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

Les auteurs remercient Leif Oxburgh et Derek Adams de partager généreusement leurs connaissances, Leif Oxburgh pour les commentaires utiles sur le manuscrit, et Stefan Wölfl et Ulrike Müller pour leur soutien technique et Saskia Schmitteckert, Julia Gobbert, Sascha Weyer et Viola Mayer pour l&#39;aide dans le laboratoire. Ce travail a été soutenu par Development, The Company of Biologists (to CP).

Les souris ont été maintenues selon la réglementation suédoise et la législation de l&#39;Union européenne (2010/63 / UE). Toutes les procédures ont été effectuées selon les directives du Comité suédois d&#39;éthique (permis C79 / 9, C248 / 11 et C135 / 14). Les procédures à l&#39;Université de Heidelberg portant sur des matières animales ont été approuvées par le Regierungspräsidium Karlsruhe et les agents de protection des animaux à l&#39;Université de Heidelberg. 1. Préparation des réactifs et des matériaux pour la culture REMARQUE: Utilisez un capot à flux laminaire pour minimiser la contamination. <ol><li> Le jour de la dissection, la protéine d&#39;éphrine chimère recombinante Fc seule ou recombinante est fusionnée à Fc humaine en la mélangeant avec des anticorps contre la chèvre Fc anti-humains à un rapport molaire de 1: 5 dans une solution saline stérile tamponnée au phosphate (PBS). Incuber pendant 1 h à 37 ° C 5 . </li><li> Préparer le milieu de culture en complétant l&#39;Eagle Eagle modifié par Dulbecco: NutrientMélange F-12 (DMEM / F12) avec 1% de pénicilline / streptomycine (v / v), 1% de glutamine (v / v), 1% d&#39;insuline-transferrine-sélénium (v / v), 50 μg / mL d&#39;holo- La transferrine et 1,5 μg / ml d&#39;amphotéricine. REMARQUE: 10 ml de milieu sont suffisants pour 16 embryons. </li><li> Ajouter une solution d&#39;éphrine recombinante en grappe à une concentration finale de 20 μg / ml sur le milieu de culture. Utilisez le Fc humain en cluster comme contrôle. </li><li> Préparer des plaques de culture en ajoutant 500 μl de milieu de culture à chaque puits d&#39;une plaque stérile, non revêtue, à base de polystyrène à fond plat et à 4 puits. </li><li> En utilisant des pinces stériles, placez soigneusement un filtre à membrane en polycarbonate avec une taille de pore de 5 μm par puits sur le dessus du milieu. Transférer la plaque préparée à l&#39;incubateur (37 ° C / 5% de CO 2 ). Assurez-vous que le filtre reste sec sur la surface supérieure et les flotteurs. REMARQUE: Un filtre suffit pour deux anilènes rénaux. </li><li> En utilisant une lame de rasoir, couper environ 30 pointes de pipette (volume: 100 μL) approximatelY 1 mm de la pointe pour aboutir à une ouverture plus large. Placez les embouts dans une crémaillère à pipette. </li></ol> 2. Dissection de Rudiments Metanephriques à E11.5 <ol><li> Sacrifiez une souris enceinte chronométrée à E11.5 par dislocation cervicale (ou utilisez une méthode approuvée par le comité local d&#39;éthique). </li><li> Supprimer l&#39;utérus et les embryons, comme décrit par Brown et al 6 . Sur ce point, travailler sous un microscope à dissection. </li><li> En utilisant la pince à horlogerie n ° 5, couper l&#39;embryon directement sous les pattes antérieures. Utilisez un plat de Petri frais pour chaque embryon. Retirer les jambes et la queue de l&#39;embryon. Pour ce faire, prenez le tissu à enlever avec une paire de pince à horlogerie n ° 5 et utilisez les pointes d&#39;une deuxième paire pour couper. </li><li> Pour enlever la masse d&#39;organe ventrale, utilisez les pointes d&#39;une pince pour ouvrir latéralement la paroi du corps de l&#39;embryon dans une direction rostrale à caudale. <ol><li> Couper superficiellement, parallèlement à tL&#39;aorte dorsale. Utilisez l&#39;aorte dorsale remplie de sang et donc visible pour l&#39;orientation. Séparez soigneusement la partie ventrale de la paroi du corps de la partie dorsale en utilisant les pointes de la pince fermée et retournez l&#39;embryon. Coupez la paroi