Name: dissection and culture of mouse embryonic kidney
Uploaded: 2017-05-17T15:00:00.000+00:00
Duration: 8 min 30 s
Description: Watch this Scientific Journal Video about Dissection and Culture of Mouse Embryonic Kidney at JoVE.com

Os autores agradecem a Leif Oxburgh e Derek Adams por compartilhar generosamente seus conhecimentos, Leif Oxburgh pelos comentários úteis sobre o manuscrito, e Stefan Wölfl e Ulrike Müller pelo seu apoio técnico e Saskia Schmitteckert, Julia Gobbert, Sascha Weyer e Viola Mayer pela ajuda na Laboratório Este trabalho foi apoiado por Desenvolvimento, A Empresa de Biólogos (a CP).

Os ratinhos foram mantidos de acordo com a regulamentação sueca ea legislação da União Europeia (2010/63 / UE). Todos os procedimentos foram realizados seguindo as diretrizes do Comitê de Ética Sueco (autorizações C79 / 9, C248 / 11 e C135 / 14). Procedimentos na Universidade de Heidelberg envolvendo animais sujeitos foram aprovados pelo Regierungspräsidium Karlsruhe e os agentes de bem-estar animal na Universidade de Heidelberg. 1. Preparação de Reagentes e Materiais para a Cultura NOTA: Use uma capa de fluxo laminar para minimizar a contaminação. <ol><li> No dia da dissecção, Fc humano pré-agrupamento ou proteína de efrina quimérica recombinante fundida a Fc humano por mistura com anticorpos de cabra Fc anti-humanos numa razão molar de 1: 5 em solução salina tamponada com fosfato estéril (PBS). Incubar durante 1 h a 37 ° C 5 . </li><li> Preparar o meio de cultura suplementando Dulbecco Modified Eagle Medium: NutrientMistura F-12 (DMEM / F12) com penicilina / estreptomicina a 1% (v / v), glutamina a 1% (v / v), insulina transferrina selénio (v / v) a 50 μg / Transferrina e 1,5 μg / mL de anfotericina. NOTA: 10 mL de meio é suficiente para 16 embriões. </li><li> Adicionar solução de efrínina recombinante agrupada a uma concentração final de 20 μg / mL ao meio de cultura. Use o Fc humano agrupado como controle. </li><li> Preparar placas de cultura por adição de 500 μL de meio de cultura a cada poço de uma placa de 4 poços de poliestireno estéril, não revestido, de fundo plano. </li><li> Usando pinças esterilizadas, coloque cuidadosamente um filtro de membrana de policarbonato com um tamanho de poro de 5 μm por poço em cima do meio. Transferir a placa preparada para a incubadora (37 ° C / 5% CO 2 ). Certifique-se de que o filtro permanece seco na superfície superior e flutua. NOTA: Um filtro é suficiente para dois anlagen de rim. </li><li> Usando uma lâmina de barbear, corte aproximadamente 30 pontas de pipeta (volume: 100 μL) approximatelY 1 mm da ponta para resultar numa abertura mais larga. Coloque as pontas de volta em um rack de ponta de pipeta. </li></ol> 2. Dissecção de Rudimentos Metanefricos em E11.5 <ol><li> Sacrifique um rato com gestação cronometrada em E11.5 por deslocamento cervical (ou usando um método aprovado pelo comitê de ética local). </li><li> Remova o útero e os embriões, como descrito por Brown et al 6 . Deste ponto em diante, trabalhar sob um microscópio de dissecção. </li><li> Usando pinças de relojoeiro No. 5, corte o embrião diretamente sob as patas dianteiras. Use um prato de Petri fresco para cada embrião. Remova as pernas ea cauda do embrião. Para fazer isso, pegue o tecido a ser removido com um par de fórceps de relojoeiro No. 5 e use as pontas de um segundo par para cortar. </li><li> Para remover a massa do órgão ventral, use as pontas de um par de fórceps para abrir lateralmente a parede do corpo do embrião em uma direção rostral para caudal. <ol><li> Corte superficialmente, paralelo a tAorta dorsal. Use a aorta dorsal cheia de sangue e portanto visível para orientação. Cuidadosamente