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.

<ol>
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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. 24</td>
		</tr>
		<tr>
			<td>B27 supplement (50X)</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td>Component of ENR media; see McCraken et al. 24</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. 24</td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Life Technologies</td>
			<td>15630080</td>
			<td>Component of ENR media; see McCraken et al. 24</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 C. difficile Toxin A/TcdA Protein</td>
			<td>R&#38;D Systems</td>
			<td>8619-GT-020</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>R&#38;D Systems</td>
			<td>236-EG</td>
			<td>Component of ENR media; see McCraken et al. 24</td>
		</tr>
		<tr>
			<td>R-spondin 1</td>
			<td>R&#38;D Systems</td>
			<td>4645-RS</td>
			<td>Component of ENR media; see McCraken et al. 24</td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>R&#38;D Systems</td>
			<td>6057-NG</td>
			<td>Component of ENR media; see McCraken et al. 24</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

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JoVE Journal

Developmental Biology

14.0K 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

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease phenotypes. Recent advances have identified more efficient and safe methods for introduction of reprogramming factors. However, there are few tools to monitor and track the progression of reprogramming. Current methods for monitoring reprogramming rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. Tools to dissect the progression of iPSC generation can help better understand the process under different conditions from diverse cell sources. 
This study presents key approaches for kinetic measurement of reprogramming progression using flow cytometry as well as real-time monitoring via imaging. To measure the kinetics of reprogramming, flow analysis was performed at discrete time points using antibodies against positive and negative pluripotent stem cell markers. The combination of real-time visualization and flow analysis enables the quantitative study of reprogramming at different stages and provides a more accurate comparison of different systems and methods. Real-time, image-based analysis was used for the continuous monitoring of fibroblasts as they are reprogrammed in a feeder-free medium system. The kinetics of colony formation was measured based on confluence in the phase contrast or fluorescence channels after staining with live alkaline phosphatase dye or antibodies against SSEA4 or TRA-1-60. The results indicated that measurement of confluence provides semi-quantitative metrics to monitor the progression of reprogramming.

The protocol presented in this study describes methods for the real-time monitoring of reprogramming progression via the kinetic measurement of positive and negative pluripotent stem cell markers using flow cytometry analysis. The protocol also includes the imaging-based assessment of morphology, and marker or reporter expression during iPSC generation.

Somatik yeniden programlanması Kinetik Ölçümü ve Gerçek Zamanlı Görselleştirme

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease ...

Gelişim Biyolojisi

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

IMMUNO florasan mikrotübüller ve Ex Vivo bağırsak dokusu ve 3D Vitro bağırsak Organoids Centrosomal proteinler etiketleme

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to manipulate gene expression in organoids have made limited progress in this arena. While CRISPR/Cas9 technology allows for precise genome editing of cells for organoid generation, this strategy requires extensive selection and screening by sequence analysis, which is both time-consuming and costly. Here, we provide a detailed protocol for efficient viral transduction of intestinal organoids. This approach is rapid and highly efficient, thus decreasing the time and expense inherent in CRISPR/Cas9 technology. We also present a protocol to generate frozen sections from intact organoid cultures for further analysis with immunohistochemical or immunofluorescent staining, which can be used to confirm gene expression or silencing. After successful transduction of viral vectors for gene expression or silencing is achieved, intestinal stem cell and crypt function can be rapidly assessed. Although most organoid studies employ in vitro assays, organoids can also be delivered to mice for in vivo functional analyses. Moreover, our approaches are advantageous for predicting therapeutic responses to drugs because currently available therapies generally function by modulating gene expression or protein function rather than altering the genome.

We describe step-by-step instructions to: 1) efficiently engineer intestinal organoids using magnetic nanoparticles for lenti- or retroviral transduction, and, 2) generate frozen sections from engineered organoids. This approach provides a powerful tool to efficiently alter gene expression in organoids for investigation of downstream effects.

