Die Autoren möchten DRS. Stephanie Spohn und Basel Abuaita für viele nützliche Diskussionen über organoide Mikroinjektion danken. JRS wird vom Darm Stem Cell Consortium (U01DK103141), eine Kooperative Forschungsprojekt finanziert durch das National Institute of Diabetes und Magen-Darm und Niere-Krankheiten (NIDDK) und National Institute of Allergy und Infectious Diseases (NIAID) unterstützt. JRS und VBY werden durch den Roman NIAID, Alternative Modellsysteme für enterischen Erkrankungen (NAMSED) Consortium (U19AI116482) unterstützt. DRH unterstützt die Mechanismen der mikrobiellen Pathogenese Training Zuschuss von National Institute of Allergy und Infektionskrankheiten (NIAID, T32AI007528) und der klinischen und translationalen Science Award an der Michigan-Institut für klinische und Gesundheit Forschung (UL1TR000433).
Die komplette Datendateien und Daten-Analyse-Code verwendet in dieser Handschrift sind an https://github.com/hilldr/HIO_microinjection erhältlich.

<ol>
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Die Autoren haben keinen Interessenkonflikt finanzielle oder sonstige Interessenkonflikte offen zu legen.

Dieses Protokoll stellt eine allgemeine Methode für die Mikroinjektion von Chios hPSC abgeleitet und Darm Organellen Gewebe abgeleitet und die Messung der epitheliale Barriere Permeabilität in Echtzeit. Wir haben auch unser Ansatz zur Analyse und Interpretation der Daten generiert, mit Hilfe dieser Methoden gezeigt. Angesichts der wachsenden Annahme Darm Organellen Modellsysteme16,20,21,28 und das langjährige Interesse an Darmbarriere Durchlässigkeit als physiologisch relevanten funktionellen Ergebnis3,4,5,6, erwarten wir, dass andere auf diesem Gebiet tätig werden können anwenden und auf diese Methoden aufbauen.
Es gibt mehrere Schritte, die für die Anwendung dieser Technik entscheidend sind. Zugang zu qualitativ hochwertigen hPSC- oder Gewebe abgeleiteten HIO Gewebe vor umfangreiche Experimente mit Mikroinjektion geschaffen werden sollte. HIO Makrostruktur möglicherweise heterogen, mit Variationen in Größe und Form, obwohl Gewebe Identität und zellulären Morphologie ist sehr reproduzierbar, bei der Verwendung von etablierten Methodik um Chios24zu generieren. Sphärische Chios, bestehend aus einer einzigen halbtransparenten Lumen und messen ca. 1 mm im Durchmesser sind ideal für Mikroinjektion und Messung der luminalen Fluoreszenz in Echtzeit. In einigen Fällen fehl, Mikroinjektion zu einem Zusammenbruch der HIO oder offensichtliche Undichtigkeit des injizierten Materials. Gescheiterten Chios können die Kultur gut Ermessen des Benutzers mithilfe einer standard Mikropipette entnommen werden. Auswahl von Chios für Mikroinjektion und Bildgebung der Objektive auf einer imaging Plattform berücksichtigen. Im Allgemeinen 2-4 X Objektive sind ideal für die Erfassung der kompletten HIO Fluoreszenzsignal, obwohl ein 10 X-Objektiv verwendet werden kann, wenn low-Power-Objektive nicht verfügbar sind oder die verfügbaren Chios sind &#60; 1 mm im Durchmesser. Imaging-Software darf für die automatisierte Erfassung von fluoreszierenden Bilder an definierten Stellen im Laufe der Zeit.
Mehrere Änderungen dieses Protokolls sind möglich, um die experimentellen Anforderungen anzupassen. Zum Beispiel die Ergebnisse der Barriere Funktionstests der Molekülgröße der Verbindungen im Einsatz43 abhängig gemacht werden und es kann zweckmäßig sein, Dextran-Vorbereitungen von unterschiedlichem Molekulargewicht zu testen. Darüber hinaus kann Hellfeld Bildgebung zusätzlich Fluoreszenz-Bildgebung als Indikator für die strukturelle Integrität der Gewebe25durchgeführt werden. Bei der Mikroinjektion von lebender Bakterien25,28,31,32,33,44ggf. Penicillin hinzufügen und Streptomycin oder Gentamicin HIO Kultur Medien vor oder nach Mikroinjektion. Außen an der Microcapillary wird bei der Befüllung mit der Bakterienkultur Aussetzung kontaminiert werden und dies kann den HIO Medien übertragen werden. Alternativ kann Mikroinjektion erfolgen auf Chios im extrazellulären Matrix (z. B. Matrigel) ohne Medien ausgesetzt die Medien hinzufügen, nachdem die Mikroinjektion abgeschlossen ist. Dies kann Kontamination der extrazellulären Matrix und Außenseite der HIO einschränken. Bei der Planung von mikrobiellen Wachstums Assays es möglicherweise notwendig, Antibiotika in den Medien nach 1-2 h zu vermeiden, verlangsamt oder verhindert Wachstum von mikroinjiziert Organismen zu entfernen.
Schließlich ist in der Erkenntnis, dass nicht alle Forscher auf Mikroskopie Ausrüstung geeignet für in-vitro- Bildgebung zugreifen können, es wichtig, darauf hinzuweisen, dass die Anweisungen in diesem Protokoll für die Fluoreszenz Datenerhebung auf Bilder angewendet werden können an festen Zeitpunkten mit standard Epifluorescent Mikroskopie ohne automatische Bildaufnahme oder Umweltkontrolle. Beispiele dafür finden Sie in den Berichten von Leslie und Huang Et al. 25, die C. Difficile Toxin Aktivität im Darm Organellen hPSC abgeleitet und Karve und Pradan Et Al. untersuchten 44, die epitheliale Barriere Permeabilität in ähnlichen hPSC abgeleitet Darm Organellen mikroinjiziert mit live E. Coliuntersucht. Manuelle Bedienung der bildgebenden Geräten führen größere Variation und Schwierigkeiten bei der Normalisierung der Fluoreszenzsignal. Bei der Durchführung manueller Bildgebung der FITC-Dextran Chios injiziert unbedingt feste Vergrößerung, fluoreszierende Anregung Intensität und Belichtungszeiten während das Experiment nicht zu verfälschen die Fluoreszenz Intensität Messungen zu gewährleisten.