separar a parte ventral da parede do corpo da parte dorsal usando as pontas da pinça fechada e virar o embrião sobre. Corte a parede do corpo lateralmente em uma direção rostral-para-caudal e usando a aorta dorsal para a orientação, como para o lado anterior. </li></ol></li><li> Uma vez que a parede do corpo ventral foi separada do corpo dorsal, agarrá-lo com um par de fórceps e puxe cuidadosamente para removê-lo, juntamente com a massa do órgão. NOTA: O anlagen metanefric permanece geralmente unido à parede do corpo dorsal. São estruturas ovais, ~ 200 μm de comprimento, localizadas ao nível dos membros posteriores. </li><li> Segure a parede do corpo dorsal com um par de fórceps, com o lado ventral voltado para cima. Usando o outro par de fórceps, pegue a aorta dorsal em sua extremidade rostral e cuidadosamente descascar a aorta dorsal da parede do corpo dorsal. NOTA: Ambos os rins metanefric geralmente permanecem anexarÀ aorta dorsal. </li><li> Usando um par de fórceps, corte rostralmente para o rim anlagen para remover a aorta. Em seguida, cuidadosamente separar o anlagen dois rins usando as pontas do fórceps. </li><li> Cuidadosamente descascar tecido não desejado do rim anlagen usando as pontas do fórceps. Tome cuidado para não danificar o mesênquima, uma vez que os danos podem resultar em crescimento fraco e exclusão do explante de uma análise posterior. </li><li> Transferir o anlagen renal separadamente usando uma pipeta de microlitro e uma ponta de pipeta aberta em 10 μL de PBS para o filtro de membrana de policarbonato em uma placa de 4 poços. NOTA: Devido à tensão superficial, o explante será coberto com uma fina camada de meio. Um filtro é suficiente para dois rim anlagen. <ol><li> Coloque cada um dos dois anlagen renal no centro de cada respectiva metade do filtro. Transferir a placa de volta para a incubadora a 37 ° C / 5% de CO2. Deixar a placa na incubadora até que o metanephric rudImensos do próximo embrião estão prontos para serem colocados no filtro. </li></ol></li><li> Repita os passos 2.3-2.9.1 para cada embrião. NOTA: Com alguma prática, o procedimento de dissecção levará cerca de 5 min por embrião. </li><li> Incubar o anlagenio renal durante 3 dias a 37 ° C / 5% CO 2 . </li></ol> 3. Preparação de Reagentes para Fixação e Coloração <ol><li> Para preparar paraformaldeído a 4% (PFA) em PBS sem cálcio e magnésio (p / v), dissolver a PFA a 60 ° C em PBS, agitando continuamente. Arrefecer até à temperatura ambiente e utilizar um filtro de papel para remover as restantes partículas. Alíquota e manter a 4 ° C para uso imediato ou congelar para armazenamento de longo prazo. CUIDADO: PFA é tóxico. Usar o equipamento de proteção individual especificado nas normas de segurança locais e trabalhar em um exaustor. Descarte resíduos de acordo com as diretrizes institucionais e usando recipientes de resíduos designados. </li><li> Para preparar a solução de permeabilização, diluir Triton X-100Em PBS sem cálcio e magnésio até 0,3% (v / v). </li><li> Para preparar a solução de bloqueio, adicionar 5% (v / v) de soro de cabra e 0,1% de Triton X-100 a PBS sem cálcio e magnésio. Adicionar azida de sódio a uma concentração final de 0,02% (p / v). Armazenar a 4 ° C. CUIDADO: A azida sódica é tóxica. Manipular azida de sódio de acordo com os regulamentos locais de segurança e protecção ambiental. NOTA: Quando se utilizam anticorpos para coloração, utilizar soro a 5% da espécie a partir da qual se derivou o anticorpo secundário para se obter uma solução de bloqueio. </li><li> Diluir a aglutinina de Dolichorus biflorus biotinilada 1: 200 em solução de bloqueio. Diluir estreptavidina conjugada com Alexa488 1: 200 em solução de