Primer fare bağırsak Organoidlerin genetik mühendisliği dondurulmuş bölümleme için Manyetik Nano madde dönüştürücü viral vektörler kullanma

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

Multicellular Human Primer Endometrial Organoidlerin Üretimi

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Murin Nörogenezi Sırasında Çentik Sinyal Dinamiğinin Gerçek Zamanlı Biyolüminesans Görüntülemesi

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to digest solid food. This process is termed suckling-to-weaning transition and results in the replacement of neonatal epithelium with adult epithelium which goes hand in hand with metabolic and morphological adjustments. These complex developmental changes are the result of a genetic program that is intrinsic to the intestinal epithelial cells but can, to some extent, be modulated by extrinsic factors. Prolonged culture of mouse primary intestinal epithelial cells from late fetal period, recapitulates suckling-to-weaning transition in vitro. Here, we describe a detailed protocol for mouse fetal intestinal organoid culture best suited to model this process in vitro. We describe several useful assays designed to monitor the change of intestinal functions associated with suckling-to-weaning transition over time. Additionally, we include an example of an extrinsic factor that is capable to affect suckling-to-weaning transition in vivo, as a representation of modulating the timing of suckling-to-weaning transition in vitro. This in vitro approach can be used to study molecular mechanisms of the suckling-to-weaning transition as well as modulators of this process. Importantly, with respect to animal ethics in research, replacing in vivo models by this in vitro model contributes to refinement of animal experiments and possibly to a reduction in the use of animals to study gut maturation processes.

This protocol describes how to mimic suckling-to-weaning transition in vitro using mouse late fetal intestinal organoids cultured for 30 days.

Fetal Intestinal Organoidler Kullanılarak Tüp Bebek Teyidi--Weaning Geçiş

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

İnsan iPSC Tabanlı Kan-Beyin Bariyer Chip Üretimi

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.&#160; Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.

This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy.

Farelerde Periosteal İskelet Kök Hücrelerinin CCL5 Kaynaklı Göçünün Gerçek Zamanlı Görüntülenmesi

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of ...

Establishing Organoids from Human Tooth as a Powerful Tool Toward Mechanistic Research and Regenerative Therapy

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development and biology is scarce. In particular, not much is known about the tooth's epithelial stem cells and their function. We succeeded in developing a novel organoid model starting from human tooth tissue (i.e., dental follicle, isolated from extracted wisdom teeth). The organoids are robustly and long-term expandable and recapitulate the proposed human tooth epithelial stem cell compartment in terms of marker expression as well as functional activity. In particular, the organoids are capable to unfold an ameloblast differentiation process as occurring in vivo during amelogenesis. This unique organoid model will provide a powerful tool to study not only human tooth development but also dental pathology, and may pave the way toward tooth-regenerative therapy. Replacing lost teeth with a biological tooth based on this new organoid model could be an appealing alternative to the current standard implantation of synthetic materials.

We present a protocol to develop epithelial organoid cultures starting from human tooth. The organoids are robustly expandable and recapitulate the tooth's epithelial stem cells, including their ameloblast differentiation capacity. The unique organoid model provides a promising tool to study human dental (stem cell) biology with perspectives for tooth-regenerative approaches.

İnsan Dişinden Organoidlerin Mekanik Araştırma ve Rejeneratif Terapiye Doğru Güçlü Bir Araç Olarak Kurulması

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development ...

Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish with fluorescently-marked plasma membranes can be scanned in a confocal laser scanning microscope in order to determine the volume of muscle tissue and the number of muscle fibers present. Efficient approaches for the measurement of cell number and size in live animals over time are described and validated against more arduous segmentation methods. Methods are described that permit the control of muscle electrical, and thus contractile, activity. Loss of skeletal muscle contractile activity greatly reduced muscle growth. In larvae, a protocol is described that allows reintroduction of patterned electrical-evoked contractile activity. The described methods minimize the effect of inter-individual variability and will permit analysis of the effect of electrical, genetic, drug, or environmental stimuli on a variety of cellular and physiological growth parameters in the context of the living organism. Long-term follow-up of the measured effects of a defined early-life intervention on individuals can subsequently be performed.

Optical clarity is a major advantage for cell biological and physiological work in zebrafish. Robust methods for measurement of cell growth in individual animals are described that permit novel insights into how growth of skeletal muscle and neighboring tissues are integrated with whole body growth.

Değiştirilmiş elektriksel aktiviteye maruz kalan bireysel canlı zebra balıklarında iskelet kası büyümesinin gerçek zamanlı ve tekrarlanan ölçümü

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish ...