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

Die Autoren danken Leif Oxburgh und Derek Adams für ihre Kenntnisse, Leif Oxburgh für die hilfreichen Kommentare zum Manuskript und Stefan Wölfl und Ulrike Müller für ihre technische Unterstützung und Saskia Schmitteckert, Julia Gobbert, Sascha Weyer und Viola Mayer für die Hilfe in der Labor. Diese Arbeit wurde von der Entwicklung, der Gesellschaft der Biologen (nach CP) unterstützt .

Die Mäuse wurden nach den schwedischen Vorschriften und den Rechtsvorschriften der Europäischen Union (2010/63 / EU) aufrechterhalten. Alle Verfahren wurden nach den Richtlinien des schwedischen Ethikausschusses durchgeführt (Genehmigungen C79 / 9, C248 / 11 und C135 / 14). Die Verfahren an der Universität Heidelberg mit Tierfächern wurden vom Regierungspräsidium Karlsruhe und den Tierschutzbeauftragten an der Universität Heidelberg genehmigt. 1. Vorbereitung von Reagenzien und Materialien für Kultur HINWEIS: Verwenden Sie eine laminare Strömungshaube, um die Kontamination zu minimieren. <ol><li> Am Tag der Sezierung wurde das menschliche Fc allein oder rekombinantes chimäres Ephrinprotein, das mit menschlichem Fc fusioniert ist, durch Mischen mit anti-humanen Fc-Ziegen-Antikörpern in einem Molverhältnis von 1: 5 in steriler phosphatgepufferter Kochsalzlösung (PBS) hergestellt. 1 Stunde bei 37 ° C inkubieren 5 . </li><li> Bereiten Sie das Kulturmedium vor, indem Sie Dulbecco&#39;s Modified Eagle Medium: Nutrient ergänzenMischung F-12 (DMEM / F12) mit 1% Penicillin / Streptomycin (v / v), 1% Glutamin (v / v), 1% Insulin-Transferrin-Selen (v / v), 50 μg / ml menschliches Holo- Transferrin und 1,5 μg / ml Amphotericin. HINWEIS: 10 ml Medium reicht für 16 Embryonen aus. </li><li> Fügen Sie gruppierte rekombinante Ephrinlösung in einer Endkonzentration von 20 μg / ml zum Kulturmedium hinzu. Verwenden Sie Clustered Human Fc als Kontrolle. </li><li> Bereiten Sie Kulturplatten vor, indem Sie 500 &amp; mgr; l Kulturmedium zu jeder Vertiefung einer sterilen, unbeschichteten, flachen Boden-Polystyrol-4-Well-Platte hinzufügen. </li><li> Mit sterilen Pinzetten sorgfältig einen Polycarbonat-Membranfilter mit einer Porengröße von 5 μm pro Well auf das Medium legen. Übertragen Sie die vorbereitete Platte in den Inkubator (37 ° C / 5% CO 2 ). Stellen Sie sicher, dass der Filter auf der Oberseite trocken bleibt und schwimmt. HINWEIS: Ein Filter reicht für zwei Nierenanlagen aus. </li><li> Mit einer Rasierklinge schneiden Sie ca. 30 Pipettenspitzen (Volumen: 100 μl) approximatelY 1 mm von der Spitze, um zu einer breiteren Öffnung zu führen. Legen Sie die Spitzen wieder in ein Pipetten-Tip-Rack. </li></ol> 2. Dissektion von metanephrischen Rudimente bei E11.5 <ol><li> Opfer eine zeitlich-schwangere Maus bei E11.5 durch zervikale Versetzung (oder mit einer Methode, die von der lokalen Ethik-Ausschuss genehmigt). </li><li> Entfernen Sie den Uterus und die Embryonen, wie von Brown et al. 6 beschrieben. Von diesem