Este trabalho foi apoiado pela Deutsche Forschungsgemeinschaft SFB688 (para A.G.).

Reprodução de rato foi aprovada pelo Regierung Unterfranken, e todas as análises foram realizadas em estrita conformidade com todos os alemão e da União Europeia leis e regulamentos aplicáveis relativos ao uso de animais de laboratório e cuidados.
1. revestimento de microtitulação placas com α4β1 integrina para Ex Utero Allantois explante cultura
<ol><li>Reconstitua a liofilizado murino α4β1 integrina a 200 µ g/mL em salina tampão fosfato (PBS). Dilua para uma concentração final de 10 µ g/mL em pré-aquecido (37 ° C) PBS. Pipete 20 µ l/poço da solução diluída α4β1 integrina para o número necessário de poços de uma placa de microtitulação (1 alantoide/poço). Nota: Evite a formação de bolhas de ar quando o revestimento dos poços da placa de microtitulação.</li>
	<li>Incube as placas de microtitulação para 60 min a 37 ° C, colocando-os em uma incubadora de cultura de tecidos umidificado.</li>
	<li>Aspire cuidadosamente o sobrenadante com uma pipeta. Usar sucção suave e inclinar a placa para evitar tocar bem fundo com a ponta da pipeta. Lavar os poços duas vezes pela adição de 50 µ l de meio de cultura previamente aquecido (37 ° C) como de Dulbecco modificado águia médio (DMEM) e remover o meio de lavagem por sucção suave.</li>
	<li>Passo fundamental: bloco de ligação grátis sites pela adição de 50 µ l/poço de 0,1% (p/v) albumina de soro bovino (BSA) / DMEM e incube por ≥ 30 min a 37 ° C, colocando as placas de microtitulação numa incubadora umidificado cultura de tecidos. Nota: Explantes alantoide podem anexar várias superfícies na presença de soro. Portanto, é imperativo para bloquear sites de ligação não-específica, após o revestimento dos poços com integrina e antes de adicionar o alantoide dissecado por explantes cultura no soro contendo médio (consulte a etapa 4.4).</li>
	<li>Manter os Poços revestidos na solução de bloqueio a 37 ° C, até que seja necessário na etapa 4.4.</li>
</ol>2. o útero dissecação de um rato grávida na Dpc 8
Nota: Reduza taxas de falso-positivo de gravidez, de discriminação de ganho de peso15.
<ol><li>Sacrifica o mouse por deslocamento cervical. Desinfecte o casaco na região abdominal por pulverização com etanol a 70% (v/v). Nota: Utilize luvas quando manusear os ratos. Eutanásia por deslocamento cervical sem anestésicos impede a contaminação química do alantoide, que pode interferir com o acessório de alantoide.</li>
	<li>Abra a pele abdominal, fazendo uma pequena incisão na linha com uma tesoura. Use os dedos com luvas para manter a pele acima e abaixo da incisão e separar a pele em direção ao peito e a cauda do rato.</li>
	<li>Agarre o peritônio com fórceps. Com a outra mão, faça uma incisão em forma de V de anterior para posterior usando a tesoura e mover o tecido para expor na cavidade abdominal. Use a pinça para empurrar os intestinos de um lado para obter acesso ao útero.</li>
	<li>Localize os dois cornos uterinos. Segure o colo do útero (o segmento caudal) com fórceps e corte os ligamentos com uma tesoura bem. Então retire ambos os cornos uterinos por separando os ovidutos com uma tesoura.</li>
	<li>Usar fórceps para transferir o útero em um prato de 10cm estéril contendo estéril, temperatura (RT) PBS. Remova o tecido adiposo, vasos e nervos que cercam os cornos uterinos com pinça fina e/ou tesoura (figura 1A).</li>
</ol>3. embrião dissecação
Nota: Execute as seguintes etapas sob um microscópio estereoscópico equipado com uma gama de zoom de 8:1.
<ol><li>Separe os embriões por corte entre os locais de implantação com uma tesoura. Segure o revestimento do útero muscular dos brotos útero com pinça e puxá-lo distante para expor a decídua (figura 1B).</li>
	<li>Usando fórceps, transferi os gomos do útero para uma nova placa de 10 cm contendo estéril, RT PBS.</li>
	<li>Corrigi a decidua em torno do embrião com as pontas da pinça.</li>
	<li>Fazer uma incisão no lado anti-mesometrial da decidua com pinça fina (Figura 1) e descascar a decidua com fórceps para expor o embrião (Figura 1). Nota: O polo mesometrial da decidua é altamente vascularizado e pode ser mais largo na base que a parte de anti-mesometrial, que contém o embrião e aparece mais pálida.</li>
	<li>Disse o embrião com pinça (Figura 1E, F).</li>
	<li>Usando uma microcolher, transferi o embrião para uma gota de PBS estéril contido em um prato de estéril de 3,5 cm.</li>
	<li>Transferir o embrião para uma gota fresca de PBS no mesmo prato 3,5 cm usando uma microcolher e dissecar o embrião do saco vitelino usando fórceps (Figura 1, H).</li>
