Name: real-time measurement of epithelial barrier permeability in human intestinal organoids
Uploaded: 2017-12-18T15:00:00.000+00:00
Duration: 8 min 4 s
Description: Watch this Scientific Journal Video about Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids at JoVE.com

Yazarlar Drs. Stephanie Spohn ve Basel Abuaita organoid mikroenjeksiyon birçok yararlı tartışmalar için teşekkür etmek istiyorum. Solumasını bağırsak kök hücre Konsorsiyumu (U01DK103141), diyabet Ulusal Enstitüsü ve sindirim ve böbrek hastalıkları (NIDDK) ve Ulusal Enstitüsü alerji ve bulaşıcı hastalıklar (NIAID) tarafından finanse edilen bir ortak araştırma projesi tarafından desteklenir. Solumasını ve VBY NIAID roman, alternatif Model sistemleri için enterik hastalıklar (NAMSED) Konsorsiyumu (U19AI116482) tarafından desteklenmektedir. DRH desteklenmektedir mikrobiyal Patogenez mekanizmaları eğitim hibe Ulusal Enstitüsü alerji ve enfeksiyon hastalıkları (NIAID, T32AI007528) ve klinik ve çevirim Bilim Ödülü Michigan Enstitüsü Klinik ve sağlık için Araştırma (UL1TR000433).
Tam veri dosyaları ve bu el yazması kullanılan veri analizi kodu https://github.com/hilldr/HIO_microinjection mevcuttur.

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Yazarlar hiçbir rakip mali çıkarlarının veya diğer çatışmaları ifşa etmek var.

Bu protokol, gerçek zamanlı olarak hPSC elde edilen HIOs ve bağırsak organoids doku kaynaklı mikroenjeksiyon ve epitel bariyer geçirgenliği ölçümü için genel amaçlı bir yöntem oluşturur. Biz de analiz ve bu yöntemleri kullanan oluşturulan verilerin yorumlanması bizim yaklaşım göstermiştir. Bağırsak organoids büyüyen kabulü model sistemleri verilen16,20,21,28 ve fonksiyonel bağırsak bariyer geçirgenliği gibi fizyolojik ilgili uzun süredir ilgi sonuç3,4,5,6, biz diğerleri bu alanda çalışan uygulamak ve bu yöntemler kurmak mümkün olacak tahmin.
Bu teknik uygulama için kritik olan birkaç adım vardır. Yüksek kalite hPSC - veya türetilmiş HIO doku mikroenjeksiyon ile kapsamlı deneme önce kurulmalıdır doku erişim. Doku kimlik ve hücresel Morfoloji olmasına rağmen son derece tekrarlanabilir HIOs24üretmek için kurulan metodoloji kullanan zaman HIO macrostructure hànzú, her iki boyutu ve şekli, varyasyon ile olabilir. Tek bir yarı saydam Lümen oluşan ve yaklaşık 1 mm çapında ölçme küresel HIOs mikroenjeksiyon ve gerçek zamanlı olarak luminal Floresans ölçümü için idealdir. Bazı durumlarda, mikroenjeksiyon, HIO çöküşü enjekte malzeme açık kaçağı sonuçlanabilecek başarısız olur. Başarısız HIOs-ebilmek var olmak çıkarmak ile kültür de kullanıcının isteği doğrultusunda standart bir micropipette kullanarak. Objektif lensler bir görüntüleme platformu üzerinde kullanılabilir HIOs mikroenjeksiyon ve görüntüleme için seçerken göz önünde bulundurun. 10 X amaç düşük güç lensler kullanılabilir değilse veya mevcut HIOs varsa kullanılabilir, ancak genel olarak, 2-4 X objektif lens tam HIO floresan sinyal yakalamak için idealdir &#60; 1 mm çapında. Disk görüntüsü oluşturma yazılımı için tanımlanmış noktalarda floresan görüntülerin otomatik yakalama zaman içinde izin vermeniz gerekir.
Bu protokol birçok değişikliği deneysel gereksinimlerine uymak için mümkündür. Örneğin, bariyer fonksiyonu testlerinin sonuçlarını kullanım43 bileşiklerin moleküler boyutu bağımlı olabilir ve değişen molekül ağırlığı dextran hazırlıkları test etmek uygun olabilir. Buna ek olarak, aydınlık alan görüntüleme Floresans görüntüleme doku25genel olarak yapısal bütünlüğünü bir göstergesi olarak ek olarak gerçekleştirilebilir. Canlı bakteri25,28,31,32,33,44mikroenjeksiyon işlemi sırasında penisilin eklemek gerekli olabilir ve streptomisin veya gentamisin önce veya sonra mikroenjeksiyon HIO kültür medyaya. Microcapillary dışına doldurma ile bakteri kültürü Süspansiyon sırasında kontamine ve bu HIO medyaya devredilebilir. Alternatif olarak, mikroenjeksiyon mikroenjeksiyon tamamlandıktan sonra medya ekleme medya, hücre dışı Matriks (örneğin, Matrigel) askıya HIOs üzerinde gerçekleştirilebilir. Bu kirlenme hücre dışı matriks ve dış yüzü HIO sınırlayabilir. Mikrobiyal büyüme deneyleri planlarken, antibiyotik medyada yavaşlama veya microinjected organizmaların büyümesini engelleyerek önlemek için 1-2 h sonra kaldırmak gerekli olabilir.
Son olarak, tüm araştırmacılar mikroskobu ekipman vitro görüntüleme için uygun erişimi kabul ederek, Floresans veri toplamak için bu protokolü bölümünde özetlenen yordamlar çekilen görüntülere uygulanabilir işaret etmek önemlidir otomatik görüntü yakalama veya çevresel kontroller standart epifluorescent mikroskobu kullanarak sabit timepoints. Bu yaklaşımın örnekleri raporlarda Leslie ve Huang vd tarafından bulunabilir C. difficile toksin aktivite bağırsak organoids hPSC elde edilen Karve ve Pradan ve ark. , muayene 25, epitel bariyer geçirgenliği ile canlı E. colimicroinjected benzer hPSC elde edilen bağırsak organoids içinde muayene 44. Ekipman görüntüleme el ile işlem daha fazla değişim ve floresan sinyal normalize zorluk neden olabilir. El ile görüntüleme FITC dextran performans HIOs enjekte edildiğinde sabit büyütme, floresan uyarma yoğunluğu ve pozlama süreleri floresan yoğunluk ölçümleri bozan önlemek için deney boyunca korumak için önemlidir.