du corps latéralement dans une direction rostral à caudale et utilisez l&#39;aorte dorsale pour l&#39;orientation, comme pour le côté précédent. </li></ol></li><li> Une fois que la paroi du ventral a été séparée du corps dorsal, saisissez-la avec une paire de pince et tirez soigneusement pour l&#39;enlever, avec la masse de l&#39;organe. REMARQUE: l&#39;anilène métanéphrique reste habituellement attaché à la paroi du corps dorsal. Ce sont des structures ovales, de 200 μm de long situées au niveau des membres postérieurs. </li><li> Tenez la paroi du corps dorsal avec une paire de pince, avec le côté ventral vers le haut. À l&#39;aide de l&#39;autre pince, saisissez l&#39;aorte dorsale à son extrémité rostrale et épluchez soigneusement l&#39;aorte dorsale de la paroi du corps dorsal. NOTE: Les deux reins métasphériques restent habituellement attachésÀ l&#39;aorte dorsale. </li><li> À l&#39;aide d&#39;une paire de pinces, coupez vers le riz anlagen pour éliminer l&#39;aorte. Ensuite, séparez soigneusement les deux anols du rein en utilisant les pointes de la pince. </li><li> Éloignez soigneusement les tissus indésirables du rein anlagen en utilisant les pointes de la pince. Veillez à ne pas endommager le mésenchyme, car les dommages peuvent entraîner une mauvaise croissance et l&#39;exclusion de l&#39;explant à partir d&#39;analyses ultérieures. </li><li> Transférer le rein anlagen séparément à l&#39;aide d&#39;une pipette à microlitres et d&#39;une pointe de pipette large-ouverte dans 10 μL de PBS au filtre à membrane en polycarbonate dans une plaque à 4 puits. REMARQUE: En raison de la tension de surface, l&#39;explant sera recouvert d&#39;une mince couche de milieu. Un filtre suffit pour deux anilènes rénaux. <ol><li> Placez chacun des deux anilènes rénaux au centre de chaque moitié respective du filtre. Transférer la plaque à l&#39;incubateur à 37 ° C / 5% de CO 2 . Laisser la plaque dans l&#39;incubateur jusqu&#39;à ce que la rud metanephricLes imites du prochain embryon sont prêtes à être placées sur le filtre. </li></ol></li><li> Répétez les étapes 2.3-2.9.1 pour chaque embryon. REMARQUE: avec une certaine pratique, la procédure de dissection prend environ 5 min par embryon. </li><li> Incuber le rein anlagen pendant 3 jours à 37 ° C / 5% de CO 2 . </li></ol> 3. Préparation des réactifs pour la fixation et la coloration <ol><li> Pour préparer 4% de paraformaldéhyde (PFA) dans du PBS sans calcium et magnésium (p / v), dissoudre le PFA à 60 ° C dans du PBS, en agitant continuellement. Refroidissez à température ambiante et utilisez un filtre en papier pour enlever les particules restantes. Aliquote et conservez à 4 ° C pour une utilisation immédiate ou un gel pour un stockage à long terme. ATTENTION: le PFA est toxique. Portez l&#39;équipement de protection individuelle spécifié dans les normes de sécurité locales et travaillez dans une hotte aspirante. Jeter les déchets en suivant les directives institutionnelles et en utilisant les conteneurs de déchets désignés. </li><li> Pour préparer une solution de perméabilisation, diluer Triton X-100Dans du PBS sans calcium et magnésium à 0,3% (v / v). </li><li> Pour préparer une solution de blocage, ajouter 5% (v / v) de sérum de chèvre et 0,1% de Triton X-100 à PBS sans calcium et magnésium. Ajouter de l&#39;azoture de sodium à une concentration finale de 0,02% (p / v). Conserver à 4 ° C. ATTENTION: L&#39;azide de sodium est toxique. Manipuler l&#39;azide de sodium conformément à la réglementation locale sur la sécurité et la protection de l&#39;environnement. NOTE: Lorsque vous utilisez des anticorps pour la coloration, utilisez un sérum de 5% parmi les espèces dont l&#39;anticorps secondaire a été dérivé pour obtenir une solution de blocage. </li><li> Diluer l&#39;agglutinine biotinylée de Dolichorus biflorus 1: 200 dans la solution bloquant. Diluer la streptavidine conjuguée Alexa488 1: 200 dans la solution de blocage. </li><li> Pour préparer un milieu d&#39;incorporation