Punkt aus, arbeiten Sie unter einem Sektionsmikroskop. </li><li> Mit der Nr. 5 Uhrmacherzange, schneide den Embryo direkt unter die Vorderbeine. Verwenden Sie für jeden Embryo eine frische Petrischale. Entfernen Sie die Beine und den Schwanz des Embryos. Um dies zu tun, greifen Sie das Gewebe mit einem Paar von Nr. 5 Uhrmacher Pinzette entfernt werden und verwenden Sie die Spitzen eines zweiten Paares zu schneiden. </li><li> Um die ventrale Organmasse zu entfernen, benutze die Spitzen einer Pinzette, um die Körperwand des Embryos in einer rostral-to-caudalen Richtung zu öffnen. <ol><li> Schnitt oberflächlich, parallel zu tEr dorsale aorta Verwenden Sie die Blut-gefüllte und daher sichtbare dorsale Aorta zur Orientierung. Sorgfältig trennen Sie den ventralen Teil der Körperwand von der dorsalen Teil mit den Spitzen der geschlossenen Pinzette und drehen Sie den Embryo über. Schneiden Sie die Körperwand seitlich in einer rostralen bis kaudalen Richtung und verwenden Sie die dorsale Aorta zur Orientierung, wie für die vorherige Seite. </li></ol></li><li> Sobald die ventrale Körperwand vom dorsalen Körper getrennt ist, packt sie mit einer Pinzette und zieht sie sorgfältig zusammen, um sie zusammen mit der Orgelmasse zu entfernen. HINWEIS: Die metanephrischen Anlagen bleiben gewöhnlich an der dorsalen Körperwand befestigt. Sie sind ovale, 200 μm lange Strukturen, die sich auf der Höhe der Hinterbeine befinden. </li><li> Halten Sie die dorsale Körperwand mit einer Pinzette, mit der ventralen Seite nach oben. Mit dem anderen Paar Pinzette, greifen die dorsale Aorta an seinem rostralen Ende und sorgfältig schälen die dorsale Aorta aus der dorsalen Körperwand. ANMERKUNG: Beide metanephrischen Nieren bleiben in der Regel angebrachtAuf die dorsale aorta </li><li> Mit einem Paar Pinzette, schneiden rostral auf die Niere anlagen, um die Aorta zu entfernen. Dann trennen Sie sorgfältig die beiden Niere anlagen mit den Spitzen der Pinzette. </li><li> Vorsichtig vor unerwünschtes Gewebe aus der Niere anlagen mit den Spitzen der Pinzette abziehen. Achten Sie darauf, das Mesenchym nicht zu beschädigen, da Schäden zu einem schlechten Wachstum und Ausschluss des Explantats aus einer weiteren Analyse führen können. </li><li> Übertragen Sie die Niere anlagen separat mit einer Mikroliterpipette und einer weit geöffneten Pipettenspitze in 10 μl PBS zum Polycarbonat-Membranfilter in einer 4-Well-Platte. HINWEIS: Durch die Oberflächenspannung wird das Explantat mit einer dünnen Schicht von Medium bedeckt. Ein Filter reicht für zwei Nierenanlagen aus. <ol><li> Legen Sie jedes der beiden Nieren-Anlagen in die Mitte jeder jeweiligen Hälfte des Filters. Übertragen Sie die Platte wieder auf den Inkubator bei 37 ° C / 5% CO 2 . Verlasse den Teller im Inkubator bis zum metanephrischen RudIments aus dem nächsten Embryo sind bereit, auf den Filter gelegt zu werden. </li></ol></li><li> Wiederholen Sie die Schritte 2.3-2.9.1 für jeden Embryo. ANMERKUNG: Bei