo as seções vibratome. </li><li> Adicionar uma gota de meio de montagem aquoso no topo das secções de vibratome e depois lamela. </li><li> Sele as lamelas para as lâminas com derreteu valap aplicado com um cotonete. </li><li> Imagem seções vibratome em ambos um microscópio confocal vertical ou invertida. Consulte a discussão para mais detalhes sobre a seleção de um sistema de imagem. </li></ol></li></ol> 3 Imagens ao vivo de intestinos inteiros Intestinos colhidas a partir de fetoss depois de E12.5, com o omento ligado deixado intacto, desenvolver com sucesso vilosidades em cultura usando o sistema acima. Portanto, este sistema ex vivo permite a captura de imagens de secção z vivo do intestino em desenvolvimento, que podem ser reconstituídas para uma vista tridimensional da morfogénese ao longo do tempo. <ol><li> Configurando imagens ao vivo <ol><li> Use cola instantânea para aderir uma membrana de policarbonato indivíduo a uma tela de malha fina. Isto proporciona uma estrutura de suporte de apoio para segurar o intestino em lugar abaixo da lente de imersão do microscópio confocal vertical. Por favor, consulte a seção de discussão para mais detalhes sobre a seleção de microscópios de imagem ao vivo. </li><li> Coloque a tela de malha no centro do poço de uma placa de cultura. </li><li> Inserir o intestino dissecados sobre a membrana de policarbonato e adicionar uma gota de cola instantânea em cada extremidade. Use apenas cola suficiente para apor o intestino, mas não revesti-la! </li><li> Adicionar cultura de mídia livre de fenol vermelho abaixo do policarbonatomembrana no centro de uma das cavidades da placa de cultura. </li><li> Adicionar água para a borda exterior da placa de cultura para fornecer humidade. </li><li> Uma vez que o intestino fetal sofre ondas peristálticas semelhantes de contracção da cultura, adicionar 100 ml de uma solução a 1: 5 de xilazina em 1x PBS a 3 ml de meio para controlar o movimento do intestino, enquanto imagiologia. Esta dosagem vai controlar os movimentos intestinais por cerca de 8-10 horas, sem comprometer o desenvolvimento das vilosidades. </li><li> Para um corte transversal do intestino delgado, o intestino incorporar vivos em uma solução de 3-4% de agarose e cortou secções espessas (150-500 um) sobre um vibratome. Combinar a rigidez da agarose com a rigidez do intestino que está sendo cortada e empiricamente determinada fase de desenvolvimento. Geralmente, uma solução de agarose a 3% funciona bem para os intestinos menores de E14.5 e uma solução a 4% funciona bem para os intestinos E15-E16. </li><li> Cole as seções vibratome a uma membrana de policarbonato aposta em uma tela de malha (como na Seção 3.1.1) ecultura como descrito na Seção 3.1.2-3.1.6. </li><li> Conduzir a imagens ao vivo usando um de dois fótons microscópio confocal de fluorescência (excitação laser entre 690-1,040 nm) em uma posição vertical com uma mesa ajustável. </li><li> Dois fótons de laser sintonizado 900 nm proporciona uma penetração profunda com a melhor resolução para sinais de GFP. Reduz danos aos tecidos, enquanto Imaging. Além disso, a configuração da potência do laser de emissão mais baixa possível reduzir o dano tecidual. Tipicamente, para obter uma boa excitação de GFP, usar 12% de energia em que o laser de dois fotões. </li></ol></li></ol>

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The authors have no financial conflicts to disclose.

A natureza dinâmica e interações complexas de tecido do intestino desenvolvimento requer visualização 3D para ter uma apreciação plena desses eventos morfogenéticos. Com a evolução da tecnologia de imagem, a capacidade de examinar vilosidades morfogênese em detalhe está a desenvolver / melhorar e, com isso, a compreensão da comunicação espacial e interação durante a organogênese é bastante reforçada. Os métodos alternativos para a cultura de intestinos inteiros foram tam...

