Diese Studie wurde unterstützt durch Zuschüsse aus dem chinesischen Sonderfonds für Postdocs (Nr. 2014T70392, YF), der National Natural Science Foundation of China (Nr. 81673766, YF), der neue Lehrer Grundieren Fonds, das Zuoxue Fundament der Fudan-Universität und die Entwicklung Projekt der Shanghai Peak Disziplinen-Integrative Medizin (20150407).

Alle Verfahren im Zusammenhang mit tierischen Themen folgen die Richtlinien der Tier-Ethik-Kommission am Shanghai Medical College, Fudan-Universität (Zulassungsnummer 20160225-013).
1. Vorbereitung des Eierstocks Transparent Maus
<ol><li>
Vorbereitung der Lösungen
	<ol>
<li>Bereiten Sie Phosphat-gepufferte Kochsalzlösung (PBS) Lösung (1 M, pH 7.6) mit 0,1 % Triton x-100 (PBST). Um 1 L 10 X PBS-Stammlösung zu machen, mischen Sie 87 g NaCl, 3,1 g NaH2PO4 · 2H2O, 28,7 g Na2HPO4 ·12H2O. dH2O 1 L und Rühren ~ 2 h. anpassen auf pH 7.4 mit 1 N NaOH und bei Raumtemperatur lagern. Diese Stammlösung 10 X 1/10 verdünnen mit dH2O und dann 0,1 % Triton x-100 mit PBS-Puffer.</li>
		<li>Hydrogel-Lösung (pH 7,5) durch Vermischungen 4 % (wt/Vol) Paraformaldehyd (PFA) Lösung, 4 % (wt/Vol) Acrylamid, 0,05 % (wt/Vol) Bisacrylamide und 0,25 % (wt/Vol) VA-044-Initiator in bidestilliertem Wasser auf Eis zubereiten.</li>
		<li>Bereiten Sie die Clearing-Lösung (pH 8,5) durch das Mischen von 200 mM Natrium Borat Puffer, 4 % (wt/Vol) Sodium Dodecyl Sulfat (SDS) in bidestilliertem Wasser enthält.</li>
	</ol>
</li>
	<li>
Transcardial Perfusion und Eierstock Vorbereitung
	<ol>
<li>Halten Sie alle Lösungen auf Eis, während alle Verfahren.</li>
		<li>Erwachsene weibliche Wildtyp C57BL/6 Maus mit eine intraperitoneale Injektion von Pentobarbital (0,35 mg/kg) Lösung zu betäuben. Sobald es keine Zehe-Prise Reaktion zeigt, ist die Maus richtig betäubt.</li>
		<li>Verwendung Klebeband, die Maus mit Gliedmaßen zu beheben ausgestreckt auf einem flachen Hartschaumplatten.</li>
		<li>Setzen Sie das Herz durch einen Schnitt auf dem Xiphoid-Prozess durch die Brust, Haut und Knochen, mit einer Schere.</li>
		<li>Einen Einschnitt in das Tier rechten Vorhof, und dann Durchspülen des linken Ventrikels mit 20 mL eiskaltes 1 X PBS mit 10 mL/min.</li>
		<li>Durchspülen Sie das Tier mit mindestens 20 mL Hydrogel-Lösung mit 10 mL/min.</li>
		<li>Einschneiden der Haut über den Bauch entlang der Medioventral Linie mit einer Schere, die die ganze Enterocoelia macht, und den Darm entfernen. Sammeln Sie die Eierstöcke sowie der Gebärmutter, die Integrität der Eierstöcke Morphologie.</li>
		<li>Entfernen Sie das Fettgewebe um den Eierstock unter dem Mikroskop.</li>
	</ol>
</li>
	<li>
Entgasung
	<ol>
<li>Legen Sie die Eierstöcke in 10 mL Hydrogel Lösung bei 4 ° C zur Fixierung für 3 Tage.</li>
		<li>Übertragen Sie des Eierstocks in einer 5-mL-Tube und füllen Sie das Rohr mit Hydrogel-Lösung. Dehnen Sie Parafilm über der Oberseite des Rohres zu und dann wickeln Sie Parafilm um den Hals des Rohres. Achtung: Sicherstellen Sie, dass keine Luftblasen zwischen den Parafilm und der Hydrogel-Lösung gibt.</li>
		<li>Legen Sie das Rohr auf einen Shaker im Inkubator bei 37 ° C für maximal 3 h erlauben das Hydrogel zu polymerisieren.</li>