Bağırsak epitel besinler, H2O, iyonlar ve atık ürünler yönlü taşımacılığının diğer parçacıklar Lümen arasında Difüzyon-aracılı nonspesifik alışverişini en aza indirerek aracılık eder seçici bir bariyer oluşturur ve Mezenkimal doku veya kan kaynağı1,2. Bağırsak epitel bariyer nonspesifik geçirgenliği uzun oranı yansıtan sağlık ve hastalık3,4,5,6, bir anahtar işlev parametre kabul edilmiştir Difüzyon yolu ile paracellular alan epitel boyunca küçük moleküllerin. Epitel bariyer geçirgenliği ölçümü içinde yürütülen hayvan modelleri7 ve insan hasta8 ' de laktuloz sindirim yoluyla, gastrointestinal sistem ve sonraki koleksiyonu belirli hiçbir ışınlama sahip olduğu ve laktuloz konsantrasyonlarda periferik kan ölçümü. Alternatif alınan işaretleri fluorescently etiketli karbonhidratlar gibi bariyer fonksiyonunun da mevcut9,10vardır. Bu yaklaşım Transwell destekler11, daha fazla deneysel kontrol için izin verir, ancak aynı zamanda içinde vivo zavallı bir tahmin eleştirilmiştir basitleştirilmiş bir yaklaşım üzerinde yetiştirilen bağırsak epitel hücre kültürleri için adapte edilmiş geçirgenliği farklılaştırılmış epitel alt türlerini ve doku yapısı12yokluğu nedeniyle. Chambers'ı kullanarak yeni bir yaklaşım temsil ve epitel bariyer fonksiyonu tüm bağırsak mukoza ex vivo13' te ölçümü için izin verir. Bu tekniğin uygulanması sık doku kullanılabilirlik durumu13,ve14tarafından sınırlıdır. Böylece tekrarlanabilirlik ve işlem hacmi fizyolojik alaka ile dengeleyen yeni yöntemler gereklidir.
Vitro organogenesis son gelişmeler 3D doku kültürü model sistemleri kabulü karmaşık doku15,16,17 dinamikleri recapitulating için sofistike bir platform olarak yol açmıştır ,18,19,20,21,22,23. Özellikle, insan pluripotent kök hücre (hPSC) elde edilen insan bağırsak organoids (HIOs)19,24 konak-mikrobiyal etkileşim incelenmesi için bir tekrarlanabilir ve deneysel olarak uysal modeli sistem olarak çıkmıştır ve epitel bariyer dynamics25,26,27,28. Benzer şekilde, insan doku kaynaklı organoids (enteroids olarak da bilinir) bir basit biyopsi yordamla elde ve uysal bir sistem olarak insan fizyolojisi ve hastalık15,29,30eğitim için kullanılabilir. İnsan bağırsak organoids mikroenjeksiyon deneysel bileşikler25 teslimi için izin verir veya mikropların25,31,32,33 ' e apikal epitel yaşamak organoid Lümen yüzeyine. Leslie ve Huang vd. 25 son zamanlarda bakteriyel toksinler maruz takip dextran etiketli floresein isothiocyanate (FITC) ile microinjected HIOs bariyer geçirgenliği ölçmek için bu tekniği adapte.
Bu iletişim kuralı hPSC elde edilen HIOs ve floresan mikroskopi kullanarak doku kaynaklı HIOs epitel bariyer geçirgenliği ölçüm için bir kılavuz olarak hazırlanmıştır. Küçük değişiklikler ile o da HIOs mikroenjeksiyon deneysel bileşikler ile genel bir astar olarak hizmet verebilir. Kullanıcılar bu iletişim kuralı ek veya alternatif aşağı akım uygulamaları ile mikroenjeksiyon sonra ek.