Each intestinal villus is composed of two main tissue compartments: an epithelial surface layer and a mesenchymal core. The mouse small intestine is formed at embryonic day 10 when a sheet of endoderm closes and seals to form a tube of epithelium surrounded by mesenchymal cells3. This flat tube of epithelium undergoes rapid proliferation, growing both in length and girth and undergoes dramatic rearrangements involving dynamic cell shape changes1. At the same time, the surrounding mesenchyme also undergoes many developmental processes including the formation of the vascular plexus, differentiation of smooth muscle and recruitment of enteric neurons4. In the proximal small intestine at embryonic day 14.5, condensations (clusters) of Hedgehog- and PDGF-responsive cells begin to form adjacent to the epithelium2,5. Formation of mesenchymal clusters continues to spread along the length of the intestine so that they cover the entirety of the small intestine by embryonic day 16.52. As mesenchymal clusters form, the epithelial cells closest to the clusters begin to withdraw from the cell cycle, while the other epithelial cells continue to proliferate. Those cells directly above the mesenchymal cluster that have withdrawn from the cell cycle begin to change shape as the emerging villus buckles into the lumen. Further growth of the villus is driven in part by the continued proliferation of the epithelium between the emerging villi. The mesenchymal clusters remain tightly adhered to the epithelium of the growing villus and continue to express a variety of signaling molecules. The wave of villus emergence propagates along the length of the small intestine following the formation of mesenchymal clusters. As the intestine continues to grow and the intervillus region extends between emerging villi, new mesenchymal clusters form adjacent to the intervillus epithelium and further rounds of villus emergence and growth ensue6.
Synchronized development of the epithelium and mesenchyme is essential for villus morphogenesis. Signaling molecules are secreted from one layer to the other where receptors receive and transduce the signal message in order to coordinate development between the epithelium and mesenchyme. Mesenchymal clusters act as signaling centers and express a variety of developmental morphogens7-10. Disruption of cluster formation or pattern results in loss of villus emergence and pattern. Inhibition of PDGF signaling results in fewer clusters and fewer villi and those villi that do form are misshapen following the abnormal clusters11. Loss of Hedgehog signaling results in complete loss of cluster formation and failure of villus emergence2,12. Together, these data demonstrate that clusters coordinate development of the villus epithelium with its mesenchymal core.
Using this whole organ culture system, we are able to alter signaling involved in epithelial-mesenchymal cluster cross-talk to determine the role of those signals in villus morphogenesis. Two-photon confocal optical sectioning and reconstruction afford the ability to visualize cluster formation and villus emergence in three-dimensions and better understand the spatial relationships between the mesenchymal clusters and their overlying epithelium. Extending the culture system to four dimensions, we can capture z-stacks of developing clusters and villi over time and observe these interactions. Ultimately, the ability to follow villus development in this manner and observe changes that occur with altered signaling will revolutionize the understanding of epithelial mesenchymal interactions in villus morphogenesis.