		<li>Entfernen Sie der Eierstöcke aus dem Gel mit einem Spatel und entfernen Sie vorsichtig das überschüssige Gel aus um den Eierstock durch Tissue-Papier. Waschen Sie der Fruchtknoten viermal mit 1 X PBS.</li>
	</ol>
</li>
	<li>
Passive clearing
	<ol>
<li>Tauchen Sie den Eierstock in 10 mL der Clearing-Lösung. Schütteln Sie bei 80 u/min bei 37 ° C zu den Clearing-Prozess zu starten.</li>
		<li>In der ersten Woche wechseln Sie die Lösung alle 3 Tage. Aktualisieren Sie nach einer Woche die Clearing-Lösung einmal wöchentlich bis zu den Eierstöcken deutlich wird. Dieser Prozess dauert in der Regel ca. 2-3 Wochen. Hinweis: Der Eierstock kann direkt Immunostained oder in verstaubar Natriumazid (0,02 %) mit PBS-Puffer bei 4 ° C für mehrere Wochen vor Immunostaining.</li>
	</ol>
</li>
</ol>(2) Immunostaining
<ol><li>Waschen Sie den Eierstock zweimal in PBST am 1. Tag bei 37 ° C auf einem Boston-Shaker.</li>
	<li>Inkubieren Sie der Fruchtknoten auf einem Shaker in primären Antikörper/PBST Lösung (Tabelle 1) am Tag 2-3 bei 37 ° c In der Arbeit präsentierten hier die primäre Antikörper Thrombozyten Endothelzellen Adhäsionsmolekül 1 (CD31) und Tyrosin-Hydroxylase 01:10 und 01:50 in PBST, bzw. verdünnt. Achtung: Wenn zwei oder mehr Arten von primären Antikörper verwendet werden, stellen Sie sicher gibt es verschiedene tierische Quellen.</li>
	<li>Die primären Antikörper mit PBST Puffer bei 37 ° C am Tag 4 auf einem Boston-Shaker abwaschen.</li>
	<li>Inkubieren Sie den Eierstock mit der gewünschten Sekundärantikörper in PBST am Tag 5-6 bei 37 ° C in einen Shaker geben. In der hier vorgestellten Arbeit wurden der Sekundärantikörper Alexa Mehl 488 für Tyrosin-Hydroxylase und Alexa Mehl 594 für CD31 01:50 in PBST verdünnt.</li>
	<li>Abwaschen der Sekundärantikörper mit PBST Puffer bei 37 ° C am Tag 7 in einen Shaker geben.</li>
	<li>Übertragen Sie das Gewebe in Brechungsindex für Brechungsindex Homogenisierung am 8. Tag passende Lösung. Legen Sie das Gewebe auf einem Papier mit Gitternetzlinien seine visuelle Klarheit zu überprüfen, indem Sie überprüfen, ob die Gitternetzlinien gesehen werden können. Sobald das Gewebe vollständig geklärt ist, kann es abgebildet werden.</li>
	<li>Machen Sie einem Kitt Zylinder, der nur Umgebung könnte das Gewebe in einer Hufeisenform Formen Sie und drücken Sie die äußeren Grat fest auf einen Glasobjektträger um einen versiegelten Raum zu bilden. Die Höhe der Kitt sollte ein wenig größer als der Fruchtknoten.</li>
	<li>Legen Sie die geklärten Eierstock vorsichtig in die Mitte der Kitt und fügen Sie Brechungsindex, die passende Lösung. Eine Glasboden Gericht über den Kitt fest und verwenden Sie Kitt, um die Schale zu fixieren.</li>
</ol>(3) konfokalen Mikroskopie
<ol><li>Für die Bildgebung mit einem konfokalen Mikroskop, im Bereich "Ni-E" wählen Sie das Objektiv (in diesem Fall Wasser eintauchen 25 X Objektiv, 1.1-NA, 2 mm-WD) mit der richtigen Arbeitsabstand (in diesem Fall 2,0 mm).</li>
	<li>
Wählen Sie im Bereich "Acquisition" die Anzahl der Kanäle. Hier die verwenden Sie drei Kanäle.
	<ol>
<li>Verwenden Sie zuerst die Registerkarte "Auge Port" "und die XY-Controller Joystick das Gewebe in die Mitte des Feldes verschieben, indem die Überprüfung durch das Mikroskop Okulare.</li>