<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

Biyopsi ile elde edilen veya pluripotent kök hücreler yönlendirilmiş farklılaşma yoluyla üretilen bağırsak dokuların 3D kültür gelişmeler bağırsak mukoza sofistike vitro modellerinde sonuçlandı. Ortaya çıkan bu model sistemleri yararlanarak araçlar ve teknikler 2D kültür sistemleri ve hayvanlar için geliştirilen uyum gerektirir. Burada, gerçek zamanlı olarak insan bağırsak organoids epitel bariyer geçirgenliği ölçüm tekniği tarif. Bu mikroenjeksiyon fluorescently etiketli dextran ve görüntüleme epifluorescent filtre ile donatılmış bir ters mikroskop Tarih tarafından gerçekleştirilir. Bu teknik aynı zamanda yaklaşımlar Imaging sabit timepoint birlikte gerçek zamanlı bağırsak organoids bariyer geçirgenliği ölçümü insan bağırsak epitel dokusu, yüksek çözünürlüklü zamansal veri nesil kolaylaştırır. Farmakolojik ajanlar, bakteriyel ürünler veya toksinler veya canlı mikroorganizmalar maruz kalma takip epitel bariyer geçirgenliği ölçümü için kolayca uyarlanabilir bir protokoldür. Küçük değişikliklerle bu protokolü de bağırsak organoids mikroenjeksiyon genel bir astar olarak hizmet verebilir ve kullanıcılar bu protokolü mikroenjeksiyon aşağıdaki ek veya alternatif aşağı akım uygulamaları ile tamamlamak tercih edebilir.