bloqueio. </li><li> Para fazer um meio de incorporação pronto a usar (ver a tabela de materiais), adicione 0,1 g de mucoadesivo / ml a base de álcool polivinílico solúvel em água a 25% (p / v) de glicerol em Tris-HCl 0,1 M (pH 8,5) . Aquecer a 50 ° C sob agitação contínua durante 1 h, arrefecer e ajustar o pH para 8,0-8,5 usando 1M NaOH. Evitar concentrações mais elevadas de NaOH. Adicionar 100 μg / mL de N-propilgalato e timerosal até uma concentração final de 0,02% (p / v). Agitar durante 30 min à temperatura ambiente. CUIDADO: O timerosal é tóxico. Manuseie-o de acordo com os regulamentos locais de segurança e protecção ambiental. <ol><li> Encher o meio de incorporação em tubos de 50 mL, equilibrar o rotor e centrifugar a 3.200 xg e à temperatura ambiente durante 10 min. Decantar a solução límpida em tubos de 15 mL e descartar a pastilha. Alicuotar e armazenar a -20 ° C durante várias semanas. Uma vez descongelado, mantenha a solução a 4 ° C. Aquecer a ~ 30 ° C imediatamente antes da utilização. </li></ol></li><li> Preparar como muitos slides de vidro como filtros com explantes devem ser montados. Para isso, cola dois pequenos lamínulas, 18 x 18 mm, em ambos os lados da lâmina de vidro usando adesivo instantâneo, deixe um espaço de ~ 15 mm para o filtro entre. </li></ol> 4. Fixação e coloração <ol><li> Após um período de cultura de 3Dias in vitro (div), remover as placas da incubadora e aspirar cuidadosamente o meio de cultura de cada poço utilizando uma micropipeta. Certifique-se de não tocar no filtro e manter a abertura da ponta para a parede do poço. </li><li> Adicionar 500 μL de PFA a 4% / PBS a cada poço. Os filtros flutuam no fixador. Usando uma micropipeta, adicione cuidadosamente o fixador PFA gota a gota para submergir os filtros. Incubar à temperatura ambiente durante 30 min. NOTA: Para todas as etapas seguintes, deve-se ter cuidado para não lavar os explantes do filtro. Mantenha a abertura da ponta da pipeta em direção à parede do poço. </li><li> Remover o PFA e enxaguar duas vezes com 500 μL de PBS, submergindo o filtro. Adicionar 500 μL de solução de permeabilização a cada poço e incubar à temperatura ambiente durante 1 h. </li><li> Enxaguar duas vezes com 500 μL de PBS e adicionar 500 μL de solução de bloqueio a cada poço. Incubar à temperatura ambiente durante 1 h. </li><li> Substitua a solução de bloqueio por250 μL de aglutinina biotinilada de Dolichorus biflorus diluída a 1: 200 em solução de bloqueio e incubar a 4 ° C durante 24 h. </li><li> Remover a solução de aglutinina de Dolichorus biflorus e enxaguar duas vezes com 500 μL de PBS por poço. </li><li> Adicionar 250 μL de estreptavidina conjugada com Alexa488 diluída a 1: 200 em solução de bloqueio e incubar a 4 ° C durante 24 h. Alternativamente, incubar à temperatura ambiente durante 2 h. NOTA: Uma melhor relação sinal-ruído é alcançada quando incubada a 4 ° C. </li><li> Enxágüe duas vezes com 500 μL de PBS por poço. Usando pinças, transfira os filtros para as lâminas de vidro preparadas, com os explantes voltados para cima. Monte com ~ 50-100 μL de meio de incorporação. Cubra com os lamínulas No. 1.5 de 60 mm de comprimento. Permitir que a solução de meio de incorporação solidifique durante 2 h à temperatura ambiente no escuro. Proceda à imagem ou guarde os slides, envoltos em papel alumínio, a 4 ° C até à imagem. </li><li> Imagem com um microscópio de epifluorescência de campo largo 7 &lt;/ Sup&gt; acoplado a uma lâmpada de Hg e utilizar uma unidade de espelho para excitação azul (onda de excitação, 460-495 nm, espelho dicromático, 505 nm, e passagem de emissão, 510-550 nm) e lentes 20X Plan-Apochromat, 0,75 numérico abertura. </li></ol>

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Os autores não têm nada a revelar.

Este manuscrito descreve um método para isolar o desenvolvimento de metanéfric anlagen do embrião de rato e para cultivar os rudimentos de órgãos. Este método é uma técnica padrão, como desenvolvido por Grobstein 8 e Saxen 9 , 10 , e foi adaptado e modificado por muitos outros 11 , 12 . O sucesso do método depende principalmente da duração da dissecção, como sobreviv...

The mammalian kidney is derived from two primordial structures with mesodermal origin: the tubular epithelial ureteric bud and the metanephric mesenchyme. During nephrogenesis, the ureteric bud invades the metanephric mesenchyme and branches to form the collecting system. The metanephric mesenchyme gives rise to the epithelial elements of the nephrons. These processes occur in a precisely timed and spatially coordinated manner and are initiated by reciprocal inductive mechanisms. Both tissue components communicate and affect the other's cell morphogenesis. 
In the 1920s, it was Boyden who performed the in vivo obstruction of the mesonephric duct in chicken, providing the first indication of inductive interactions as separated nephric blastema fail to differentiate1. At about the same time, the first successful attempts to culture chicken nephric rudiments in a hanging drop were published. Subsequently, the organ culture was developed to study tissue interactions in mammalian organogenesis. In the 1950s, Grobstein developed a technique in which metanephric rudiments could be cultured on a filter. This technique was modified by Sax&#233;n, who placed the filter on a Trowell-type screen in a culture dish1. Over the years, many modifications and applications for organ culture have emerged. The method described here is based on Sax&#233;n's technique but is simplified, as the filters float free on the medium and the diameter of the culture well only slightly exceeds the diameter of the filter, limiting unwanted movement of the filter.
Whole-organ culture is a classical, cheap, and simple but powerful tool to investigate cellular processes and intercellular communication during organogenesis. Organ culture allows for treatment with biological agents, such as growth factors, antibodies, antisense oligonucleotides, viruses, and peptides, as well as with pharmaceutical compounds and other chemicals. Also, gene function may be studied using explants derived from genetically modified mice or using inducible gene inactivation technology, such as the Cre-loxP system. This allows for the study of genetic mutations that cause embryonic lethality prior to the development of the kidney. Organ culture can also be combined with fluorescent tagging for gene function or lineage tracing and modern imaging techniques, which enable real-time monitoring of cell behavior2.
In the specific example provided here, the effect of EphrinB2-activated Eph-receptor signaling on the branching morphology of the ureteric bud was investigated. The morphology of the EphA4/EphB2 double-knockout mice suggested several severe defects in kidney development, which were detectable as early as embryonic day 11 (E11) and involved the ureteric bud, the ureter, and the common nephric duct3. Signaling via Eph receptors requires the clustering of the ligand-receptor dimer4. To over-activate Eph signaling, the kidney rudiments from E11.5 mouse embryos were cultured in the presence of clustered recombinant EphrinB2-Fc. EphrinB2 is a known ligand for the EphA4 receptor, which is expressed in the ureteric bud tips3.