Das Darmepithel bildet eine selektive Barriere, die den gerichteten Transport von Nährstoffen, H2O, Ionen und Abfallprodukte vermittelt bei gleichzeitiger Minimierung der unspezifischen Diffusion-vermittelten Austausch von andere Partikel zwischen Lumen und der mesenchymale Gewebe oder Blut Versorgung1,2. Die unspezifische Durchlässigkeit der intestinalen epithelialen Barriere galt lange Zeit einen wichtigen funktionelle Parameter in Gesundheit und Krankheit3,4,5,6, das spiegelt die Rate der Verbreitung von kleinen Molekülen über das Epithel über den parazellulär Platz. Messung der epitheliale Barriere Permeabilität kann durchgeführt werden, Tiermodelle7 und in menschlichen Patienten8 durch die Einnahme von Lactulose, hat keine spezifischen Transporter in den Magen-Darm-Trakt und die anschließende Sammlung und Messung von Lactulose Konzentrationen im peripheren Blut. Alternative eingenommen Marker der Barrierefunktion wie Eindringmittel beschrifteten Kohlenhydrate sind auch verfügbar9,10. Dieser Ansatz wurde angepasst für intestinale epitheliale Zellkulturen auf Transwell unterstützt11, einen vereinfachten Ansatz, ermöglicht eine bessere Kontrolle der experimentellen aber auch als schlechter Prädiktor für in Vivo kritisiert worden, angebaut Durchlässigkeit aufgrund des Fehlens von differenzierten epithelialen Subtypen und Gewebe Struktur12. Mit Kammern stellen einen weiteren Ansatz dar und ermöglichen die Messung der epitheliale Barriere-Funktion im ganzen Darmschleimhaut Ex Vivo13. Anwendung dieser Technik wird häufig durch Gewebe Verfügbarkeit und Zustand13,14begrenzt. Daher sind neue Methoden, die Reproduzierbarkeit und Durchsatz mit physiologische Relevanz Gleichgewicht notwendig.
Jüngste Entwicklungen in in-vitro- Organogenese führten zu die Annahme von 3D Gewebekultur Modellsysteme als eine anspruchsvolle Plattform für Rekapitulation der Dynamik von komplexen Gewebe15,16,17 ,18,19,20,21,22,23. Vor allem menschlicher pluripotenter Stammzellen (hPSC) abgeleitet menschlichen Darm Organellen (Chios)19,24 entstanden als reproduzierbar und experimentell gefügig Modellsystem für die Untersuchung von Host-mikrobielle Wechselwirkungen und epitheliale Barriere Dynamik25,26,27,28. In ähnlicher Weise menschliche Gewebe-abgeleitete Organellen (auch bekannt als Enteroids) können aus einem einfachen Biopsie-Verfahren abgeleitet werden und können als gefügig System verwendet werden, um die menschliche Physiologie und Krankheit15,29,30studieren. Mikroinjektion der menschliche Darm Organellen ermöglicht die Lieferung von experimentellen Verbindungen25 oder Mikroben25,31,32,33 , die apikale epithelialen live Oberfläche des organoide Lumen. Leslie und Huang Et al. 25 angepasst vor kurzem diese Technik zur Messung der Barriere Permeabilität in Chios mit Fluorescein erfolgt (FITC) Dextran nach Exposition gegenüber bakteriellen Toxinen beschriftet mikroinjiziert.
Dieses Protokoll dient als Leitfaden für die Messung der epitheliale Barriere Permeabilität in Chios hPSC abgeleitet und Gewebe abgeleitet Chios mit Fluoreszenz-Mikroskopie. Mit geringfügigen Änderungen kann es auch als eine allgemeine Einführung in die Mikroinjektion von Chios mit experimentellen Verbindungen dienen. Benutzer können dieses Protokoll mit zusätzliche oder alternative downstream-Anwendungen nach Mikroinjektion ergänzen.

