Name: mouse fetal whole intestine culture system for ex vivo manipulation of signaling pathways and three-dimensional live imaging of villus development
Uploaded: 2014-09-04T14:00:00.000+00:00
Duration: 6 min 46 s
Description: Watch this Scientific Journal Video about Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development at JoVE.com

We gratefully acknowledge Dr. Deborah L. Gumucio as our advisor and for her invaluable support in defining the culture and imaging methods. We also thank Dr. Jim Brodie, Dr. Hong-Xiang Lu, Dr. Charlotte Mistretta, and Dr. Ann Grosse for their contributions to the development of the whole intestine organ culture system. Helpful discussions on imaging provided excellent advice from Dr. Chip Montrose, Michael Czerwinski and Sasha Meshinchi. All imaging was performed in the Microscopy and Image Analysis Laboratory at the University of Michigan. Funding support was provided by NIH R01 DK065850.

NOT: Tüm farenin Laboratuar Hayvan Hastalıkları Michigan Tıp Fakültesi Birimi Üniversitesi tarafından onaylanmış ve Hayvanları Kullanımı ve Bakımı Üniversite Komitesi kurallarına göre protokollerini kullanarak insanca ele alındı. 1. Tüm Organ Kültür Sistemi <ol><li> Medya ve kültür plakalarının hazırlanması <ol><li> Bir doku kültürü kaputu, stok şişe edilebilen BGJb ortamdan 5 ml kaldırmak ve Kalem / Strep 5 ml ekleyin. 5 mg / (Pen / Strep) ile edilebilen BGJb ortam 49 ml ml askorbik asit 1 ml ilave etmek suretiyle, 50 ml&#39;lik konik bir tüp içinde bir ortam çalışma stok hazırlayın. </li><li> Transwells her altında 1 ml ilave etmek suretiyle, bir 6 yuvalı kültür plakası hazırlayın. NOT: Tüm 6 transwells kullanılan değilseniz, daha sonra kullanmak üzere yeni bir steril 6 plaka ekstra transwells taşıyın. Üç adet 10 cm steril petri kutularının her birine edilebilen BGJb ortam çalışma 3 ml ilave edilir. </li></ol></li><li> Diseksiyon için Hazırlık <ol><li> 70% Etal içinde diseksiyon araçları batırınve metanol çalışırken temiz araçları tutmak için emin olun. </li><li> Büyük bir buz kovası ile diseksiyon alanı ayarlayın ve 6 kuyu kültür plaka ve buz üzerinde edilebilen BGJb medya ile 10 cm petri kapları yerleştirin. Diseksiyon ve kültür için bağırsakları hazırlarken mümkün olduğunca buz üzerinde çalışın. </li><li> Fetal bağırsakların toplama için servikal dislokasyon izofluoran (100 ul) önce euthanization için olan bir oda içinde erişkin dişi fareler anestezi. </li><li> Onların anneden fetusa hasat ettikten sonra, her fetusun dikkatli grafiği 13 evreleme Theiler göre düzenlerler. Rutin bir tek çöp içinde gözlenen gelişimsel aşamada kadar 24 saat varyasyonları olarak her fetusun yaşını belirlemek için post-koitus günlerde güvenmeyin. </li></ol></li><li> Fetal bağırsak diseksiyonu <ol><li> Bağırsakların kaldırılması için önce, başı kesilerek fetüsleri Euthanize. </li><li> Pla sonra 1x Dulbecco Fosfat Tamponlu Salin (DPBS) her bağırsak inceleyin veedilebilen BGJb ortamı çalışan bir 10 cm&#39;lik bir petri kabı içine ce. </li><li> Dikkatlice mide, dalak, mide ve oniki parmak bağırsağı üst elde edilen pankreasa, kaldırın. </li><li> Yavaşça doğruldu bağırsak izin mümkün ve yalnızca yeterli bağlantılarını ayıran olduğunca bağlı omentum bırakmak için özen omentum ayırın. Not: yavaşça lümen içeriği bağırsak yoluyla akması ve kesik uçlarından çıkarılmasına imkan vermek için 3 eşit bölümlere ayırmak bağırsak bağırsak E14.5 daha büyük veya daha fazla olanlar, 24 saat daha kültive edilir için. Bununla birlikte, kültürlerin E13.5 önce, serozayı uygun bağırsak uzunluğu boyunca büyümeye izin bağırsak 3 kısımları ayıran önce kültürlenmesi kadar 24 saat sonra başladı bekleyin. </li><li> Kültür plakası (1.1.2) transwells üzerine bağırsak parçaları aktarmak için bir ağız pipeti kullanılır. </li><li> Onlar transwel genelinde düz böylece gerektiği gibi yavaşça bağırsakları yeniden konumlandırmakl. NOT: bağırsaklar tüp yoluyla lümen içeriğini hareket başladıktan sonra, bağırsakta herhangi bir karışıklığı bağırsağa bir yedekleme ve hasara neden olur. </li></ol></li><li> Kültür ve tedavi <ol><li> , Transwell altından medyayı çıkarın transwell altında (ilave rekombinant proteinlerin