<table><tbody><tr><td>Fine dissecting forceps&amp;nbsp;</td><td>Fine Science Tools</td><td>&amp;nbsp;11254-20</td><td>2 pairs</td></tr><tr><td>70% Ethanol</td><td></td><td></td><td></td></tr><tr><td>1x sterile Dulbecco&#039;s Phosphate-Buffered Saline (DPBS)</td><td>Gibco&amp;nbsp;</td><td>14040-133</td><td>500 ml</td></tr><tr><td>6 well plates</td><td>Costar</td><td>3516</td><td></td></tr><tr><td>24 well plates</td><td>Costar&amp;nbsp;</td><td>3524</td><td></td></tr><tr><td>60 x 15 mm petri dishes</td><td>Falcon</td><td>&amp;nbsp;451007</td><td></td></tr><tr><td>Transwell plates, 24 mm inserts, 8.0 mm polycarbonate membranes</td><td>Corning Costar&amp;nbsp;</td><td>3428</td><td>6 inserts per plate</td></tr><tr><td>BGJb media</td><td>Invitrogen&amp;nbsp;</td><td>12591-038</td><td>500 ml</td></tr><tr><td>PenStrep (10,000U/ml Penicillin; 10,000 mg/ml Streptomycin)</td><td>Gibco</td><td>&amp;nbsp;15140</td><td></td></tr><tr><td>Ascorbic Acid</td><td>Sigma&amp;nbsp;</td><td>A0278</td><td>make 5 mg/ml stock, filter, aliquot and store at -20 &amp;deg;C</td></tr><tr><td>Mouth pipet (Drummond 1-15 inch aspirator tube assembly)</td><td>Fisher</td><td>&amp;nbsp;21-180-10</td><td>remove the aspirator assembly and replace it with a 1,000 &amp;micro;l pipet tip which acts as an adaptor to plug in a 6 inch glass Pasteur pipet.</td></tr><tr><td>6 inch glass pasteur pipets</td><td></td><td></td><td></td></tr><tr><td>Capillary Tubes</td><td>World Precision Instruments&amp;nbsp;</td><td>TW100F-4</td><td>pull to needles</td></tr><tr><td>4% Paraformaldehyde</td><td></td><td></td><td>made in 1 x PBS, pH to 7.3</td></tr><tr><td>Culture plates</td><td>Falcon&amp;nbsp;</td><td>353037</td><td></td></tr><tr><td>Fine mesh stainless steel screen</td><td>purchase at hardware store</td><td></td><td></td></tr><tr><td>Polycarbonate membranes</td><td>Thomas scientific&amp;nbsp;</td><td>4663H25</td><td>alternatively, cut Corning Costar 3428 membranes off of transwell supports</td></tr><tr><td>Instant glue</td><td>purchase at hardware store</td><td>gel based preferrably</td><td></td></tr><tr><td>35 x 10 mm plates</td><td>Falcon&amp;nbsp;</td><td>351008</td><td></td></tr><tr><td>7% agarose</td><td>Sigma</td><td>&amp;nbsp;A9414</td><td>prepare w/v in 1x DPBS, heating to dissolve in a waterbath</td></tr><tr><td>minutien pins</td><td>Fine Science Tools&amp;nbsp;</td><td>26002-20</td><td></td></tr><tr><td>Phenol red free media (DMEM)</td><td>Gibco&amp;nbsp;</td><td>21063-029</td><td></td></tr><tr><td>Xylazine (100 mg/ml)</td><td>AnaSed&amp;nbsp;</td><td>139-236</td><td></td></tr><tr><td>Matrigel</td><td>BD</td><td>356231</td><td>basement membrane matrix, growth factor reduced, phenol red-free</td></tr><tr><td>3-4% agarose</td><td>Sigma&amp;nbsp;</td><td>A9414</td><td>prepare w/v in 1x DPBS, heating to dissolve in a waterbath</td></tr><tr><td>Imaging of fixed intestines</td><td></td><td></td><td></td></tr><tr><td>Name of Material/Equipment</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>vaseline</td><td>purchase at pharmacy</td><td></td><td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td></tr><tr><td>lanolin</td><td>Sigma&amp;nbsp;</td><td>L7387</td><td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td></tr><tr><td>paraffin</td><td>Surgipath</td><td>39601006</td><td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td></tr><tr><td>70% glycerol in 1 x PBS</td><td></td><td></td><td></td></tr><tr><td>Focus clear and Mount Clear</td><td>CelExplorer Labs Co.</td><td>&amp;nbsp;F101-KIT</td><td></td></tr><tr><td>Modeling clay</td><td>purchase at art supply store</td><td></td><td></td></tr><tr><td>double stick tape</td><td></td><td></td><td></td></tr><tr><td>cotton applicator swabs</td><td></td><td></td><td></td></tr><tr><td>plastic molds, 10mm x 10mm x 5 mm)</td><td>Tissue Tek&amp;nbsp;</td><td>4565</td><td></td></tr><tr><td>slides</td><td></td><td></td><td></td></tr><tr><td>coverslips</td><td></td><td></td><td></td></tr><tr><td>lab wipe</td><td>Kimberly Clark</td><td>34155</td><td>lint free delicate task wipe</td></tr><tr><td>Theiler staging chart&amp;nbsp;</td><td></td><td></td><td>&lt;a href=&quot;http://www.emouseatlas.org/emap/ema/theiler_stages/%20downloads/theiler2.pdf&quot;&gt;http://www.emouseatlas.org/emap/ema/theiler_stages/ downloads/theiler2.pdf&lt;/a&gt;</td></tr><tr><td>Leica SP5X confocal microscope&amp;nbsp;</td><td>Leica</td><td></td><td>Used to conduct the live imaging&amp;nbsp;</td></tr><tr><td>Leica DMI 6000 stand&amp;nbsp;</td><td>Leica</td><td></td><td>Used to conduct the live imaging&amp;nbsp;</td></tr><tr><td>Aqueous mounting medium (Prolong Gold)</td><td>Molecular Probes&amp;nbsp;</td><td>P36930</td><td></td></tr><tr><td>Name of Material/Equipment</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>24 well plate</td><td>Costar</td><td>&amp;nbsp;3524</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma&amp;nbsp;</td><td>T-8787</td><td>used to make Permeabilization solution: 0.5% Triton X-100 in 1 x PBS</td></tr><tr><td>Goat serum</td><td></td><td></td><td>used to make Blocking Solution: 4% Goat serum, 0.1% Tween20 in 1x PBS</td></tr><tr><td>Tween20&amp;nbsp;</td><td>Sigma</td><td>&amp;nbsp;P9416</td><td></td></tr></tbody></table>

mouse fetal whole intestine culture system for ex vivo manipulation of signaling pathways and three-dimensional live imaging of villus development