	<li>Opcionalmente, se necessário (por exemplo, para a genotipagem por reação em cadeia da polimerase), transferir o saco vitelino em um tubo estéril aspirando com ~ 5 µ l PBS em uma ponta da pipeta orifício grande 200 µ l. Loja a-20 ° C. Nota: Tecido materno (ou seja, o saco vitelino parietal, incluindo a membrana do Reichert e o cone de ectoplacental) que possam contaminar a preparação do saco vitelino pode levar a resultados errôneos de genotipagem. Remoção desses tecidos é mais fácil antes a dissecação do embrião do saco vitelino.</li>
</ol>4. o alantoide dissecção e cultura Ex Utero no imobilizado α4β1 integrina
<ol><li>Corte o alantoide do seu local de inserção basal na extremidade posterior do embrião usando pinça fina (Figura 1 H).</li>
	<li>Opcionalmente, se a plataforma do desenvolvimento é necessária, conta os pares somite do embrião por inspeção visual, usando um microscópio equipado com ótica de contraste de fase e um 5 × 10 × objetivo. Nota: O embrião pode ser estendido com fórceps para facilitar a contagem do somite. Fusão e apego corioalantoicas ocorrem entre E 8.0 e 9.0 E em embriões com pares de 6 somite ≥. Giro começa em embriões com 6-8 pares de somite.</li>
	<li>Pipeta de 40 µ l/poço de DMEM suplementado com 10% (v/v) de soro fetal bovino, L-glutamina e antibióticos no número necessário de poços de placa do microtiter pré-revestida (ver seção 1).</li>
	<li>Recolher o alantoide flutuante aspirando com ~ 5 µ l PBS, em um grande orifício de uma ponta de pipeta de 200 µ l. Transferi 1 alantoide/bem.</li>
	<li>Incubar as allantoides colocando as placas de microtitulação numa incubadora de cultura de tecidos umidificado (37 ° C, 5% CO2) para , por exemplo, 6, 12 e 24 h. Para evitar interferência com o acessório de alantoide, não mova as placas de microtitulação entre esses pontos de tempo.</li>
	<li>Em cada ponto do tempo, marcar acessório alantoide (por exemplo, nenhum acessório/flutuante alantoide alantoide parcialmente ou totalmente anexada; totalmente propagação alantoide) usando um microscópio equipado com ótica de contraste contraste ou diferencial do interferência fase e um × 5 ou 10 × objectivo (Figura 2). Nota: Exemplos representativos de allantoides recém isoladas e totalmente anexadas são mostrados na Figura 2. Ex utero, ponta distal do allantoides isolados de camundongos C57BL/6J de sua anexa dentro de 4-6 h (isto é referido como penhora parcial da alantoide), e acessório completo do alantoide inteira é alcançado dentro de ~ 12 h de cultura. Por 18-24 h, o explante tem aplainado e assumiu uma forma circular (referida para como totalmente espalhar alantoide) com um CD31 positivo do plexo vascular9,16.</li>
</ol>5. confirmação dos coloração o alantoide explantado para o CD31 marcador de células endoteliais
<ol><li>Aspire o meio de cultura de explantes de propagação totalmente e corrigir as células adicionando 40 µ l/poço de 4% (p/v) paraformaldeído/PBS. Nota: Paraformaldehyde é tóxico. Use luvas e óculos de segurança para evitar o contato de olho e pele.</li>
	<li>Incubar durante 10 minutos a RT e, em seguida, aspirar o sobrenadante. Lavar três vezes com a adição de 40 µ l/poço de PBS e remover o meio de lavagem por sucção suave.</li>
	<li>Permeabilize as células e bloquear sites inespecificas adicionando 40 µ l/poço de 0,1% (v/v) de soro de cabra normal BSA/10% Triton X-100/0.1% (w/v) (v/v) em PBS por 30 min em RT. Wash duas vezes adicionando 40 µ l/poço de PBS e remover o meio de lavagem por sucção suave. Nota: O protocolo pode ser pausado a noite neste ponto.</li>
	<li>Mancha de células, adicionando 20 µ l/poço de anticorpos anti-CD31 primário diluído 01:50 em 0,1% (p/v) BSA/PBS durante a noite a 4 ° C. Lavar três vezes com a adição de 40 µ l/poço de PBS e remover o meio de lavagem por sucção suave.</li>
	<li>Detecta anticorpos primários ligados pela adição de 20 µ l/poço de cabra secundário fluorescente etiquetado anticorpos anti-rato diluído 1: 200 em 0,1% (p/v) BSA/PBS por 1h em placas de microtitulação RT. Protect da luz por envolvimento em papel de alumínio ou colocando em uma câmara escura. Lavar três vezes com a adição de 40 µ l/poço de PBS e remover o meio de lavagem por sucção suave.</li>
	<li>Inserir células, adicionando 20 µ l/poço de meio de montagem. Encube por ~ 1h no RT até o meio de montagem tem solidificado. Armazenar placas de microtitulação no RT ou a 4 ° C, protegido da luz.</li>
	<li>Imagem do plexo vascular do alantoide explants sob um microscópio de fluorescência, equipado com um × 5 ou 10 × objetivo e filtros de fluorescência apropriado. Nota: Imagens representativas do plexo vascular CD31 positivo de uma totalmente propagação alantoide são mostradas na Figura 3. Outros marcadores para o plexo vascular, tais como anticorpos dirigidos contra Flk-1, Flt, gravata-1 ou Tie-2 pode ser usado como bem16.</li>
</ol>