<table><tbody><tr><td>DMEM/F-12</td><td>Thermo Fisher Scientific</td><td>21331020</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Thermo Fisher Scientific</td><td>15140148</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>Thermo Fisher Scientific</td><td>35050061</td><td></td></tr><tr><td>DPBS, calcium, magnesium</td><td>Thermo Fisher Scientific</td><td>14040117</td><td>use for dissection</td></tr><tr><td>holo-Transferrin human</td><td>Sigma-Aldrich</td><td>T0665</td><td></td></tr><tr><td>Insulin-Transferrin-Selenium (ITS -G) (100x)</td><td>Thermo Fisher Scientific</td><td>41400045</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Amphotericin B solution</td><td>Sigma-Aldrich</td><td>A2942</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S8032</td><td></td></tr><tr><td>Thimerosal</td><td>Sigma-Aldrich</td><td>T5125</td><td></td></tr><tr><td>Propyl gallate</td><td>Sigma-Aldrich</td><td>2370</td><td></td></tr><tr><td>Mowiol 4-88</td><td>Sigma-Aldrich</td><td>81381</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Biotinylated Dolichorus Biflorus Agglutinin</td><td>Vector Laboratories</td><td>B-1035</td><td></td></tr><tr><td>Alexa488 conjugated Streptavidin</td><td>Jackson Immuno Research</td><td>016-540-084</td><td></td></tr><tr><td>Recombinant Mouse Ephrin-B2 Fc Chimera Protein, CF</td><td>R&amp;amp;D Systems</td><td>496-EB</td><td></td></tr><tr><td>Recombinant Human IgG1 Fc, CF</td><td>R&amp;amp;D Systems</td><td>110-HG-100</td><td></td></tr><tr><td>Goat Anti-Human IgG Fc Antibody</td><td>R&amp;amp;D Systems</td><td>G-102-C</td><td></td></tr><tr><td>Phosphate buffered saline tablets</td><td>Sigma-Aldrich</td><td>P4417</td><td>use for fixation and immunostaining</td></tr><tr><td>Dumont #5, biologie&lt;br/&gt; tips, INOX, 11 cm</td><td>agnthos.se</td><td>0208-5-PS</td><td>2 pairs of forceps are needed</td></tr><tr><td>Iris scissors, straight, 12 cm</td><td>agnthos.se</td><td>03-320-120</td><td></td></tr><tr><td>Dressing Forceps,&lt;br/&gt; straight, delicate, 13 cm</td><td>agnthos.se</td><td>08-032-130</td><td></td></tr><tr><td>Petri dishes Nunclo Delta treated</td><td>Thermo Fisher Scientific</td><td>150679</td><td></td></tr><tr><td>TMTP01300 Isopore Membrane Filter, polycarbonate, Hydrophilic, 5.0 &amp;micro;m, 13 mm, white, plain</td><td>MerckMillipore</td><td>TMTP01300</td><td></td></tr><tr><td>Nunclon Multidishes&lt;br/&gt; 4 wells, flat bottom</td><td>Sigma-Aldrich</td><td>D6789-1CS</td><td></td></tr><tr><td>Microscope cover glass 24 x 50 mm thickn. No.1.5H 0.17+/-0.005 mm</td><td>nordicbiolabs</td><td>107222</td><td></td></tr><tr><td>Cover glasses No.1.5, 18 mm x 18 mm</td><td>nordicbiolabs</td><td>102032</td><td></td></tr><tr><td>Slides ~76 x 26 x 1, 1/2-w. ground plain</td><td>nordicbiolabs</td><td>1030418</td><td></td></tr><tr><td>VWR Razor Blades</td><td>VWR</td><td>55411-055</td><td></td></tr><tr><td>50 mL centrifuge tubes</td><td>Sigma-Aldrich</td><td>CLS430828</td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td>Sigma-Aldrich</td><td>CLS430055</td><td></td></tr><tr><td>Whatman prepleated qualitative filter paper, Grade 113V, creped</td><td>Sigma-Aldrich</td><td>WHA1213125</td><td></td></tr><tr><td>Fixed stage research mircoscope</td><td>Olympus</td><td>BX61WI</td><td></td></tr><tr><td>Black 6 inbred mice, male, C57BL/6NTac</td><td>Taconic</td><td>B6-M</td><td></td></tr><tr><td>Black 6 inbred mice,female, C57BL/6NTac</td><td>Taconic</td><td>B6-F</td><td></td></tr><tr><td>Greenough Stereo Microscope</td><td>Leica</td><td>Leica S6 E</td><td></td></tr></tbody></table>