<table><tbody><tr><td>EGTA 0.5 M sterile (pH 8.0)</td><td>Bioworld</td><td>405200081</td><td></td></tr><tr><td>Cell matrix solution (Matrigel)</td><td>Corning</td><td>354230</td><td></td></tr><tr><td>Deltavision RT live cell imaging system</td><td>GE Life Sciences</td><td>29065728</td><td>http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/brands/deltavision/</td></tr><tr><td>Camera</td><td>GE Life Sciences</td><td>29065728</td><td>Included with Deltavision system</td></tr><tr><td>softWoRx Imaging software</td><td>GE Life Sciences</td><td>29065728</td><td>Included with Deltavision system</td></tr><tr><td>Biosafety cabinet</td><td>Labconco</td><td>Cell Logic+</td><td>http://www.labconco.com/product/purifier-cell-logic-class-ii-type-a2-biosafety-cabinets-2/4262</td></tr><tr><td>1X PBS</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>Advanced DMEM-F12</td><td>Life Technologies</td><td>12634-010</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>B27 supplement (50X)</td><td>Life Technologies</td><td>17504044</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>L-glutamine (100X)</td><td>Life Technologies</td><td>25030-081</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>HEPES buffer</td><td>Life Technologies</td><td>15630080</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>Manipulator</td><td>Narshge</td><td>UM-3C</td><td></td></tr><tr><td>Micromanipulator</td><td>Narshge</td><td>UM-1PF</td><td></td></tr><tr><td>Pipette Holder</td><td>Narshge</td><td>UP-1</td><td>Alternate to Xenoworks pipette holder</td></tr><tr><td>Magnetic stand</td><td>Narshge</td><td>GJ-1</td><td></td></tr><tr><td>Dissecting scope</td><td>Olympus</td><td>SX61</td><td>Recommended scope, although other models are likely compatible</td></tr><tr><td>Olympus IX71 Fluorescent microscope</td><td>Olympus</td><td>IX71</td><td>Included with Deltavision system</td></tr><tr><td>CoolSNAP HQ2</td><td>Photometrics</td><td>29065728</td><td>Included with Deltavision system</td></tr><tr><td>Recombinant &lt;em&gt;C. difficile&lt;/em&gt; Toxin A/TcdA Protein</td><td>R&amp;amp;D Systems</td><td>8619-GT-020</td><td></td></tr><tr><td>EGF</td><td>R&amp;amp;D Systems</td><td>236-EG</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>R-spondin 1</td><td>R&amp;amp;D Systems</td><td>4645-RS</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>Noggin</td><td>R&amp;amp;D Systems</td><td>6057-NG</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>Mineral oil</td><td>Sigma-Aldrich</td><td>M8410</td><td></td></tr><tr><td>FITC-dextran (4 kDa)</td><td>Sigma-Aldrich</td><td>46944</td><td></td></tr><tr><td>Micropipette puller</td><td>Sutter Instruments</td><td>P-30</td><td></td></tr><tr><td>Nunc Lab-Tek II Chamber Slides</td><td>ThermoFisher Scientific</td><td>154526PK</td><td></td></tr><tr><td>Glass filaments</td><td>WPI</td><td>TW100F-4</td><td></td></tr><tr><td>Micropipette holder</td><td>Xenoworks</td><td>BR-MH2</td><td>Preferred device</td></tr><tr><td>Analog Tubing kit</td><td>Xenoworks</td><td>BR-AT</td><td></td></tr><tr><td>1/16 in clear ferrule</td><td>Xenoworks</td><td>V001104</td><td></td></tr><tr><td>1-1.2 mm O-ring</td><td>Xenoworks</td><td>V300450</td><td></td></tr></tbody></table>