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Os autores não têm nada para divulgar.

Na presença de soro, que é uma fonte rica de moléculas pro-adesivo da matriz extracelular como a fibronectina, explantes alantoide prontamente irão anexar vários plástico, vidro ou superfícies de filtro9,16. Assim, se o objectivo do ensaio realizado é especificamente investigar o efeito de uma modificação genética ou qualquer outro tratamento no acessório alantoide para imobilizado α4β1, é fundamental para bloquear sites de ligação não-específi...

A placenta é indispensável para o crescimento embrionário e desenvolvimento no ambiente uterino. Constitui a interface para o oxigênio, metabólito e troca de nutrientes entre a circulação fetal e materna e também funções como órgão endócrino e imunológico. Qualquer insulto endógeno ou exógeno que prejudica o desenvolvimento da placenta pode levar a retardo de crescimento intra-uterino, perda fetal ou complicações na gravidez, tanto em ratos e em seres humanos1. Enquanto o número de mutações específicas do mouse que causam fisiologia placentária anormal continua a aumentar2, com 219 genótipos atualmente listados no site da informática do genoma do rato (MGI) (http://www.informatics.jax.org/vocab/ mp_ontology / MP:0010038), muitos destes fenótipos não são bem compreendidos, do ponto de vista mecanicista. Além de alterações genéticas, drogas pequena molécula, anticorpos monoclonais, toxinas, agentes patogénicos, metabolitos em excesso ou vários agentes ambientais podem afetar desenvolvimento de placenta e função3,4,5 , 6 , 7. ainda, simples em vitro ensaios que passos críticos do modelo em desenvolvimento de placenta são escassos.
Desenvolvimento placentário murino é iniciado no início midgestation, quando o alantoide (o precursor do desenvolvimento do cordão umbilical) emerge da extremidade posterior do embrião como um broto que cresce em direção do córion8. Chorioadhesive células na camada exterior do bud alantoico mediam o apego e a difusão do alantoide na superfície do cório (corioalantoicas "fusão"). O córion dobra-se posteriormente em vilosidades no qual vasculatura do alantoide cresce para formar o labirinto, a camada interna da placenta onde nutrientes e oxigênio são trocados entre o sangue materno intimamente justapostos sinusoides e vasos embrionários9 ,10.
O anexo do alantoide para córion é o passo inicial e fundamental na formação do labirinto, e defeitos neste processo estão entre as causas mais comuns de letalidade embrionária em midgestation1. Embora um número de mutantes de rato tem sido descrito onde corioalantoicas ceda a ocorrer10, e o banco de dados do MGI atualmente lista 108 mutantes de rato que caracterizam-se por fusão corioalantoicas anormal (http:// www.Informatics.Jax.org/Vocab/mp_ontology/MP:0002824), a interação intercelular entre as células vasculares adesão molécula-1 (VCAM-1, expressado na mesoderme alantoico) e α4β1 integrina (expressado no mesotélio gonadotrofina coriônica, que é derivado do mesoderma extraembryonic) parece ser indispensável para a ligação corioalantoicas e fusão11,12,13. Coloração imuno-histoquímica de todo-monte selvagem-tipo embriões tem mostrado que o VCAM-1 é expresso dentro os dois terços distais do caule alantoico no dia aproximadamente embrionário (E) 7,513 (palco do somite 1-4), e que sua expressão permanece elevada durante corioalantoicas anexo e - fusão11,12. Falha de alantoico apego ao córion normalmente é detectada por análise histológica de botões do útero. No entanto, alantoide tamanhos são fisiologicamente altamente variáveis entre e mesmo dentro de ninhadas do mesmo grau de desenvolvimento14, e o alantoide só pode anexar o córion quando atingiu um tamanho suficiente para fazer contato físico. Reflectindo esta variabilidade natural, anexo corioalantoicas ocorre em algum momento entre ~ E 8.0 E 9.0 e no uteroe uma avaliação estatisticamente confiável deste processo pela histologia depende, portanto, a análise de um grande número de conceptuses para obter suficiente amostras na fase do desenvolvimento apropriado.
Aqui, descrevemos um método para avaliar fixação alantoico ex utero que é menos dependente do tamanho do alantoide. Demonstramos a dissecação de embriões do rato e seus allantoides desde o útero de ratos grávidas ~ 8 dias post coitum (dpc; dpc 0.5 designa a detecção de um tampão vaginal), e a cultura subsequente do alantoide explants na α4β1 imobilizado integrinas. Este método permite uma avaliação rápida, funcional de acessório e espalhando o comportamento de vários explantes alantoide em paralelo e em pontos diferentes de tempo sucessivos. O protocolo pode ser usado para tela para efeitos de diversos fatores ambientais, drogas ou mutações específicas no alantoide acessório ex vivo.

<table><tbody><tr><td>C57BL/6J wildtype mice</td><td>The Jackson Laboratory</td><td>Stock No:&amp;nbsp;000664</td><td></td></tr><tr><td>70 % (v/v) Ethanol</td><td>VWR International</td><td>930031006</td><td></td></tr><tr><td>Standard pattern forceps</td><td>Fine Science Tools</td><td>11000-12</td><td>Forceps used to open the abdominal cavity.</td></tr><tr><td>Micro dissecting forceps</td><td>Fine Science Tools</td><td>11254-20</td><td>Dumont # 5 medical biology forcepts with fine, sharp tip.&amp;nbsp;</td></tr><tr><td>Fine scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>14094-11</td><td>Straight blades.</td></tr><tr><td>Spring scissors&amp;nbsp;</td><td>Fine Science Tools</td><td>15012-12</td><td>Noyes spring scissors with 14 mm blades for the dissection of uterus buds.</td></tr><tr><td>Microspoon</td><td>Carl Roth</td><td>AT18.1</td><td>5 mm Spoon diameter.</td></tr><tr><td>Microtiter plates</td><td>ibidi</td><td>81501</td><td>We use uncoated, sterile angiogenesis slides (internal volume 50 &amp;micro;L) with a hydrophobic surface that is not tissue-culture treated to reduce non-&amp;#945;4&amp;#946;1-mediated binding to plastic.</td></tr><tr><td>10 cm dish</td><td>Nunc</td><td>150350</td><td>Sterile tissue culture dishes.</td></tr><tr><td>3.5 cm dish</td><td>Nunc</td><td>150288</td><td>Sterile tissue culture dishes.</td></tr><tr><td>Large orifice pipette tips&amp;nbsp;</td><td>Biozym</td><td>VT0140X</td><td>Low binding pipette tips, 200 &amp;micro;L.</td></tr><tr><td>&amp;#945;4&amp;#946;1 integrin</td><td>R&amp;amp;D Systems</td><td>6054-A4</td><td>Murine &amp;#945;4&amp;#946;1 integrin recombinantly expressed and purified from a Chinese hamster ovary cell line.</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline (PBS)</td><td>Sigma Aldrich</td><td>D8537</td><td>Without Ca&lt;sup&gt;2+&lt;/sup&gt; and Mg&lt;sup&gt;2+&lt;/sup&gt;.</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>PAN Biotech</td><td>P04-03600</td><td>The formulation contains 4.5 g/L glucose, 110 mg/L sodium pyruvate and 3.7 g/L NaHCO&lt;sub&gt;3&lt;/sub&gt; but no L-glutamine, which is added separately.</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco (ThermoFisher Scientific)</td><td>15140-122</td><td>100 x (10,000 U/mL Penicillin and 10,000 &amp;micro;g/mL streptomycin) stock solution.&amp;nbsp;Prepare and store aliquots at -20 &amp;deg;C to avoid freeze/thaw.</td></tr><tr><td>L-Glutamine</td><td>PAN Biotech</td><td>P04-80100</td><td>100 x (200 mM) Stock solution. Prepare and store aliquots at -20 &amp;deg;C to avoid freeze/thaw.</td></tr><tr><td>Fetal bovine serum&amp;nbsp;</td><td>Biochrom</td><td>S 0115</td><td>We use heat-inactivated FBS (heated to 56 &amp;deg;C for 30 min with mixing to inactivate complement).