		<li>Nach dem Ausschalten der Auslöser Licht, klicken Sie auf "live"-Fenster anpassen "Laser-Energie", "HV (Gain)" und "Offset" für jeden Kanal separat. Höhere Laserleistung und HV führen zu höheren Signale und höhere Versatz kann die Hintergrundgeräusche reduzieren.</li>
		<li>Wählen Sie die richtige "Scan-Größe" und "Count". Verwenden Sie eine Scangröße von 1024 x 1024 µm2 und eine Anzahl von 4 bis 8.</li>
	</ol>
</li>
	<li>
Klicken Sie nach der Einstellung der Bildqualität für die Definition der Tiefe des Gewebes im Bereich "Z-Intensität-Korrektur", und nach Auffinden der Linse auf die oberste Schicht des Eierstocks (in der Mitte des Gewebes) und die Übernahme für die oberste Schicht anpassen auf das "Plus" Taste, um die oberste Schicht zu definieren.
	<ol>
<li>Unten Sie das Objektiv nach und legen Sie die entsprechenden HV und Laserleistung für die Unterseite des Gewebes und fügen Sie die niedrigste Schicht Informationen in die Z-Intensität-Korrektur-Tabelle hinzu. Klicken Sie auf ", ND", die Einstellung mit dem "ND Erwerb" Panel zu synchronisieren.</li>
	</ol>
</li>
	<li>
In die "ND-Übernahme" panel, erste Reihe, die die Anzahl der Scan-Schritte. Dann kreuzen Sie die Registerkarten "Z" und "Großbild".
	<ol>
<li>Gehen Sie zum Fenster "Große Bild", die Größe des Feldes nach der Grenze des Gewebes mit Hilfe von Gewebe Visualisierung durch die Okulare zu definieren. Gehen Sie zurück zum Fenster "ND Erwerb" geben Sie die Größe des Scanbereichs ("Felder") und wählen die Überlappung zwischen 10 % und 15 %.</li>
		<li>Klicken Sie auf "Run Z Korrektur", nicht "Run jetzt", um automatisch die richtige HV, Laserleistung und Offset für jede Schicht gesetzt, und warten auf das Bild, um zu verarbeiten.</li>
	</ol>
</li>
</ol>(4) 3D-Rekonstruktion der einzelnen Follikel und ihre Vasculatures
<ol><li>Erstens verwenden Sie ImageJ5 , um die ursprünglichen NIS-Datei zu öffnen. Klicken Sie auf die Schaltfläche "Bild" und wählen Sie "Farbe" und die "Split-Kanäle". Dann klicken Sie auf "Datei" und speichern Sie die Kanäle separat als TIFF-Datei Reihe mithilfe der "Bild-Sequenzen" Option in "Save as".</li>
	<li>Verwenden Sie Imaris, um eines der Baureihe TIFF-Datei zu öffnen. Klicken Sie auf die Schaltfläche "Bearbeiten" und wählen Sie "Add Channels", den Rest der Kanäle hinzuzufügen. Den Namen und die Farbe des Kanals können auf den Registerkarten "Display-Einstellung" geändert werden. Die Dicke des Gewebes kann im Bereich "Bildeigenschaften" in der Dropdown-Liste unter "Bearbeiten" korrigiert werden müssen.</li>
	<li>Klicken Sie für die 3D Zuschneiden der fertigen Bilder und Entfernung der überschüssigen Teile auf die Schaltfläche "Bearbeiten" und wählen Sie "Ernte 3D". Die Größe des Feldes kann durch die Grenze zu ziehen.</li>
	<li>
Um die Schiff-Signale mit der Filamente Algorithmus im Bedienfeld "Surpass" zu rekonstruieren, klicken Sie auf "Filamente" zum Erstellen einer neuen Filament und klicken Sie auf weiter.
	<ol>
<li>Um die größte und die dünnste Durchmesser zu definieren, gehen Sie zum Fenster "Scheibe" und finden Sie die Ablaufverfolgung Schiff zu. Der Abstand wird automatisch im Feld "Maß" indem Sie zwei Punkte auf die maximale Breite des Schiffes auswählen angezeigt.</li>
		<li>Gehen Sie zurück zum Fenster "Surpass" und geben Sie die gemessenen Breite und klicken Sie auf weiter.</li>
	</ol>
</li>
	<li>Passen Sie die Anfangs- und Saatgut Punkt-Schwelle. Wählen Sie im Bereich "Kamera" "Select". Wenn einige der automatisch erzeugten Kugeln entfernt werden müssen, drücken Sie die Umschalttaste und klicken Sie auf den Punkt. Durch die Auswahl "Navigate" und bewegen Sie die Maus, kann die Struktur gedreht werden, um sicherzustellen, dass die richtigen Punkte wurden beibehalten, und klicken Sie dann auf weiter.</li>
	<li>Wählen Sie den höchsten Schwellenwert für den lokalen Kontrast und klicken Sie auf weiter. Nicht wählen Sie "Erkennen Dornen", und weiterhin die Rekonstruktion des Zylinders Blutgefäß abzuschließen. Die Farbe kann bearbeitet werden, indem Sie auf die Registerkarte "Farbe". Die statistischen Daten können im Bereich "Statistik" des rekonstruierten Filaments extrahiert werden. Im Bereich "Bearbeiten" können überflüssige Teile entfernt werden. Hinweis: Andere Algorithmen können für die Messung des Volumens des gesamten Follikel oder follikulären Wände verwendet werden. In der hier vorgestellten Arbeit wurden die Eizellen der Autofluoreszenz der beiden Sekundärantikörper geprägt. Wegen der großen Menge von Daten war ein Workstation-Server für die Datenanalyse verwendet.</li>
</ol>5. statistische Analyse
Hinweis: Analyse der Bilddaten werden vorgestellt, wie ± Standardfehler bedeutet.
<ol><li>Führen Sie Vergleiche zwischen den Follikeln mit unabhängigen Stichproben t-Test aus und verwenden Sie Pearson Korrelation Test Korrelationskoeffizienten von follikulären Wand- und Kapillarlänge zu vergleichen. Legen Sie einen Wert von P < 0,05 als Grenze für statistische Signifikanz.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free-of-acrylamide+SDS-based+tissue+clearing+(FASTClear):+A+novel+protocol+of+tissue+clearing+for+three-dimensional+visualisation+of+human+brain+tissues.">Free-of-acrylamide SDS-based tissue clearing (FASTClear): A novel protocol of tissue clearing for three-dimensional visualisation of human brain tissues.</a> Neuropathol Appl Neurobiol. 43, 346-351 (2016).</li><li>Xu, N., 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=Fast+free-of-acrylamide+clearing+tissue+(FACT)-an+optimized+new+protocol+for+rapid,+high-resolution+imaging+of+three-dimensional+brain+tissue.">Fast free-of-acrylamide clearing tissue (FACT)-an optimized new protocol for rapid, high-resolution imaging of three-dimensional brain tissue.</a> Sci Rep. 7, 9895 (2017).</li><li>Migone, F. F., Cowan, R. G., Williams, R. M., Gorse, K. J., Zipfel, W. R., Quirk, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+reveals+an+essential+role+of+vasoconstriction+in+rupture+of+the+ovarian+follicle+at+ovulation.">In vivo imaging reveals an essential role of vasoconstriction in rupture of the ovarian follicle at ovulation.</a> Proc Natl Acad Sci U S A. 113, 2294-2299 (2016).</li><li>Malki, S., Tharp, M. E., Bortvin, 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+whole-mount+approach+for+accurate+quantitative+and+spatial+assessment+of+fetal+oocyte+dynamics+in+mice.">A whole-mount approach for accurate quantitative and spatial assessment of fetal oocyte dynamics in mice.</a> Biol Reprod. 93 (113), (2015).</li></ol>