dissection and culture of mouse embryonic kidney

O objetivo deste protocolo é descrever um método para a dissecção, isolamento e cultura de rastreios metanefric do rato. Durante o desenvolvimento do rim mamífero, os dois tecidos progenitorais, o broto ureterico e o mesênquima metanéfrico, comunicam-se e induzem reciprocamente mecanismos celulares para formar eventualmente o sistema colector e os nefrões do rim. À medida que os embriões de mamíferos crescem intra-uterinos e, portanto, são inacessíveis ao observador, desenvolveu-se uma cultura de órgãos. Com este método, é possível estudar interações epitélio-mesenquimais e comportamento celular durante a organogênese do rim. Além disso, a origem das malformações congênitas do rim e do trato urogenital pode ser investigada. Após dissecção cuidadosa, os rudimentos metanéfricos são transferidos para um filtro que flutua no meio de cultura e podem ser mantidos numa incubadora de cultura de células durante vários dias. Contudo, deve ter-se em conta que as condiçõesArtificial e poderia influenciar o metabolismo no tecido. Além disso, a penetração das substâncias de teste poderia ser limitada devido à matriz extracelular e à membrana basal presentes no explante. Uma vantagem principal da cultura do órgão é que o experimentador pode ganhar o acesso direto ao órgão. Esta tecnologia é barata, simples e permite um grande número de modificações, como a adição de substâncias biologicamente ativas, o estudo de variantes genéticas e a aplicação de técnicas de imagem avançadas.

O anlagenio metanï¿½rico de rim foi derivado de murganhos consanguï¿½eos Black-6 grï¿½idos a E11,5 e foram cultivados. Após 3 dias, o botão ureteriano ramificou-se até 5 vezes, resultando em uma ramificação do botão ureter inicialmente em forma de T. Cada explante foi fotografado e os números de segmentos e pontos finais foram quantificados para determinar as gerações de ramificação e calcular o número de pontos finais por ramo ( Figura 1 ). ( <stron...

Watch this Scientific Journal Video about Dissection and Culture of Mouse Embryonic Kidney at JoVE.com

Dissection and Culture of Mouse Embryonic Kidney

Este protocolo descreve um método para isolar e cultivar rudimentos metanefricos de embriões de ratinho.

Dissecção e cultura do rim embrionário do rato

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

dissection-and-culture-of-mouse-embryonic-kidney

Research

JoVE Journal

Developmental Biology

11.9K visualizações.  Uppsala University.  Este protocolo descreve um método para isolar e cultivar rudimentos metanefricos de embriões de ratinho. 

Vídeo: Dissection and Culture of Mouse Embryonic Kidney

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.
Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.

A method allowing for direct pharmacological manipulation of mouse embryos during neurulation that bypasses maternal metabolism is described. The technique can be adapted to study different aspects of neurulation by varying the time point and pharmacological agent.

Fechamento do tubo neural no embrião do rato Cultura Whole

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray &amp; Ross, 2009; Ross, 2010).  However, the study of mouse ...

Neurociência

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

We present a method for isolation and culture of the mouse Wolffian duct (WD). We also demonstrate a detailed procedure for whole mount immunostaining of cultured/freshly isolated WDs with fluorescently tagged antibodies. Together, these techniques enable the study of WD development, coiling, and differentiation.

Órgão Cultura e Whole Mount Imunofluorescência Coloração de rato Wolff Dutos

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an ...