</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Sigma Aldrich</td><td>A7030</td><td>We use protease- and fatty acid-free albumin. Prepare a 0.1 % (w/v) solution in DMEM. Sterile filter the solution through a disposable cell culture filter with a 0.22 &amp;micro;m pore size and low protein-binding membrane, and store aliquots at -20 &amp;deg;C.</td></tr><tr><td>&lt;em&gt;para&lt;/em&gt;-Formaldehyde</td><td>Carl Roth</td><td>0335.2</td><td>To make a formaldehyde solution in PBS, weigh &lt;em&gt;para&lt;/em&gt;-formaldehyde powder in a ventilated hood, and add it to PBS preheated to ca. 60 &amp;deg;C in a beaker on a stir plate. Add 1 N NaOH dropwise until solution clears. Let solution cool to room temperature, and filter through 0.22 mm filter syringe. Adjust pH with diluted HCl to ca. 6.9, and adjust to final volume with PBS. We aliquot and freeze the solution, but it can also be stored at 4 &amp;deg;C for approximately four weeks.&amp;nbsp;</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>X100</td><td>Use wide-orifice pipette tips to handle undiluted Triton X-100.</td></tr><tr><td>Normal goat serum</td><td>Sigma Aldrich</td><td>G9023</td><td>To block non-specific binding of antibodies in immunofluorescence analyses.</td></tr><tr><td>&amp;#945;-CD31 Antibodies</td><td>BD Biosciences</td><td>550274</td><td>Purified rat anti-mouse monoclonal antibodies, clone MEC 13.3.</td></tr><tr><td>Secondary goat anti-rat antibodies</td><td>Thermo Fisher</td><td>A-11006</td><td>Goat anti-rat IgG (heavy and light chains), cross-adsorbed, fluorescently labeled with Alexa Fluor 488. Other fluorescent labels are also possible.</td></tr><tr><td>Mowiol 4-88</td><td>Sigma Aldrich</td><td>81381</td><td>Mounting medium for cytochemistry. MW ~31,000</td></tr><tr><td>Labovert&amp;nbsp;</td><td>Leitz</td><td>Labovert&amp;nbsp;</td><td>Inverted phase contrast microscope equipped with a 4 x and 10 x objective for somite pair counting. Other phase contrast microscopes (such as those that are routinely used in tissue culture) are also suitable.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>M80</td><td>Leica Microsystems</td><td>M80</td><td>Stereomicroscope with 8 : 1 zoom range (yielding a magnification between 7.5 x and 60 x) for embryo and allantois dissection.</td></tr><tr><td>DM4000B</td><td>Leica Microsystems</td><td>DM4000B</td><td>Microscope system for histology with LED illumination. We use HCX PL FLUOTAR 5 x/0.15 and HC PL FLUOTAR 10 x/0.30 objectives. Other microscopes equipped phase contrast and fluorescence optics can be used to score allantois attachment.&amp;nbsp;</td></tr><tr><td>Digital camera</td><td>JVC</td><td>KY-F75U</td><td>3-CCD digital capture camera attached to the DM4000B LED microscope. We use Diskus software (Hilgers) to operate both the microscope and the camera.</td></tr><tr><td>Confocal microscope</td><td>Leica Microsystems</td><td>SP5</td><td>Confocal microscope for higher-resolution imaging of endothelia in the vascular plexus of allantois explants. Immunofluorescence images were taken with a 40 x objective.</td></tr></tbody></table>