Die Autoren haben nichts preisgeben.

In der aktuellen Studie präsentieren wir 3D imaging um die Beziehungen zwischen den Kapillaren und einzelnen wachsenden Follikel zu bewerten. In unserer bisherigen Arbeit mit dem gleichen Protokoll 9haben wir die Rollen von großen Gefäßsystem, Wechselwirkungen zwischen Follikel und der Position der Follikel in den Eierstöcken intakt Maus untersucht. Die passive Klarheit Ansatz erlaubt uns, Mikro und Makro-Vasculatures, Folliculogenesis und die Zusammenhänge zwischen der Corpora Lutea und Fol...

Die Follikel ist die strukturelle und funktionelle Grundeinheit des Eierstocks, und seine Entwicklung bezieht sich stark auf das Gefäßsystem innerhalb des Eierstocks. Blutgefäße versorgen, Ernährung und Hormone zu den Follikeln und somit eine wichtige Rolle für das Wachstum und die Reifung der Follikel1.
Eine Kombination von Technologien, einschließlich selektive Blutgefäß Marker, transgenen Mausmodellen und pharmazeutische Entwicklung, haben unser Wissen über Eierstockkrebs vaskulären Netzwerken, Angiogenese und die Funktion der Blutgefäße im erhöht Folliculogenesis. Der Fruchtknoten ist als aktives Organ bekannt, weil es verschiedenen Geweben und vaskulären Netzwerken während Folliculogenesis und Ovulation umbaut. Solche aktiven Umbau in der Größe und Struktur der Schiffe ist erforderlich für die biologische Funktion der Entwicklung und der Rekrutierung Follikel.
Traditionelle histologische und Histomorphometric Methoden mit Eierstockkrebs Abschnitte und Immunolabeling der Blutgefäße beschränken sich auf zweidimensionale (2D) Bilder2. Mit der Entwicklung von dreidimensionalen (3D) Rekonstruktion Technologien, 2D-Bilder Gewebe Scheiben können überlappt werden, um eine 3D-Struktur zu machen, aber diese Methode hat noch einige Einschränkungen – Schneiden des Gewebes zerstören die Mikrostrukturen, einige Teile der Gewebe sind oft fehlen, und bedeutende Arbeit engagiert sich bei der Herstellung von 3D Rekonstruktionen aus Bilder von Scheiben. Gesamte Gewebe 3D-Bildgebung mit konfokalen Mikroskopie kann viele dieser Beschränkungen überwinden, aber diese Methoden beschränken sich auf die Bewertung der Angiogenese in der embryonalen Eierstock-3. Mit ganzen Gewebe clearing-Methoden wie Klarheit4 können Sie die Lautstärke visualisierte um diese Probleme postnatale und Erwachsenen Eierstöcke und solche Methoden bieten optische Freiraum des Eierstocks ohne strukturelle Verformungen. Bildgebung der 3D Architektur der intakten Eierstöcke bietet eine genaue Bilddatenbank für Bildanalyse-Software, wie z. B. die Imaris-Software-Paket in dieser Arbeit verwendet.
Umbau des Eierstocks im gesamten Erwachsenenalter ist Teil eines dynamischen physiologischen Systems, und dadurch dem Eierstock ein hervorragendes Modell für Untersuchungen über die Regulierung der Angiogenese. Darüber hinaus kann die Bewertung der Rolle der Eierstöcke Blutgefäße unter pathologischen Bedingungen des weiblichen Fortpflanzungssystems wie PCO-Syndrom oder Eierstockkrebs durch ganze Eierstockgewebe Bildgebung untersucht werden. Die Entwicklung der passiven Klarheit-Methode und die Verwendung von modernen Bildanalyse-Software haben detaillierte Rauminformationen über die Beziehungen zwischen Blutgefäßen und ovarielle Strukturen wie Follikel zur Verfügung gestellt.