einigen Übungen dauert das Dissektionsverfahren etwa 5 min pro Embryo. </li><li> Inkubation der Niere anlagen für 3 Tage bei 37 ° C / 5% CO 2 . </li></ol> 3. Vorbereitung der Reagenzien zur Fixierung und Färbung <ol><li> Zur Herstellung von 4% Paraformaldehyd (PFA) in PBS ohne Calcium und Magnesium (w / v), löst man die PFA bei 60 ° C in PBS, kontinuierlich rühren. Auf Raumtemperatur abkühlen und mit einem Papierfilter die restlichen Partikel entfernen. Aliquot und bei 4 ° C zur sofortigen Anwendung aufbewahren oder zur Langzeitlagerung einfrieren. ACHTUNG: PFA ist giftig. Tragen Sie die persönliche Schutzausrüstung, die in den örtlichen Sicherheitsnormen angegeben ist, und arbeiten Sie in einer Abzugshaube. Verwerfung von Abfällen nach institutionellen Richtlinien und Verwendung von Abfallbehältern. </li><li> Zur Vorbereitung der Permeabilisierungslösung verdünnen Sie Triton X-100In PBS ohne Kalzium und Magnesium bis 0,3% (v / v). </li><li> Zur Herstellung der Blockierungslösung 5% (v / v) Ziegenserum und 0,1% Triton X-100 zu PBS ohne Calcium und Magnesium zugeben. Füge Natriumazid zu einer Endkonzentration von 0,02% (w / v) hinzu. Bei 4 ° C aufbewahren. ACHTUNG: Natriumazid ist giftig. Behandeln Sie Natriumazid gemäß den örtlichen Sicherheits- und Umweltschutzbestimmungen. HINWEIS: Bei der Verwendung von Antikörpern für die Färbung, verwenden Sie 5% Serum aus der Spezies der sekundäre Antikörper wurde abgeleitet, um Sperrlösung zu machen. </li><li> Verdünnen Sie biotinyliertes Dolichorus biflorus Agglutinin 1: 200 in Blockierungslösung. Verdünnen Sie Alexa488-konjugiertes Streptavidin 1: 200 in Blockierungslösung. </li><li> Zur Herstellung von gebrauchsfertigem Einbettungsmedium (siehe Tabelle der Materialien) werden 0,1 g wasserlösliches Polyvinylalkohol-basiertes Mukoadhäsiv / ml bis 25% (w / v) Glycerin in 0,1 M Tris-HCl (pH 8,5) . Unter ständigem Rühren für 1 h auf 50 ° C erhitzen, abkühlen lassen und den pH-Wert auf 8,0-8,5 einstellenM NaOH. Vermeiden Sie höhere NaOH-Konzentrationen. Füge 100 μg / ml N-Propylgallat und Thimerosal zu einer Endkonzentration von 0,02% (w / v) hinzu. 30 min bei Raumtemperatur umrühren. ACHTUNG: Thimerosal ist giftig. Behandeln Sie es gemäß den örtlichen Sicherheits- und Umweltschutzbestimmungen. <ol><li> Füllen Sie das Einbettungsmedium in 50 ml Röhrchen, balancieren Sie den Rotor und zentrifugieren Sie bei 3.200 xg und Raumtemperatur für 10 min. Die klare Lösung in 15 ml Röhrchen dekantieren und das Pellet entsorgen. Aliquot und bei -20 ° C für mehrere Wochen aufbewahren. Einmal aufgetaut, halten Sie die Lösung bei 4 ° C. Warm bis ~ 30 ° C sofort vor Gebrauch. </li></ol></li><li> Bereiten Sie so viele Glasrutschen vor, wie Filter mit Explantaten montiert werden sollen. Dazu kleben Sie zwei kleine Deckgläser, 18 x 18 mm, auf beiden Seiten des Glasschiebers mit Instant-Klebstoff, lassen Sie einen Durchmesser von 15 mm für den Filter dazwischen. </li></ol> 4. Fixierung und Färbung <ol><li> Nach einer Kultivierungszeit von 3Tage in vitro (div), entfernen Sie die Platten aus dem Inkubator und sorgfältig aspirieren das Kulturmedium aus jeder Vertiefung mit einer Mikropipette. Achten Sie darauf, den Filter nicht zu berühren und halten Sie die Spitze in Richtung der Wand des Brunnens. </li><li> Fügen Sie 500 μl 4% PFA / PBS zu jeder Vertiefung hinzu. Die Filter schweben auf dem Fixiermittel. Verwenden Sie eine Mikropipette, fügen Sie vorsichtig das PFA-Fixiermittel hinzu, um die Filter zu tauchen. Inkubieren bei Raumtemperatur für 30 min. HINWEIS: Für alle folgenden Schritte ist darauf zu achten, dass die Explantate nicht aus dem Filter abgewaschen werden. Halten Sie das Öffnen der Pipettenspitze zur Wand des Brunnens. </li><li> Entfernen Sie die PFA und spülen Sie zweimal mit 500 μl PBS, tauchen Sie den Filter ein. Füge 500 μl Permeabilisierungslösung zu jeder Vertiefung hinzu und inkubiere bei Raumtemperatur für 1 h. </li><li> Spülen Sie zweimal mit 500 &amp; mgr; l PBS und fügen Sie 500 &amp; mgr; l Blockierungslösung zu jeder Vertiefung hinzu. Inkubieren bei Raumtemperatur für 1 h. </li><li> Ersetzen Sie die Sperrlösung durch250 μl biotinyliertes Dolichorus biflorus Agglutinin verdünnt 1: 200 in Blockierungslösung und inkubieren bei 4 ° C für 24 h. </li><li> Die Dolichorus biflorus Agglutininlösung entfernen und zweimal mit 500 μl PBS pro Vertiefung ausspülen. </li><li> Füge 250 μl Alexa488-konjugiertes Streptavidin hinzu, verdünnt 1: 200 in Blockierungslösung und inkubiere bei 4 ° C für 24 h. Alternativ bei Raumtemperatur für 2 h inkubieren. HINWEIS: Ein besseres Signal-Rausch-Verhältnis wird erreicht, wenn es bei 4 ° C inkubiert wird. </li><li> Spülen Sie zweimal mit 500 μl PBS pro Vertiefung. Mit Pinzette, die Filter auf die vorbereiteten Glasrutschen übertragen, mit den Explantaten nach oben. Montage mit ~ 50-100 μl Einbettmedium. Deckel mit 60 mm langen Nr. 1,5 Deckgläschen. Man läßt die Einbettmediumlösung 2 h bei Raumtemperatur im Dunkeln erstarren. Gehen Sie zum Abbilden oder lagern Sie die Folien, in Folie verpackt, bei 4 ° C bis zum Bild. </li><li> Bild mit einem Weitfeld-Epifluoreszenzmikroskop 7 &lt;/ Sup&gt; gekoppelt an eine Hg-Lampe und verwenden eine Spiegeleinheit für die blaue Anregung (Anregungsbandpass, 460 - 495 nm, dichromatischer Spiegel, 505 nm und Emissionsbandpass, 510 - 550 nm) und 20X Plan-Apochromat-Linsen, 0,75 numerisch Öffnung. </li></ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+cold+storage+and+transport+of+viable+embryonic+kidney+rudiments.">A method for cold storage and transport of viable embryonic kidney rudiments.</a> Kidney Int. 70 (11), 2031-2034 (2006).</li><li>Batourina, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distal+ureter+morphogenesis+depends+on+epithelial+cell+remodeling+mediated+by+vitamin+A+and+Ret.">Distal ureter morphogenesis depends on epithelial cell remodeling mediated by vitamin A and Ret.</a> Nat Genet. 32 (1), 109-115 (2002).</li></ol>