real-time measurement of epithelial barrier permeability in human intestinal organoids

Fortschritte in der 3D Culture der intestinalen Gewebe durch Biopsie gewonnen oder aus pluripotenten Stammzellen über gezielte Differenzierung generiert haben dazu geführt, dass anspruchsvolle in-vitro- Modelle der Darmschleimhaut. Nutzt diese aufstrebenden Modellsysteme erfordert Anpassung der Werkzeuge und Techniken für 2D Kultur-Systeme und Tiere entwickelt. Hier beschreiben wir eine Technik zur Messung der epitheliale Barriere Permeabilität im menschlichen Darm Organellen in Echtzeit. Dies geschieht durch Mikroinjektion von Eindringmittel beschriftet Dextran und Bildgebung auf einem inversen Mikroskop mit Epifluorescent Filter ausgestattet. Echtzeit-Messung der Durchlässigkeit Barriere im Darm Organellen erleichtert die Generation der hochauflösenden Zeitdaten im menschlichen Darm Epithelgewebe, obwohl diese Technik auch auf festen Timepoint imaging Ansätze angewendet werden kann. Dieses Protokoll ist leicht anpassbar für die Messung der epitheliale Barriere Permeabilität nach Exposition gegenüber pharmakologische Agenten, Bakterienprodukte oder Giftstoffe oder lebenden Mikroorganismen. Mit geringfügigen Änderungen dieses Protokolls dient auch als eine allgemeine Einführung in die Mikroinjektion der intestinalen Organellen und Benutzer können beschließen, dieses Protokoll durch zusätzliche oder alternative downstream-Anwendungen nach Mikroinjektion zu ergänzen.