<table border="0" cellpadding="0" cellspacing="0" width="1664">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Acrylamide</td>
			<td style="width:189px;">Vetec</td>
			<td style="width:127px;">v900845</td>
			<td style="width:861px;">http://www.sigmaaldrich.com/catalog/product/vetec/v900845</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Alexa Flour 488 (Dilution 1:50)&#160;</td>
			<td style="width:189px;">Life Technologies</td>
			<td style="width:127px;">A11039</td>
			<td style="width:861px;">https://www.thermofisher.com/antibody/product/Goat-anti-Chicken-IgY-H-L-Secondary-Antibody-Polyclonal/A-11039</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Alexa Flour 594 (Dilution 1:50)</td>
			<td style="width:189px;">Life Technologies</td>
			<td style="width:127px;">A11012</td>
			<td style="width:861px;">https://www.thermofisher.com/antibody/product/Goat-anti-Rabbit-IgG-H-L-Cross-Adsorbed-Secondary-Antibody-Polyclonal/A-11012</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Bisacrylamide</td>
			<td style="width:189px;">Amresco</td>
			<td style="width:127px;">172</td>
			<td style="width:861px;">http://www.amresco-inc.com/BIS-ACRYLAMIDE-0172.cmsx</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Black wall glass bottom dish (Willco-Dish)</td>
			<td style="width:189px;">Ted Pella</td>
			<td style="width:127px;">14032</td>
			<td style="width:861px;">http://www.tedpella.com/section_html/706dish.htm#black_wall</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Boric acid</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">10004818</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=10004818</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:487px;">Disodium hydrogen phosphate dodecahydrate (Na2HPO4 12H2O)</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">10020318</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=10020318</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">FocusClear</td>
			<td style="width:189px;">Celexplorer</td>
			<td style="width:127px;">FC-102</td>
			<td style="width:861px;">http://www.celexplorer.com/product_list.asp?MainType=107&#38;BRDarea=1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Parafilm</td>
			<td style="width:189px;">Bemis</td>
			<td style="width:127px;">PM996</td>
			<td style="width:861px;">http://www.parafilm.com/products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Paraformaldehyde</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">80096618</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=80096618</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">PECAM1/CD31, platelet-endothelial cell adhesion molecule 1 (Dilution 1:10)</td>
			<td style="width:189px;">Abcam</td>
			<td style="width:127px;">ab28364</td>
			<td style="width:861px;">http://www.abcam.com/cd31-antibody-ab28364.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Photoinitiator VA044</td>
			<td style="width:189px;">Wako</td>
			<td style="width:127px;">va-044/225-02111</td>
			<td style="width:861px;">http://www.wako-chem.co.jp/specialty/waterazo/VA-044.htm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium azide</td>
			<td style="width:189px;">Sigma</td>
			<td style="width:127px;">S2002</td>
			<td style="width:861px;">http://www.sigmaaldrich.com/catalog/product/sial/s2002?lang=en&#38;region=US</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium chloride (NaCl)</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">10019318</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=10019318</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:487px;">Sodium dihydrogen phosphate dihydrate (NaH2PO4 2H2O)</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">20040718</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=20040718</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium dodecyl sulfate</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">30166428</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=30166428</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium hydroxide (NaOH)</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">10019718</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=10019718</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Triton X-100</td>
			<td style="width:189px;">Sinopharm Chemical Reagent</td>
			<td style="width:127px;">30188928</td>
			<td style="width:861px;">http://en.reagent.com.cn/enshowproduct.jsp?id=30188928</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:487px;">Tyrosine hydroxylase (TH, Dilution 1:50)</td>
			<td style="width:189px;">Abcam</td>
			<td style="width:127px;">ab76442</td>
			<td style="width:861px;">http://www.abcam.com/tyrosine-hydroxylase-phospho-s40-antibody-ab51206.html</td>
		</tr>
	</tbody>
</table>

three-dimensional reconstruction of the vascular architecture of the passive clarity-cleared mouse ovary