HIOs insan pluripotent kök hücreleri ayrıştırılan ve hücre matris çözüm olarak daha önce açıklanan19,24kültürlü. Kültür 4 hafta sonra HIOs mikroenjeksiyon için izin vermek için yeterince genişletti. HIOs 4 kDa FITC Birleşik dextran PBS veya Clostridium difficile toksin TcdA içeren PBS askıya ile microinjected. C. difficile HIOs25epitel toksisite toksin-aracılı sergileyen bir fırsatçı gastrointestinal hastalık var. Pozitif kontrol, EGTA bir alt kümesini PBS ve FITC dextran ile enjekte HIOs HIO kültür medyada eklenmiştir. EGTA hızlı de-polimerizasyon aktin sitoiskeleti42neden olur bir kalsiyum şelatör var. FITC Floresans gerçek zamanlı canlı görüntüleme mikroskop içinde kontrollü bir çevre odası olarak izlenmeli ve görüntüleri 10 dakikalık aralıklarla ele geçirildi.
Görüntüleme veri Post-hoc çözümlemesi FITC floresan (şekil 5) tutma önemli farklılıkları ortaya koydu. PBS ile enjekte HIOs muhafaza neredeyse tüm floresan sinyal t mevcut Ayrıca TcdA ile EGTA ile tedavi enjekte edildi HIOs 8 saat sonrası mikroenjeksiyon (şekil floresan yoğunluğu önemli bir düşüş sergiledi ancak = 0, 5A). görüntüleme veri göreceli değişiklik floresan yoğunluğu zamanla deneysel her durumda (şekil 5B) temsil eden yüksek çözünürlüklü bir veri kümesi oluşturmak tüm HIOs tüm zaman noktalarda için sayısal. Epitel geçirgenliği farklılıkları her tedavi grubu (Tablo 1) FITC (t1/2 ) ortalama eliminasyon zaman hesaplama ve t t1/2 gruplar arasındaki farkları karşılaştırarak değerlendirildi kullanarak öğrenci t-test. Denetimi değerlendirilmesi HIOs muhafaza FITC floresan sinyal 16 saatten fazla çoğunluğu (t1/2 = 17 ± 0,3 h). EGTA ile tedavi azaltılacağını FITC dextran eleme zaman denetime HIOs göre (t1/2 = 2.6 ± 0.2 h; P = 1.3 x 10-8). Daha önce yayımlanmış sonuçları25ile tutarlı, TcdA mikroenjeksiyon önemli ölçüde epitel bariyer geçirgenliği kontrol tedavi göre artar (t1/2 = 7,6 ± 0.6 h; P = 5.4 x 10-4). Böylece, dış (EGTA) ve microinjected (TcdA) bileşikleri HIOs epitel bariyer geçirgenliği içinde önemli değişiklikler inducing yeteneğine sahiptirler. Bu sonuçlar epitel bariyer fonksiyonu çok çeşitli farmakolojik ajanlar, metabolitleri, bakteriyel ürünleri, sitokinler, büyüme faktörleri ve diğer bileşikler etkileri bu yaklaşımı kullanarak değerlendirilecek öneririz.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tedavi</td>
			<td>Half-Life (h)</td>
			<td>SEM (h)</td>
			<td>% 95 CI (h) indir</td>
			<td>Üst %95 CI (h)</td>
			<td>n</td>
		</tr>
		<tr>
			<td>Denetim</td>
			<td>17.11</td>
			<td>0,3</td>
			<td>16,45</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>TcdA</td>
			<td>7.56</td>
			<td>0,63</td>
			<td>5.93</td>
			<td>9,18</td>
			<td>6</td>
		</tr>
	</tbody>
</table>
Tablo 1: demek eleme zaman (t1/2) FITC-dextran HIOs içinde EGTA veya TcdA ile tedavi için. Saat sonrası mikroenjeksiyon birimleridir.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56960/56960fig1.jpg"> 
Şekil 1: microinjector ve micromanipulator HIO mikroenjeksiyon için temel düzenini. Bu görüntü cihazları HIOs mikroenjeksiyon gerçekleştirmek için tam tamamlayıcı gösterir. Anahtar bileşenleri olarak etiketlenir ve sipariş bilgileri malzemeleri tablosunda bulunabilir. <a href="//ecsource.jove.com/files/ftp_upload/56960/56960fig1large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56960/56960fig2.jpg"> 
Resim 2: Micropipette çektirme. Bakır bobin hc, üst kelepçe c1, alt kelepçe (c2), Isıtma çektirme kol (pa), ısı Seçimi Değiştir (ht) oklarla tanımlanır. Doğru ayarlar için ısı 1 ve çekme kırmızı metin olarak gösterilir. İlave bir doğru şekilde monte edilmiş cam filament Isıtma için hazır gösterir. <a href="//ecsource.jove.com/files/ftp_upload/56960/56960fig2large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56960/56960fig3.jpg"> 
Şekil 3: FITC-dextran ile enjekte bir doku kaynaklı HIO temsilcisi görüntülerini. (A)görüntü cam microcapillary hemen önce HIO ve mikroenjeksiyon içine ekleme konumlandırma gösteren. (B) bir doku elde edilen insan bağırsak organoids FITC dextran mikroenjeksiyon sonra aydınlık alan görüntü. Floresans sinyal bile bir süzgeç kümesi kullanımı belirgin olduğunu unutmayın. Bu renklendirme mikroenjeksiyon hassas yardımcı olur. 3 X büyütme <a href="//ecsource.jove.com/files/ftp_upload/56960/56960fig3large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56960/56960fig4.jpg"> 
Şekil 4: görüntüleme iş akışı yaşamak. (A)mikroskop parametrelerini ayarlamak slayt üzerinde HIOs bulup her HIO yansıması için sahne konumunu kaydetmek gereken adımları gösteren ekran görüntüsü. (B) öncesi görüntüleme ayarları her konumları düzenli aralıklarla FITC kanal yakalamak için DV formatında veri A. (C) sonrası görüntüleme dışa aktarma işlemine belirtilen dosyaları TIFF görüntüleri ile her zaman noktası HIO başına bir TIFF görüntü olarak.<a href="//ecsource.jove.com/files/ftp_upload/56960/56960fig4large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56960/56960fig5.jpg"> 
Şekil 5: temsilcisi sonuçları. (A)kök hücre elde edilen 2 mg/mL ile FITC dextran microinjected insan bağırsak organoids (HIO) (4 kDa) görüntüsü 20 saattir. HIOs da PBS (kontrol) veya Clostridium difficile toksin TcdA ile microinjected (12,8 ng/µL) veya 2 EGTA dış kültür ortamına eklenen mM ile tedavi. 4 X büyütme. (B) arsa zaman HIOs içinde ortalama normalleştirilmiş FITC yoğunluğunun PBS (kontrol), TcdA veya EGTA ile tedavi. Hata çubukları temsil S.E.M. ve n = 11 HIOs (kontrol), 3 HIOs (EGTA), 6 HIOs (TcdA). <a href="//ecsource.jove.com/files/ftp_upload/56960/56960fig5large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</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