A maioria dos processos morfogenéticos no intestino fetal têm sido inferida a partir de lâminas finas de tecidos fixados, oferecendo instantâneos de mudanças ao longo do estágio de desenvolvimento. Informação tridimensional de cortes seriados finos podem ser difíceis de interpretar por causa da dificuldade de reconstrução de cortes seriados perfeitamente e manter a orientação adequada do tecido sobre cortes seriados. Recentes descobertas por Grosse et al., 2011 destacam a importância de três informações dimensional na compreensão morfogênese das vilosidades desenvolvimento do intestino 1. A reconstrução tridimensional de células intestinais isoladamente marcadas demonstraram que a maioria das células epiteliais intestinais em contato com ambas as superfícies apicais e basais. Por outro lado, a reconstrução tridimensional do citoesqueleto de actina para a superfície apical do epitélio demonstrado que o lúmen intestinal é contínua e que lúmens secundários são um artefacto sectioning. Estes dois pontos, juntamente com a demonstração de migração nuclear interkinetic no epitélio intestinal, definido o epitélio intestinal em desenvolvimento como um epitélio pseudo estratificado e não como se pensava anteriormente 1. A capacidade de observar o epitélio tridimensionalmente foi seminal para demonstrar este ponto e redefinindo morfogênese epitelial no intestino fetal. Com a evolução da tecnologia de imagem multi-fotão e software de reconstrução tridimensional, a capacidade de visualizar intacta, o desenvolvimento de órgãos está melhorando rapidamente. Dois fótons de excitação permite a penetração mais profunda menos prejudiciais para os tecidos com alta resolução. Dois fótons e reconstrução 3D de todo o intestino do feto de rato em Walton et al., 2012 ajudou a definir o padrão de vilo conseqüência 2. Aqui nós descrevemos um sistema de cultura de órgão inteiro que permite ex vivo desenvolvimento de vilosidades e extensões desse sistema de cultura para permitiros intestinos para ser tridimensionalmente fotografada durante o seu desenvolvimento.

Culturas de explantes de intestino fetal inteiros permite a análise da localização, distribuição e duração das moléculas de sinalização que coordenam o desenvolvimento intestinal, como este sistema permite a manipulação da sinalização com reagentes farmacológicos ou proteínas recombinantes. O sistema de cultura Transwell (Figura 1A, reproduzido a partir de Walton et al., 2012) 2 fornece uma interface ar-líquido, o que faz com que seja possível colocar droga ou prote...

Watch this Scientific Journal Video about Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development at JoVE.com

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Rato Fetal Whole Intestino Sistema de Cultura para<em&gt; Ex Vivo</em&gt; Manipulação de vias de sinalização e tridimensional Imagens ao vivo de Desenvolvimento Villus

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

mouse-fetal-whole-intestine-culture-system-for-ex-vivo-manipulation

Research

JoVE Journal

Biology

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

14.9K visualizações.  University of Michigan. Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine. 

Vídeo: Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

Usando<em&gt; Ex Vivo</em&gt; Culturas Vertical Gota de toda fetais Órgãos para estudar processos de desenvolvimento durante Rato Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Biologia do Desenvolvimento

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs likely involves other differentiated cells within the niche, as it has previously been demonstrated that the &#8216;&#8216;stemness&#8217;&#8217; of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

Intestinal epithelial stem cells (ISCs) are intermingled with Paneth cells. These cells are differentiated progeny of the ISC, which support the ISCs and provide antibacterial protection. Here we demonstrate how we used transgenic conditional mouse models to establish that Paneth cells play a crucial role in maintaining the intestinal epithelia.