Der Fruchtknoten ist das wichtigste Organ des weiblichen Fortpflanzungssystems und ist unerlässlich für die Produktion von weiblichen Gameten und für die Steuerung des endokrinen Systems, aber die strukturellen Zusammenhänge und dreidimensionale (3D) Gefäßsystem Architekturen von der Eierstöcke sind nicht gut beschrieben. Um die 3D Verbindungen und Architektur der Blutgefäße im Eierstock intakt zu visualisieren, ist der erste wichtige Schritt, den Eierstock optisch klar zu machen. Um Gewebe Schrumpfung zu vermeiden, wir nutzten das Hydrogel Fixierung-basierte passive Klarheit (klar Lipid ausgetauscht Acrylamid-hybridisiert Rigid Imaging / Immunostaining/In Situ-Hybridisierung-kompatible Gewebe Hydrogel) Protokoll Methode um ein intaktes Eierstock zu löschen . Immunostaining, erweiterte multiphoton confocal Mikroskopie und 3D-Rekonstruktionen Bild wurden dann für die Visualisierung der ovariellen Gefäße und follikulären Kapillaren verwendet. Mit diesem Ansatz, wir zeigten eine signifikante positive Korrelation (P < 0,01) zwischen der Länge des follikulären Kapillaren und Volumen der follikulären Wand.

Wir haben die passive Klarheit-Methode in eine schnelle und einfache Methode für passive Eierstock clearing-unter Wahrung der follikulären und vaskulären Architektur und Erlangung der höchsten Fluoreszenzsignal von beschrifteten Marker von Schiffen und Follikel angepasst. Die 3D Architektur des follikulären Gefäßsystems wurde durch Immunostaining für CD31, ein Marker für die Endothelzellen6bestimmt. CD31 Färbung in den Eierstöcken erwachsener Mäuse wurd...

Watch this Scientific Journal Video about Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary at JoVE.com

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

Hier präsentieren wir eine Anpassung der passiven Klarheit und 3D-Rekonstruktion Methode zur Visualisierung der Eierstöcke Gefäßsystem und follikulären Kapillaren in intakten Maus Eierstöcke.

Dreidimensionale Rekonstruktion der vaskulären Architektur des Eierstocks Passive Klarheit geclearte Maus

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

three-dimensional-reconstruction-vascular-architecture-passive

Research

JoVE Journal

Biology

8.6K Aufrufe.  Fudan University. Hier präsentieren wir eine Anpassung der passiven Klarheit und 3D-Rekonstruktion Methode zur Visualisierung der Eierstöcke Gefäßsystem und follikulären Kapillaren in intakten Maus Eierstöcke.

Serie: Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Hallo-C: eine Methode, um die dreidimensionale Architektur des Genoms Study.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Forschung

Biologie

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generation der Mäuse aus induzierten pluripotenten Stammzellen

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

An Analytical Tool that Quantifies Cellular Morphology Changes from Three-dimensional Fluorescence Images

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for inclusion and exclusion of data points, and computer-aided pattern recognition to support the interpretation and findings of the analysis. It has become increasingly important to be able to measure fluorescence images constructed from three-dimensional (3D) datasets in order to be able to capture the complexity of cellular dynamics and understand the basis of cellular plasticity within biological systems. Sophisticated microscopy instruments have permitted the visualization of 3D fluorescence images through the acquisition of multispectral fluorescence images and powerful analytical software that reconstructs the images from confocal stacks that then provide a 3D representation of the collected 2D images. Advanced design-based stereology methods have progressed from the approximation and assumptions of the original model-based stereology1 even in complex tissue sections2. Despite these scientific advances in microscopy, a need remains for an automated analytic method that fully exploits the intrinsic 3D data to allow for the analysis and quantification of the complex changes in cell morphology, protein localization and receptor trafficking. 
Current techniques available to quantify fluorescence images include Meta-Morph (Molecular Devices, Sunnyvale, CA) and Image J (NIH) which provide manual analysis. Imaris (Andor Technology, Belfast, Northern Ireland) software provides the feature MeasurementPro, which allows the manual creation of measurement points that can be placed in a volume image or drawn on a series of 2D slices to create a 3D object. This method is useful for single-click point measurements to measure a line distance between two objects or to create a polygon that encloses a region of interest, but it is difficult to apply to complex cellular network structures. Filament Tracer (Andor) allows automatic detection of the 3D neuronal filament-like however, this module has been developed to measure defined structures such as neurons, which are comprised of dendrites, axons and spines (tree-like structure). This module has been ingeniously utilized to make morphological measurements to non-neuronal cells3, however, the output data provide information of an extended cellular network by using a software that depends on a defined cell shape rather than being an amorphous-shaped cellular model. To overcome the issue of analyzing amorphous-shaped cells and making the software more suitable to a biological application, Imaris developed Imaris Cell. This was a scientific project with the Eidgen&ouml;ssische Technische Hochschule, which has been developed to calculate the relationship between cells and organelles. While the software enables the detection of biological constraints, by forcing one nucleus per cell and using cell membranes to segment cells, it cannot be utilized to analyze fluorescence data that are not continuous because ideally it builds cell surface without void spaces. To our knowledge, at present no user-modifiable automated approach that provides morphometric information from 3D fluorescence images has been developed that achieves cellular spatial information of an undefined shape (Figure 1). 
We have developed an analytical platform using the Imaris core software module and Imaris XT interfaced to MATLAB (Mat Works, Inc.). These tools allow the 3D measurement of cells without a pre-defined shape and with inconsistent fluorescence network components. Furthermore, this method will allow researchers who have extended expertise in biological systems, but not familiarity to computer applications, to perform quantification of morphological changes in cell dynamics.