prêt à l&#39;emploi (voir la table des matériaux), ajouter 0,1 g de mucoadhésif à base d&#39;alcool polyvinylique à l&#39;eau / ml à 25% (p / v) de glycerol dans du Tris-HCl 0,1 M (pH 8,5) . Chauffer à 50 ° C sous agitation continue pendant 1 h, refroidir et régler le pH à 8,0-8,5 en utilisant 1M NaOH. Évitez les concentrations de NaOH plus élevées. Ajouter 100 μg / mL de N-propylgallate et thimerosal à une concentration finale de 0,02% (p / v). Mélanger pendant 30 minutes à température ambiante. ATTENTION: Le thimerosal est toxique. Manipulez-le conformément à la réglementation locale sur la sécurité et la protection de l&#39;environnement. <ol><li> Remplissez le milieu d&#39;incorporation dans des tubes de 50 ml, équilibrez le rotor et centrifugez à 3,200 xg et la température ambiante pendant 10 min. Décrivez la solution transparente dans des tubes de 15 mL et jeter la pastille. Aliquotez et conservez à -20 ° C pendant plusieurs semaines. Une fois décongelée, maintenir la solution à 4 ° C. Chaud à ~ 30 ° C immédiatement avant utilisation. </li></ol></li><li> Préparez autant de diapositives en verre que les filtres avec des explants doivent être montés. Pour cela, collez deux petits lamelles, 18 x 18 mm, de chaque côté de la glissière en verre à l&#39;aide d&#39;un adhésif instantané, laissez un espace de ~ 15 mm pour le filtre entre les deux. </li></ol> 4. Fixation et coloration <ol><li> Après une période de culture de 3Jours in vitro (div), retirer les plaques de l&#39;incubateur et aspirer soigneusement le milieu de culture de chaque puits à l&#39;aide d&#39;une micropipette. Assurez-vous de ne pas toucher le filtre et de garder la pointe en direction de la paroi du puits. </li><li> Ajouter 500 μL de PFA à 4% / PBS à chaque puits. Les filtres flottent sur le fixateur. À l&#39;aide d&#39;une micropipette, ajoutez soigneusement le fixateur PFA pour immerger les filtres. Incuber à température ambiante pendant 30 min. REMARQUE: Pour toutes les étapes suivantes, il faut prendre soin de ne pas laver les explants du filtre. Gardez l&#39;ouverture de la pointe de la pipette vers le mur du puits. </li><li> Retirez le PFA et rincez deux fois avec 500 μL de PBS, en immergeant le filtre. Ajouter 500 μL de solution de perméabilisation à chaque puits et incuber à température ambiante pendant 1 h. </li><li> Rincer deux fois avec 500 μL de PBS et ajouter 500 μl de solution bloquant à chaque puits. Incuber à température ambiante pendant 1 heure. </li><li> Remplacez la solution de blocage par250 μL d&#39;agglutinine biotinylée de Dolichorus biflorus diluée à 1: 200 dans une solution bloquant et incubée à 4 ° C pendant 24 h. </li><li> Enlever la solution d&#39;agglutinine Dolichorus biflorus et rincer deux fois avec 500 μL de PBS par puits. </li><li> Ajouter 250 μL de streptavidine conjuguée avec Alexa488 dilué 1: 200 dans la solution de blocage et incuber à 4 ° C pendant 24 h. Alternativement, incuber à température ambiante pendant 2 h. REMARQUE: Un meilleur rapport signal / bruit est obtenu lorsque l&#39;on incube à 4 ° C. </li><li> Rincer deux fois avec 500 μL de PBS par puits. En utilisant une pince, transférez les filtres sur les lames de verre préparées, avec les explants vers le haut. Monter avec ~ 50-100 μL de support d&#39;encastrement. Couvrir avec des lamelles de 60 mm de long n ° 1.5. Laisser la solution du milieu d&#39;incorporation se solidifier pendant 2 h à température ambiante dans l&#39;obscurité. Procédez à l&#39;imagerie ou rangez les diapositives, emballées dans du papier d&#39;aluminium, à 4 ° C jusqu&#39;à ce qu&#39;elles soient prêtes à l&#39;image. </li><li> Image avec un microscope épifluorescent large champ 7 &lt;/ Sup&gt; couplé à une lampe Hg et utiliser une unité miroir pour l&#39;excitation bleue (passe-bande d&#39;excitation, 460 - 495 nm, miroir dichromatique, 505 nm et bande passante d&#39;émission, 510 à 550 nm) et 20X lentilles Plan-Apochromat, 0,75 numériques ouverture. </li></ol>