dissection and explant culture of murine allantois for the in vitro analysis of allantoic attachment

A placenta é essencial para o crescimento e o desenvolvimento de embriões de mamíferos. Por este motivo, inúmeras alterações genéticas e insultos ambientais também prováveis que perturbam o desenvolvimento da placenta ou função podem causar perda de gravidez precoce em ratos e seres humanos. No entanto, simples em vitro ensaios para a tela para potenciais efeitos sobre a formação da placenta são escassos. Aqui, focalizamos o primeiro e fundamental passo na formação da placenta, que consiste de penhora do alantoide com o cório de modelagem. Nós descrevemos um método para avaliar rapidamente o acessório de explantes alantoico na imobilizado α4β1 integrina, que serve como substrato cório-mimética. Esta abordagem in vitro permite uma avaliação qualitativa do acessório e espalhando o comportamento de vários explantes alantoide em pontos diferentes vez consecutiva. O protocolo pode ser usado para investigar o efeito de mutações específicas do mouse, drogas ou vários fatores ambientais que têm sido associados a complicações na gravidez ou perda fetal no alantoide acessório ex vivo.

Este protocolo descreve um método para isolar e explantes murino allantoides ex vivoe detalha a avaliação qualitativa do alantoide penhora a α4β1 integrina, um processo crítico durante na vivo corioalantoicas fusão. Os representante resultados mostrados na figura 1A–H demonstram as etapas sucessivas de isolamento alantoide a partir do útero gestante. Tecidos maternos que cercam os cornos uterinos, ...

Watch this Scientific Journal Video about Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment at JoVE.com

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

Descrevemos um ensaio em vitro de fixação corioalantoicas de modelo, o primeiro passo na formação da placenta. O protocolo demonstra a cultura de dissecção e explante de murino allantoides em imobilizado α4β1 integrina. Acessório de alantoide é avaliado microscopicamente em pontos de tempo pré-determinado.

Dissecção e cultura de explante de murino alantoide para a análise In Vitro de fixação alantoico

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

7.8K visualizações.  University of Würzburg. Descrevemos um ensaio em vitro de fixação corioalantoicas de modelo, o primeiro passo na formação da placenta. O protocolo demonstra a cultura de dissecção e explante de murino allantoides em imobilizado α4β1 integrina. Acessório de alantoide é avaliado microscopicamente em pontos de tempo pré-determinado.