We developed a software platform that utilizes Imaris Neuroscience, ImarisXT and MATLAB to measure the changes in morphology of an undefined shape taken from three-dimensional confocal fluorescence of single cells. This novel approach can be used to quantify changes in cell shape following receptor activation and therefore represents a possible additional tool for drug discovery.

Ein analytisches Werkzeug, das zelluläre Morphologie Änderungen von Dreidimensionale Fluorescence Images Quantifiziert

The most common software analysis tools available for measuring fluorescence images are for two-dimensional (2D) data that rely on manual settings for ...

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.

The material here describes a method developed to preserve the three-dimensional chromatin structure of testicular germ cells. This has been termed the three-dimensional (3D) slide method. This method improves sensitivity for detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH).

Folienvorbereitungsmethode zur Erhaltung der dreidimensionalen Chromatinarchitektur von Hodenkeimzellen

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

Isolation und Primärkultur von Maus Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

Die Isolierung von murinen Coronary glatten Gefäßmuskelzellen

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

FRET-Imaging im dreidimensionalen Hydrogelen

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-Clear: Ein Gewebe Clearing-Methode für die dreidimensionale Darstellung von Fettgewebe

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Quadruple-Checkerboard: A Modification of the Three-Dimensional Checkerboard for Studying Drug Combinations

The concept of drug-combination therapy is becoming very important mainly with the drastic increase in resistance to drugs. The Quadruple&#160;checkerboard, also called the Q-checkerboard, aims at maximizing the number of possible combinations that can be obtained between four drugs in one experiment to minimize the time and work needed to accomplish the same results with other protocols. This protocol is based on the simple micro dilution technique where the drugs are diluted and combined together in several 96-well plates.
In the first set of 96-well plates, Muller-Hinton broth is added followed by the first required drug (e.g., Cefotaxime here) to serially dilute it. After the first step is done, another set of 96-well plates is used to dilute the second drug (e.g., Amikaci), which will be transferred by removing a specific volume of drug 2 and put in the corresponding wells in the first set of 96-well plates that contains drug one. The third step is done by adding the required concentrations of the third drug (e.g., Levofloxacin), to the appropriate plates in the initial set containing combination of drug 1 and 2. The fourth step is done by adding the required concentrations of the fourth drug (e.g., Trimethoprim-sulfamethoxazol) into the appropriate plates in the first set. Then, E. coli ESBL bacterial inoculum will be prepared and added.
This method is important to evaluate all the possible combinations and has a wider range of possibilities to be tested furthermore for in vivo testing. Despite being a tiring technique requiring a lot of focus, the results are remarkable and time saving where a lot of combinations can be tested in a single experiment.

This protocol describes how to study all possible combinations that can be obtained between four drugs in one single experiment. This method is based on the standard 96-well plate micro dilution assay and the calculation of fractional inhibitory concentrations (FICs) to evaluate the results.

Vierfach-Schachbrett: Eine Modifikation des dreidimensionalen Schachbretts zur Untersuchung von Arzneimittelkombinationen

The concept of drug-combination therapy is becoming very important mainly with the drastic increase in resistance to drugs. The ...

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

In vitro Dreidimensionaler Keimtest der Angiogenese unter Verwendung embryonaler Stammzellen der Maus für die Modellierung von Gefäßerkrankungen und Arzneimitteltests

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for ...