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Les auteurs n&#39;ont rien à dévoiler.

Ce manuscrit décrit une méthode pour isoler l&#39;anlagen méthanephrique en développement de l&#39;embryon de souris et pour cultiver les rudiments d&#39;organes. Cette méthode est une technique standard, développée par Grobstein 8 et Saxén 9 , 10 , et a été adaptée et modifiée par beaucoup d&#39;autres 11 , 12 . Le succès de la méthode dépend principalement de la d...

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

Le but de ce protocole est de décrire une méthode de dissection, d&#39;isolement et de culture des rudiments métanéphriques de souris. Au cours du développement du rein des mammifères, les deux tissus progéniteurs, le bourgeon urétéral et le mésenchyme métasphorique, communiquent et induisent réciproquement des mécanismes cellulaires pour former éventuellement le système collecteur et les néphrons du rein. Au fur et à mesure que les embryons de mammifères se développent par voie intra-utérine et qui sont inaccessibles à l&#39;observateur, une culture d&#39;organe a été développée. Avec cette méthode, il est possible d&#39;étudier les interactions épithéliales-mésenchymateuses et le comportement cellulaire pendant l&#39;organogenèse rénale. En outre, l&#39;origine des malformations rénales congénitales et des voies urogénitales peut être étudiée. Après une dissection minutieuse, les rudiments métanéphriques sont transférés sur un filtre qui flotte sur un milieu de culture et peuvent être conservés dans un incubateur de culture cellulaire pendant plusieurs jours. Cependant, il faut savoir que les conditions sontArtificielle et pourrait influencer le métabolisme dans le tissu. En outre, la pénétration des substances d&#39;essai pourrait être limitée en raison de la matrice extracellulaire et de la membrane basale présents dans l&#39;explant. Un des principaux avantages de la culture des organes est que l&#39;expérimentateur peut avoir un accès direct à l&#39;organe. Cette technologie est peu coûteuse, simple et permet un grand nombre de modifications, telles que l&#39;ajout de substances biologiquement actives, l&#39;étude des variantes génétiques et l&#39;application de techniques avancées d&#39;imagerie.

L&#39;anlagen rénale métasphorique a été dérivé de souris enceintes enceintes Black-6 chez E11.5 et ont été cultivées. Après 3 jours, le bourgeon urétéral s&#39;est ramifié jusqu&#39;à 5 fois, entraînant une ramification du bourrelet urétéral initialement en forme de T. Chaque explant a été photographié et les nombres de segments et de points d&#39;extrémité ont été quantifiés pour déterminer les générations de ramifications et pour calculer le nombre de point...

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

Dissection and Culture of Mouse Embryonic Kidney

Ce protocole décrit une méthode pour isoler et cultiver des rudiments métasphoriques à partir d&#39;embryons de souris.

Dissection et culture du rein embryonnaire de la souris

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 vues.  Uppsala University.  Ce protocole décrit une méthode pour isoler et cultiver des rudiments métasphoriques à partir d'embryons de souris. 

Vidéo: 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.

Fermeture du tube neural dans la culture Mouse Embryo entier

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 ...

Recherche

Neurosciences

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.

Organ Culture et Mont entiers immunofluorescence de souris Conduits Wolffian

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

Biologie du développement

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.

Optimisation de la culture rénale organoïde et organotypique pour la vascularisation, le développement prolongé et l’imagerie améliorée de microscopie

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.

Culture poumon embryonnaire de la souris, un système pour évaluer les mécanismes moléculaires des Branching

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

Biologie

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 tissu rénal primaire Segments et intégration avec la technologie de nouvelle construction exempte d’échafaudage

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

Bio-ingénierie

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.

Isolement et culture de cellules de la zone néphrogénique du rein d&#39;embryon de souris

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.

Isolement et Culture des embryons de souris des cellules souches neurales

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.

Souris développement embryonnaire dans tout un système de culture d&#39;embryons sans sérum

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.

Excroissance glomerulaire en tant qu’essai Ex Vivo pour analyser les voies impliquées dans l’activation des cellules épithéliales pariétales

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.

Dissection efficace et culture des cellules épithéliales rétiniennes primaires de pigment de souris

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

Dissection et culture du rein embryonnaire de la souris

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