Vídeo: Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

Usando In Vivo e tecido e célula explante abordagens para estudar a morfogênese e a patogênese da Aorta embrionária e Perinatal

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

Biologia do Desenvolvimento

Application of Aorta-gonad-mesonephros Explant Culture System in Developmental Hematopoiesis

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine development of the embryo. Although genetic data from knockout (KO) mice are convincing, it is not realistic to generate KO mice for all genes as needed. In addition, performing in vivo rescue experiments to consolidate the data obtained from KO mice is not convenient. To overcome these limitations, the Aorta-Gonad-Mesonephros (AGM) explant culture was developed as an appropriate system to study hematopoietic stem cell (HSC) development. Especially for rescue experiments, it can be used to recover the impaired hematopoiesis in KO mice. By adding the appropriate chemicals into the medium, the impaired signaling can be reactivated or up-regulated pathways can be inhibited. With the use of this method, many experiments can be performed to identify the critical regulators of HSC development, including HSC related gene expression at mRNA and protein levels, colony formation ability, and reconstitution capacity. This series of experiments would be helpful in defining the underlying mechanisms essential for HSC development in mammals.

This protocol describes using cultured Aorta-Gonad-Mesonephros for expression analyses, colony-forming units in the culture and spleen, and long-term reconstitution to determine the effect of regulatory factors and signaling pathways on hematopoietic stem cell development. This has been demonstrated as an effective system for studying hematopoietic stem cell biology and function.

Aplicação do sistema de cultura Aorta-gônada-mesonephros explante na hematopoiese do desenvolvimento

The limitation of using mouse embryos for hematopoiesis studies is the added inconvenience in operations, which is largely due to the intrauterine ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

Isolamento de Leucócitos a partir de tecidos de murino na interface materno-fetal

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Imunologia e Infecção

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 ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

Culturas placentária e Decidual órgão humano para estudar Infecções na interface materno-fetal

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Microdissection and Dissociation of the Murine Oviduct: Individual Segment Identification and Single Cell Isolation

Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental treatments. However, because of their small body size, some structures, such as the oviduct with a diameter of 200-400 &#956;m, have proven to be relatively difficult to study except by immunohistochemistry. Recently, immunohistochemical studies have uncovered more complex differences in oviduct segments than were previously recognized; thus, the oviduct is divided into four functional segments with different ratios of seven distinct epithelial cell types. The different embryological origins and ratios of the epithelial cell types likely make the four functional regions differentially susceptible to disease. For example, precursor lesions to serous intraepithelial carcinomas arise from the infundibulum in mouse models and from the corresponding fimbrial region in the human fallopian tube. The protocol described here details a method for microdissection to subdivide the oviduct in such a way to yield a sufficient amount and purity of RNA necessary for downstream analysis such as reverse transcription-quantitative PCR (RT-qPCR) and RNA sequencing (RNAseq). Also described is a mostly non-enzymatic tissue dissociation method appropriate for flow cytometry or single cell RNAseq analysis of fully differentiated oviductal cells. The methods described will facilitate further research utilizing the murine oviduct in the field of reproduction, fertility, cancer, and immunology.

A method for microdissection of the mouse oviduct that allows collection of the individual segments while maintaining RNA integrity is presented. In addition, non-enzymatic oviductal cell dissociation procedure is described. The methods are appropriate for subsequent gene and protein analysis of the functionally different oviductal segments and dissociated oviductal cells.

Microdisseção e Dissociação do Ovídeo Murine: Identificação do Segmento Individual e Isolamento de Célula Única

Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

Um ensaio de explante para avaliar o comportamento celular do mesênquima craniana

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Biologia

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

Co-Cultura de Organoides Epiteliais do Intestino Delgado Murine com Células Linfoides Inatas

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

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

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

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 ...

Establishing Villous and Decidual Organ Cultures as Ex Vivo Models of Human Maternal-Fetal Interface

Source: Rizzuto, G. A., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/54237">Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface.</a> J. Vis. Exp. (2016).
This video demonstrates the establishment of primary human placental villous and decidual organ cultures in ex vivo conditions. These models are well-suited for studying pathogenesis at the human maternal-fetal interface.

Estabelecendo culturas de órgãos vilosos e deciduais como modelos ex vivo de interface materno-fetal humana

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...