Protocolos para analisar o papel das células Paneth em Regenerar o Intestino Murino utilizando Condicional<em&gt; Cre-lox</em&gt; Mouse Models

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

Engenharia de Tecidos do Intestino num modelo murino

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

Bioengenharia

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The present protocol describes establishing a murine colonic organoid system to study the activity and functioning of colonic stem cells in a claudin-7 knockout model.

Cultura tridimensional de criptas colônicas murinas para estudar a função das células-tronco intestinais ex vivo

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of ...

Pesquisa do câncer

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Co-cultura de Vida Microbiome com Intestinal Microengineered Humano Villi num Gut-on-a-Chip Microfluidic Dispositivo

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to digest solid food. This process is termed suckling-to-weaning transition and results in the replacement of neonatal epithelium with adult epithelium which goes hand in hand with metabolic and morphological adjustments. These complex developmental changes are the result of a genetic program that is intrinsic to the intestinal epithelial cells but can, to some extent, be modulated by extrinsic factors. Prolonged culture of mouse primary intestinal epithelial cells from late fetal period, recapitulates suckling-to-weaning transition in vitro. Here, we describe a detailed protocol for mouse fetal intestinal organoid culture best suited to model this process in vitro. We describe several useful assays designed to monitor the change of intestinal functions associated with suckling-to-weaning transition over time. Additionally, we include an example of an extrinsic factor that is capable to affect suckling-to-weaning transition in vivo, as a representation of modulating the timing of suckling-to-weaning transition in vitro. This in vitro approach can be used to study molecular mechanisms of the suckling-to-weaning transition as well as modulators of this process. Importantly, with respect to animal ethics in research, replacing in vivo models by this in vitro model contributes to refinement of animal experiments and possibly to a reduction in the use of animals to study gut maturation processes.

This protocol describes how to mimic suckling-to-weaning transition in vitro using mouse late fetal intestinal organoids cultured for 30 days.

Recapitulando a transição de amamentação in vitro usando organóides intestinais fetais

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

o<em&gt; Ex Vivo</em&gt; Cultura e Reconhecimento de Padrões Receptor Stimulation of Mouse Intestinal Organóides

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Imunologia e Infecção

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Um Sistema de<em&gt; Ex vivo</em&gt; A cultura de Pâncreas embrionárias

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Biologia

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

Culturas primárias mistas de Murino Intestino Delgado destinadas ao estudo de Gut hormonal Secreção e imagens de células vivas de Células Enteroendócrinas

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

Isolamento de criptas intestinais de ratos e geração de organoides intestinais

Generation and Manipulation of Rat Intestinal Organoids

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. Accordingly, patient-derived organoids are used for disease modeling, drug discovery, and personalized treatment screening. Mouse intestinal organoids are commonly utilized to understand aspects of both intestinal function/physiology and stem cell dynamics/fate decisions. However, in many disease contexts, rats are often preferred over mice as a model due to their greater physiological similarity to humans in terms of disease pathophysiology. The rat model has been limited by a lack of genetic tools available in vivo, and rat intestinal organoids have proven fragile and difficult to culture long-term. Here, we build upon previously published protocols to robustly generate rat intestinal organoids from the duodenum and jejunum. We provide an overview of several downstream applications utilizing rat intestinal organoids, including functional swelling assays, whole mount staining, the generation of 2D enteroid monolayers, and lentiviral transduction. The rat organoid model provides a practical solution to the need of the field for an in vitro model which retains physiological relevance to humans, can be quickly genetically manipulated, and is easily obtained without the barriers involved in procuring human intestinal organoids.

Autor em destaque: Geração e manipulação de organoides intestinais de ratos  

Here, we present a protocol to generate rat intestinal organoids and use them in several downstream applications. Rats are often a preferred preclinical model, and the robust intestinal organoid system fills the need for an in vitro system to accompany in vivo studies.

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Protocolos para analisar o papel das células Paneth em Regenerar o Intestino Murino utilizando Condicional

Engenharia de Tecidos do Intestino num modelo murino

Cultura tridimensional de criptas colônicas murinas para estudar a função das células-tronco intestinais ex vivo

Co-cultura de Vida Microbiome com Intestinal Microengineered Humano Villi num Gut-on-a-Chip Microfluidic Dispositivo

Recapitulando a transição de amamentação in vitro usando organóides intestinais fetais

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Culturas primárias mistas de Murino Intestino Delgado destinadas ao estudo de Gut hormonal Secreção e imagens de células vivas de Células Enteroendócrinas

Geração e Manipulação de Organoides Intestinais em Rato