Biologia do Desenvolvimento

Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy Imaging

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental processes and modelling kidney disease. However, the models are limited by a lack of vascularization and functionality. To address this, an improved protocol for the method of xenografting cells and tissues to the chorioallantoic membrane (CAM) of an avian embryo to gain vascularization and restoration of blood flow was developed. The grafts are overlaid with custom-made minireservoirs that fix the samples to the CAM and supply them with culture medium that protects the grafts from drying. The improved culture method allows xenografts to grow for up to 9 days. The manuscript also describes how to provide optimal conditions for long-term confocal imaging of renal organoids and organotypic cultures using the previously published Fixed Z-Direction (FiZD) method. This method gently compresses an embryonic organ or organoid between a glass coverslip and membrane in a large amount of medium and provides excellent conditions for imaging for up to 12 days. Together, these methods allow vascularization and blood flow to renal organoids and organotypic kidney cultures with improved confocal imaging. The methods described here are highly beneficial for studying fundamental and applied functions of kidneys ex vivo. Both methods are applicable to various types of tissues and organoids.

This work describes two methods for studying organ development, an improved xenotransplantation setup on chorioallantoic membrane (CAM) from avian embryos that allows for vascularization of cultured embryonic organs and organoids and a novel fixed z-direction organ culture method with modified experimental conditions that allows for high-resolution time-lapse confocal imaging.

Otimização da Cultura Organóide Renal e Organotípica para Vascularização, Desenvolvimento Estendido e Imagem Microscopia Aprimorada

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental ...

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

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

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

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

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

Biologia

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only one-fourth of these patients achieving transplantation, there is a dire need for alternatives for those with failing organs. In order to decrease the harmful consequences of dialysis along with the overall healthcare costs it incurs, active investigation is ongoing in search of alternative solutions to organ transplantation. Implantable tissue-engineered renal cellular constructs are one such feasible approach to replacing lost renal functionality. Here, described for the first time, is the microdissection of murine kidneys for isolation of living corticomedullary renal segments. These segments are capable of rapid incorporation within scaffold-free endothelial-fibroblast constructs which may enable rapid connection with host vasculature once implanted. Adult mouse kidneys were procured from living donors, followed by stereoscope microdissection to obtain renal segments 200 - 300 &#181;m in diameter. Multiple renal constructs were fabricated using primary renal segments harvested from only one kidney. This method demonstrates a procedure which could salvage functional renal tissue from organs that would otherwise be discarded.

Tissue-engineered renal constructs provide a solution for the organ shortage and deleterious effects of dialysis. Here, we describe a protocol to micro dissect murine kidneys for isolation of cortico-medullary segments. These segments are implanted into scaffold-free cellular constructs, forming renal organoids.

Microdissection de segmentos de tecido Renal primária e incorporação com tecnologia de construção romance livre de andaime

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

Bioengenharia

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Isolamento e cultura de células da zona nefrogênica do rim do rato embrionárias

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

Isolamento e cultura de células-tronco neurais embrionárias de rato

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

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

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

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

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

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

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of pathways involved in parietal epithelial cell activation could therefore be a tool to attenuate the progression of glomerular diseases. This article describes a method to culture and analyze parietal epithelial cell outgrowth of encapsulated glomeruli isolated from mouse kidney. After dissecting isolated mouse kidneys, the tissue is minced, and glomeruli are isolated by sieving. Encapsulated glomeruli are collected, and single glomeruli are cultured for 6 days to obtain glomerular outgrowth of parietal epithelial cells. During this period, parietal epithelial cell proliferation and migration can be analyzed by determining the cell number or the surface area of outgrowing cells. This assay can therefore be used as a tool to study the effects of an altered gene expression in transgenic- or knockout-mice or the effects of culture conditions on parietal epithelial cell growth characteristics and signaling. Using this method, important pathways involved in the process of parietal epithelial cell activation and consequently in glomerulosclerosis can be studied.

This article describes a method for culturing and analyzing glomerular parietal epithelial cell outgrowths of encapsulated glomeruli isolated from mouse kidney. This method can be used to study pathways involved in parietal epithelial cell proliferation and migration.

Crescimento glomerular como um ensaio ex vivo para analisar caminhos envolvidos na ativação de células epiteliais parietal

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Dissecção eficiente e cultura de células epiteliais de pigmento de retina do rato primário

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...