Chios wurden aus menschlicher pluripotenter Stammzellen differenziert und kultiviert in Zelle Matrixlösung wie zuvor beschrieben19,24. Nach 4 Wochen in Kultur hatte die Chios ausreichend erweitert, um für die Mikroinjektion zu ermöglichen. Chios wurden mit 4 kDa konjugiert FITC Dextran in PBS oder PBS mit Clostridium Difficile Toxin TDCA suspendiert mikroinjiziert. C. Difficile ist ein opportunistischer Magen-Darm-Erreger, das Toxin-vermittelte epitheliale Toxizität in Chios25aufweist. Als Positivkontrolle wurde die HIO Kulturmedien in einer Teilmenge von Chios injiziert mit PBS und FITC-Dextran EGTA hinzugefügt. EGTA ist ein Kalzium-Chelator, die schnelle de-Polymerisation der Aktin-Zytoskelett-42verursacht. FITC Fluoreszenz wurde auf dem live imaging Mikroskop innerhalb einer kontrollierten Klimakammer in Echtzeit überwacht und Aufnahmen wurden in 10-Minuten-Takt.
Post-hoc-Analyse der Bilddaten ergab erhebliche Unterschiede in der Beibehaltung der FITC Fluoreszenz (Abbildung 5). Chios mit PBS injiziert erhalten fast alle das Fluoreszenzsignal bei t = 0, aber Chios, die auch mit TDCA von mit EGTA behandelt injiziert wurden eine erhebliche Abnahme der Fluoreszenz Intensität von 8 Stunden nach Mikroinjektion (Abbildung ausgestellt 5a). Imaging-Daten wurden für alle Chios zu allen Zeitpunkten eine hochauflösende Dataset repräsentiert die relative Änderung in Fluoreszenz Intensität im Laufe der Zeit in jeder Versuchsbedingung (Abb. 5 b) generieren quantifiziert. Unterschiede in der epithelialen Durchlässigkeit ausgewertet durch Berechnung der mittleren Beseitigung Zeit (t1/2 ) der FITC für jede Behandlungsgruppe (Tabelle 1) und vergleichen Unterschiede in t t1/2 mit der Student t-test. Kontrolle-behandelten Chios behielt die Mehrheit der FITC Fluoreszenzsignal für mehr als 16 Stunden (t1/2 = 17 ± 0,3 h). Behandlung mit EGTA deutlich reduziert FITC-Dextran Beseitigung Zeit relativ zum Steuerelement Chios (t1/2 = 2,6 ± 0,2 h; P = 1,3 x 10-8). In Übereinstimmung mit den zuvor veröffentlichten Ergebnisse25, Mikroinjektion TDCA deutlich erhöht epitheliale Barriere Permeabilität gegenüber Kontrollbehandlung (t1/2 = 7,6 ± 0,6 h; P = 5,4 x 10-4). So sind externe (EGTA) und mikroinjiziert (TDCA) Verbindungen in der Lage, wesentliche Veränderungen in epitheliale Barriere Permeabilität in Chios induzieren. Diese Ergebnisse deuten darauf hin, dass die Auswirkungen einer breiten Palette von pharmakologische Agenten, Metaboliten, Bakterienprodukte, Zytokine, Wachstumsfaktoren und anderen Verbindungen auf epitheliale Barriere-Funktion mit diesem Ansatz bewertet werden können.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Behandlung</td>
			<td>Half-Life (h)</td>
			<td>SEM (h)</td>
			<td>Untere 95 % CI (h)</td>
			<td>Obermaterial 95 % CI (h)</td>
			<td>n</td>
		</tr>
		<tr>
			<td>Kontrolle</td>
			<td>17.11.</td>
			<td>0,3</td>
			<td>16.45 Uhr</td>
			<td>17,78</td>
			<td>11</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>2,58</td>
			<td>0,19</td>
			<td>1,78</td>
			<td>3.38</td>
			<td>3</td>
		</tr>
		<tr>
			<td>TDCA</td>
			<td>7.56</td>
			<td>0,63</td>
			<td>5,93</td>
			<td>9.18</td>
			<td>6</td>
		</tr>
	</tbody>
</table>
Tabelle 1: meine Beseitigung Zeit (t1/2) FITC-Dextran in Chios mit EGTA oder TDCA behandelt. Einheiten sind Stunden Post-Mikroinjektion.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56960/56960fig1.jpg"> 