Die Autoren haben nichts zu offenbaren.

Dieses Manuskript beschreibt eine Methode, um das sich entwickelnde metanephrische Anlagen aus dem Maus-Embryo zu isolieren und die Orgel-Rudimente zu kultivieren. Diese Methode ist eine Standardtechnik, wie sie von Grobstein 8 und Saxén 9 , 10 entwickelt wurde und von vielen anderen 11 , 12 angepasst und modifiziert wurde. Der Erfolg der Methode hängt hauptsächlich von der Dau...

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

Das Ziel dieses Protokolls ist es, eine Methode für die Sektion, Isolierung und Kultur der Maus metanephrischen Rudimente zu beschreiben. Während der Säugetier-Nieren-Entwicklung, die beiden Vorläufer-Gewebe, die ureterische Knospe und das metanephrische Mesenchym, kommunizieren und wechselseitig zelluläre Mechanismen induzieren, um schließlich das Sammelsystem und die Nephrone der Niere zu bilden. Da Säugetier-Embryonen intrauterin wachsen und daher für den Beobachter unzugänglich sind, wurde eine Orgelkultur entwickelt. Mit dieser Methode ist es möglich, epithelial-mesenchymale Wechselwirkungen und zelluläres Verhalten während der Nierenorganogenese zu untersuchen. Darüber hinaus kann der Ursprung von angeborenen Nieren- und Urogenitaltrakt-Fehlbildungen untersucht werden. Nach sorgfältiger Dissektion werden die metanephrischen Rudimente auf einen Filter übertragen, der auf Kulturmedium schwimmt und in einem Zellkulturinkubator für mehrere Tage gehalten werden kann. Allerdings muss man sich bewusst sein, dass die Bedingungen sindKünstlich und könnte den Stoffwechsel im Gewebe beeinflussen. Auch konnte das Eindringen von Testsubstanzen aufgrund der extrazellulären Matrix und der im Explantat vorhandenen Basalmembran begrenzt werden. Ein wesentlicher Vorteil der Orgelkultur ist, dass der Experimentator direkten Zugang zum Orgel haben kann. Diese Technologie ist billig, einfach und erlaubt eine Vielzahl von Modifikationen, wie die Zugabe von biologisch aktiven Substanzen, die Untersuchung von genetischen Varianten und die Anwendung fortschrittlicher bildgebender Verfahren.

Metanephrische Nierenanlagen wurden von schwangeren Black-6-Inzuchtmäusen bei E11.5 abgeleitet und wurden kultiviert. Nach 3 Tagen hatte sich die Harnröhre bis zu 5 Mal verzweigt, was zu einer Verzweigung der anfänglich T-förmigen Harnröhre führte. Jedes Explantat wurde fotografiert, und die Anzahl der Segmente und Endpunkte wurde quantifiziert, um die Verzweigungsgenerationen zu bestimmen und die Anzahl der Endpunkte pro Zweig zu berechnen (Abbildung 1 ). Die Erze...

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

Dissection and Culture of Mouse Embryonic Kidney

Dieses Protokoll beschreibt eine Methode zur Isolierung und Kultivierung von metanephrischen Rudimente aus Mäuseembryonen.

Dissektion und Kultur der Maus Embryonale Niere

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 Aufrufe.  Uppsala University.  Dieses Protokoll beschreibt eine Methode zur Isolierung und Kultivierung von metanephrischen Rudimente aus Mäuseembryonen. 

Serie: 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.

Neuralrohrschlusses in Maus 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 ...

Forschung

Neurowissenschaften

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.

Orgel, Kultur und Ganze Berge Immunfluoreszenzanfärbung von Maus Wolffschen Gänge

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

Entwicklungsbiologie

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.

Optimierung der Renalorganoid- und Organotypischen Kultur für Vaskularisation, erweiterte Entwicklung und verbesserte Mikroskopie-Bildgebung

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.

Embryonalen Maus-Lung Kultur, A-System, die molekularen Mechanismen des Branching Bewerten

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.

Mikrodissektion primäre renale Gewebe Segmente und Einbindung mit neuartigen Gerüst-freie Konstrukt-Technologie

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

Biotechnik

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.

Isolierung und Kultivierung von Zellen aus dem Nephrogene Zone der embryonalen Maus Niere

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.

Isolation und Kultur der embryonalen Maus neurale Stammzellen

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.

Maus embryonalen Entwicklung in einem Serum-freien 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 ...

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.

Glomeruläres Outgrowth als Ex-Vivo-Assay zur Analyse von Pfaden, die an der Parietalepithel Cell Activation beteiligt sind

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.

Effiziente Dissektion und Kultur der primären Maus retinalen Pigmentepithelzellen

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