Bu iletişim kuralı insan bağırsak organoids floresan mikroskopi kullanarak gerçek zamanlı aşağıdaki farmakolojik tedavide epitel bariyer geçirgenliği ölçümü açıklar ve cep mikroskobu canlı.

Gerçek zamanlı insan bağırsak Organoids epitel bariyer geçirgenliği ölçümü

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

Research

JoVE Journal

Developmental Biology

14.1K Görüntüleme.  University of Michigan. Bu iletişim kuralı insan bağırsak organoids floresan mikroskopi kullanarak gerçek zamanlı aşağıdaki farmakolojik tedavide epitel bariyer geçirgenliği ölçümü açıklar ve cep mikroskobu canlı.

Video: 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 Bağırsak Sacs Gastrointestinal Hastalık Modelleri mukozal geçirgenliği Değerlendirmek için

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

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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.

Bağırsak Bölgesine Özgü İşlevselliği Modellemek için İnsan Organoidlerini ve Çip Üzerinde Organ Teknolojisini Birleştirme

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

Biyomühendislik

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.

İntravital Mikroskopi Tabanlı Bir Yaklaşım Bağırsak Geçirgenliği ve Epitel Hücre Dökülme Performansını Değerlendirmek için

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

İmmünoloji ve Enfeksiyon

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.

Apikal-Out Enteroid Modelde Lucifer Sarı Kullanarak Bağırsak Geçirgenliğinin Belirlenmesi

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.

Yaşayan Organoidlerde Bağırsak Bariyerinin Bozulmasının Araştırılması

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.

Fetal Doku Kaynaklı Enteroidlerde Epitel Geçirgenliğinin Test Edilmesi

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.

Vitro ve In Vivo yaklaşımlar Bağırsak epitel hücre geçirgenliği belirlemek için

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

Biyoloji

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 Kaynaklı Epitel Tek Katmanlı: Bağırsak Bariyer Fonksiyonu için Klinik Olarak İlgili Bir In Vitro Model

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 ...

Sığır Bağırsak Organoidlerinden 2 Boyutlu Monotabaka Kültürlerin Geliştirilmesi

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.

Yazar Spotlight: Patojen Etkileşim Çalışmaları için Sığır Organoidlerinden 2D Bağırsak Tek Tabaka Modellerinin Geliştirilmesi ve Karakterizasyonu

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.

İnce ve Kalın Bağırsak Tek Katmanlı Arayüzleri Kullanılarak Sığır Organoid Teknolojisindeki Gelişmeler

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