Abbildung 1: Grundlayout einer Microinjector und Mikromanipulator für HIO Mikroinjektion. Dieses Bild zeigt die komplette Ergänzung der Ausrüstung für die Durchführung der Mikroinjektion von Chios verwendet. Schlüsselkomponenten sind beschriftet und Bestellinformationen finden Sie in der Materialtabelle. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56960/56960fig1large.jpg" target="_blank">Bitte klicken Sie hier für eine größere Version dieser Figur.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56960/56960fig2.jpg"> 
Abbildung 2: Mikropipette Puller. Das Kupfer Heizung Spule Hc, Spannfinger c1, untere Klemme (c2), Abzieher Arm (Pa), Hitze Auswahl ein/aus (ht) sind durch die Pfeile gekennzeichnet. Die richtige Einstellung für HEAT 1 und ziehen sind in roter Schrift gekennzeichnet. Der Inset zeigt eine richtig montierte Glas-Filament für Heizung bereit. <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56960/56960fig2large.jpg" target="_blank">Bitte klicken Sie hier für eine größere Version dieser Figur.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56960/56960fig3.jpg"> 
Abbildung 3: repräsentative Bilder von einem Gewebe abgeleitet HIO injiziert mit FITC-Dextran. (A) Bild zeigt die Positionierung der Glas-Microcapillary kurz vor dem Einsetzen in die HIO und Mikroinjektion. (B) Hellfeld-Bild von einem Gewebe abgeleitet menschlichen Darm Organellen nach Mikroinjektion der FITC-Dextran. Beachten Sie, dass das Fluoreszenzsignal offensichtlich auch ohne die Verwendung eines bestimmten Filter-Sets ist. Diese Färbung hilft Mikroinjektion Präzision. 3 X Vergrößerung <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56960/56960fig3large.jpg" target="_blank">Klicken Sie bitte hier, um eine größere Version dieser Figur.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56960/56960fig4.jpg"> 
Abbildung 4: Live-imaging Workflow. (A) Screenshot zeigt die notwendigen Schritte zur Mikroskop parametrisieren, die Chios auf der Folie zu finden und speichern Sie die Position der Bühne für jede HIO, abgebildet werden. (B) Pre-Imaging-Einstellungen für die Erfassung des FITC-Kanals in regelmäßigen Abständen an die Positionen jeweils angegeben in A. (C) Post-Imaging-Export von Daten im DV-Format Dateien als TIFF-Bilder mit einem TIFF-Bild pro HIO pro Zeitpunkt.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56960/56960fig4large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56960/56960fig5.jpg"> 
Abbildung 5: repräsentative Ergebnisse. (A) Stammzellen abgeleitet menschlichen Darm Organellen (HIO) mit 2 mg/mL FITC-Dextran mikroinjiziert (4 kDa) für 20 Stunden abgebildet. Chios wurden auch mit PBS (Kontrolle) oder Clostridium Difficile Toxin TDCA mikroinjiziert (12,8 ng/µL) oder mit 2 mM EGTA, um die externe Kultur Medien hinzugefügt behandelt. 4 X Vergrößerung. (B) Grundstück mittlere normalisierte FITC-Intensität im Laufe der Zeit in Chios behandelt mit PBS (Kontrolle), TDCA oder EGTA. Fehlerbalken darzustellen S.E.M und n = 11 Chios (Control), 3 Chios (EGTA), 6 Chios (TDCA). <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56960/56960fig5large.jpg" target="_blank">Bitte klicken Sie hier für eine größere Version dieser Figur.</a>

Watch this Scientific Journal Video about Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids at JoVE.com

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Dieses Protokoll beschreibt die Messung der epitheliale Barriere Permeabilität in Echtzeit folgende pharmakologische Behandlung im menschlichen Darm Organellen mit Fluoreszenz-Mikroskopie und live-Cell-Mikroskopie.

Echtzeitmessung der epitheliale Barriere Permeabilität im menschlichen Darm Organellen

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

real-time-measurement-epithelial-barrier-permeability-human

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

14.1K Aufrufe.  University of Michigan. Dieses Protokoll beschreibt die Messung der epitheliale Barriere Permeabilität in Echtzeit folgende pharmakologische Behandlung im menschlichen Darm Organellen mit Fluoreszenz-Mikroskopie und live-Cell-Mikroskopie.

Serie: Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

Ex vivo-Darm-Sacs zu beurteilen Durchlässigkeit der Schleimhaut in Modelle von Magen-Darm-Krankheit

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Forschung

Medizin

Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed.
Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand&#160;as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions.
Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure&#160;for the in vitro modeling of interferon gamma (IFN&#947;)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

Kombination von menschlichen Organoiden und Organ-on-a-Chip-Technologie zur Modellierung der intestinalen regionsspezifischen Funktionalität

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, ...

Biotechnik

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

Ein intravitaler, auf Mikroskopie basierender Ansatz zur Bewertung der Darmdurchlässigkeit und der Epithelzellvergießen-Performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. ...

Immunologie und Infektion

Determining Intestinal Permeability Using Lucifer Yellow in an Apical-Out Enteroid Model

Enteroids are an emerging research tool in the study of inflammatory bowel diseases such as necrotizing enterocolitis (NEC). They are traditionally grown in the basolateral-out (BO) conformation, where the apical surface of the epithelial cell faces the inner lumen. In this model, access to the luminal surface of enteroids for treatment and experimentation is challenging, which limits the ability to study host-pathogen interactions. To circumvent this, a neonatal apical-out (AO) model for necrotizing enterocolitis was created. Since intestinal epithelial cell permeability changes are pathognomonic for NEC, this protocol outlines using lucifer yellow (LY) as a marker of paracellular permeability. LY traverses the intestinal epithelial barrier via all three major paracellular pathways: pore, leak, and unrestricted. Using LY in an AO model allows for a broader study of permeability in NEC. Following IRB approval and parental consent, surgical samples of intestinal tissue were collected from human preterm neonates. Intestinal stem cells were harvested via crypt isolation and used to grow enteroids. Enteroids were grown to maturity and then transformed AO or left in BO conformation. These were either not treated (control) or were treated with lipopolysaccharide (LPS) and subjected to hypoxic conditions for the induction of in vitro NEC. LY was used to assess for permeability. Immunofluorescent staining of the apical protein zonula occludens-1 and basolateral protein &#946;-catenin confirmed AO conformation. Both AO and BO enteroids treated with LPS and hypoxia demonstrated significantly increased paracellular permeability compared to controls. Both AO and BO enteroids showed increased uptake of LY into the lumen of the treated enteroids compared to controls. The utilization of LY in an AO enteroid model allows for the investigation of all three major pathways of paracellular permeability. It additionally allows for the investigation of host-pathogen interactions and how this may affect permeability compared to the BO enteroid model.

The present protocol outlines a method that utilizes lucifer yellow in an apical-out enteroid model to determine intestinal permeability. This method can be used to determine paracellular permeability in enteroids that model inflammatory bowel diseases such as necrotizing enterocolitis.

Bestimmung der Darmpermeabilität mit Lucifer Yellow in einem apikalen enteroiden Modell

Enteroids are an emerging research tool in the study of inflammatory bowel diseases such as necrotizing enterocolitis (NEC). They are traditionally grown ...

Investigating Intestinal Barrier Breakdown in Living Organoids

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-&#947;) as a positive control for barrier destruction and organoids derived from IFN-&#947; receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-&#947; on the intestinal barrier integrity and the IFN-&#947; induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.

Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner.

Untersuchung des Darmbarriereabbaus bei lebenden Organoiden

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was ...

Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit polarity, consisting of a lumen with an apical border, tight junctions, and a basolateral outer layer exposed to growth media. The consequences of intestinal injuries include mucosal inflammation and increased permeability. Testing intestinal permeability in vulnerable preterm human subjects is often not feasible. Thus, an in vitro fetal tissue-derived intestinal model is needed to study intestinal injuries in preterm infants. Enteroids can be used to test changes in epithelial permeability regulated by tight junction proteins. In enteroids, intestinal stem cells differentiate into all epithelial cell types and form a three-dimensional structure on a basement membrane matrix secreted by mouse sarcoma cells. In this article, we describe the methods used for establishing enteroids from fetal intestinal tissue, characterizing the enteroid tight junction proteins with immunofluorescent imaging, and testing epithelial permeability. As gram-negative dominant bacterial dysbiosis is a known risk factor for intestinal injury, we used lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, to induce permeability in the enteroids. Fluorescein-labeled dextran was microinjected into the enteroid lumen, and serial dextran concentrations leaked into the culture media were measured to quantify the changes in paracellular permeability. The experiment showed that apical exposure to LPS induces epithelial permeability in a concentration-dependent manner. These findings support the hypothesis that gram-negative dominant dysbiosis contributes to the mechanism of intestinal injury in preterm infants.

This protocol details the establishment of enteroids, a three-dimensional intestinal model, from fetal intestinal tissue. Immunofluorescent imaging of epithelial biomarkers was used for model characterization. Apical exposure of lipopolysaccharides, a bacterial endotoxin, using microinjection technique induced epithelial permeability in a dose-dependent manner measured by the leakage of fluorescent dextran.

Testen der Epithelpermeabilität in fetalen Gewebe-abgeleiteten Enteroiden

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

In Vitro und In Vivo Ansätze Darm Epithelzelle Durchlässigkeit zu bestimmen

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

Biologie

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

Organoid-abgeleitete Epithel-Monolayer: Ein klinisch relevantes In-vitro-Modell für die Darmbarrierefunktion

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier impairment has been associated with the development of various pathologies such as inflammatory bowel diseases (IBD). In the inflamed mucosa, superficial erosions or micro-erosions that corrupt epithelial monolayers correspond to sites of high permeability. Several mechanisms have been implicated in the formation of micro-erosions including cell shedding and apoptosis. These micro-erosions often represent microscopic epithelial gaps randomly distributed in the colon. Visualization and quantification of those epithelial gaps has emerged as an important tool to investigate intestinal epithelial barrier function. Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa. Additional use of cell death markers revealed that HPR encompass apoptotic foci where epithelial extrusion/shedding occurs. The protocol described here provides a simple but effective approach to visualize and quantify micro-erosions in the intestine, which is a very useful tool in disease models, in which the intestinal epithelial barrier is compromised.

Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa.

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier ...

Entwicklung von 2D-Monolayer-Kulturen aus rinderischen Darmorganoiden

Advancements in Bovine Organoid Technology Using Small and Large Intestinal Monolayer Interfaces

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models that faithfully mimic in vivo intestinal tissues. This is particularly vital for investigating host-pathogen interactions in bovines, which are significant reservoirs for pathogens that pose serious public health risks. Traditional 3D organoids offer limited access to the intestinal epithelium's apical surface, a hurdle overcome by the advent of 2D monolayer cultures. These cultures, derived from organoid cells, provide an exposed luminal surface for more accessible study. In this research, a detailed protocol is introduced for creating and sustaining 2D monolayer cultures from cells of bovine small and large intestinal organoids. This method includes protocols for assessing membrane integrity through transepithelial electrical resistance and paracellular permeability alongside immunocytochemistry staining techniques. These protocols lay the groundwork for establishing and characterizing a 2D bovine monolayer culture system, pushing the boundaries of these method applications in biomedical and translational research of public health importance. Employing this innovative approach enables the development of physiologically pertinent in vitro models for exploring both normal and diseased states of cattle intestinal physiology. The implications for biomedical and agricultural advancements are profound, paving the way for more effective treatments for intestinal ailments in cattle, thereby enhancing both animal welfare and food safety.

Author Spotlight: Entwicklung und Charakterisierung von 2D-intestinalen Monolayer-Modellen aus rinderlichen Organoiden für Pathogen-Interaktionsstudien

This study presents a protocol for generating bovine intestinal 2D monolayers from organoids, offering improved access for studying host-pathogen interactions. It includes methods for assessing membrane integrity and functionality, advancing in vitro models that mimic cattle's gastrointestinal physiology. This approach promises significant biomedical and agricultural benefits, including enhanced treatment strategies.

Fortschritte in der Rinder-Organoid-Technologie unter Verwendung von Dünn- und Dickdarm-Monolayer-Grenzflächen

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models ...