veya farmakolojik inhibitörleri ile medya çalışma) tedavi medya 700 ul ekleyin. </li><li> Bağırsakların üstüne tedavi medya damla damla 300 ul ekleyin ve transwell yoluyla bekletin. Gerekirse bağırsakları yerleştirin. </li><li> Tedavi reaktif yarı ömrüne bağlı olarak en az günde bir kere ya da daha çok muamele ortam değiştirin. </li></ol></li><li> Tedavi reaktif lokalize bir protein kaynağı batırılmış agaroz taneleri hazırlanması <ol><li> Kendi depolama kabı agaroz boncuklar tekrar ve 1.5 ml mikrofüj tüpüne agaroz boncuklarının 100 ul transfer. </li><li> Steril 1x Ph borunun geri kalan hacim dolumunaosphate-tamponlu salin (PBS) ile yıkamak üzere, taneler üzerine karıştırın. Birkaç dakika boncuklar dibe ve daha sonra tanelerin üzerine PBS kapalı Pipeti izin vermek üzere askıya mikrofüj tüpü yerleştirin. Steril 1x PBS ile mikrofuge&#39;ye tüp dolum ve iki kez daha yıkama işlemini tekrarlayın. </li><li> Iki kez tedavi için istenen konsantrasyona ve ilgi konusu proteini hazırlayın. </li><li> 2x Protein stoklar 5 ul ile yıkanmış agaroz boncuk 5 ul karıştırın ve yavaşça protein boncuk çalkalayın. Yavaşça, 10-15 dakika daha karıştırıldı, 1 saat boyunca buz üzerinde tüp yerleştirin. </li><li> Bağırsakların boncuk yerleştirmeden önce steril 1x PBS kısaca boncuk durulayın. </li></ol></li><li> Agaroz boncukları Yerleştirme <ol><li> Yavaşça çekilmiş kılcal iğneli bir ağız pipeti kullanılarak bağırsakların üst agaroz boncuklar yerleştirin. Kaba işleme bağırsaklara zarar ve yaralı bölgede villus gelişimini önleyecek şekilde aşırı ihtiyatla boncuk yerleştirin. </li><li> Bağırsak peristaltik hareketleri ve kültür zamanla bağırsakların inecektir yerleştirilen boncuklar yaklaşık yarısını geçirecek beri analiz için gerekli iki katı kadar boncuk yerleştirin. </li></ol></li><li> Kültürlü bağırsakların Fixation <ol><li> Dikkatle transwell alttan tedavi ortamı çıkarın ve 3 dakika boyunca havada kurumaya bağırsakları izin verir. </li><li> Daha sonra tespit süresinin geri kalan kısmı için sabitleyici 2 ml bağırsağın üst kapağı, 2 dakika boyunca transwell altında fiksatif 1 ml ilave edilir. 4 ° C&#39;de ya da oda sıcaklığında 2 saat süresince% 4 paraformaldehit O / N sabitleyin. </li></ol></li></ol> Sabit Bağırsaklar 2. Görüntüleme <ol><li> Wholemount görüntüleme <ol><li> DPBS 1x 100 ul, bir lam üzerine (iç floresan ile) bütün bağırsak yerleştirin. Yavaşça bağırsaklara düzenlemek için forseps kullanın. </li><li> Mo doku yapmak için dikkatli bir şekilde% 70 gliserol, 100 ul DPBS çıkarın ve hemen eklemek için bir laboratuar doku kullanınşeffaf yeniden ve görüntüleme olası derinliğini artırır. Not: bağırsağın başka penetrasyon isteniyorsa yerine% 70 gliserol içinde bağırsak Sabitleme, 10-30 dakika boyunca Odak sonuçları çözeltisinin bir kaç damla bağırsak ıslatın. Odak Temizle çözümü kapalı pipetlemeyin Mount Clear bağırsak monte edin. </li><li> Modelleme kil içine lamel dört köşesinden her batırın ve bağırsak düzleştirme lamel tutmak için slayt ve kapak arasında ayırıcı olarak kullanmak için kil küçük bir miktar pick up. </li><li> Bir pamuklu çubukla uygulanan erimiş valap ile slayt üzerine lamel mühür. </li><li> Görüntü ya bir dik veya ters konfokal mikroskop bağırsaklar. Daha fazla ayrıntı için tartışma bakınız. </li></ol></li><li> (Bağırsak yapıların enine kesit görünümü için) sabit bağırsak vibratome kesit <ol><li> Küçük plastik kalıplar% 7 agaroz katıştırmak için bağırsak (10 x 10 x 15 mm), izin agaroz bloklarsoğumaya ve katılaşmaya. </li><li> Anlık yapıştırıcı ile Vibratome toplama aşamasına plastik kalıp ve tutkal agaroz bloklardan agaroz blokları çıkarın. </li><li> Buz gibi soğuk 1x DPBS toplama sahne doldurun. </li><li> 100-150 um arasında bir kalınlıkta kesitler halinde kesilmiştir. Kalın kesitler kesiti içine derin görselleştirmek için (bölüm 2.1.2 açıklanan) takas çözümleri kullanın. </li><li> 4 ° C de depolama için, bir 6-yuvalı plaka içinde bir kuyu içine toplama aşamasından vibratome bölümleri dökün. </li></ol></li><li> Sabit, Vibratome kesitli dokuların İmmünofloresan boyanması <ol><li> 1x DPBS ile bir 24-yuvalı plaka içine, göz başına 5-12 vibratome bölümleri yerleştirin. </li><li> 1x DPBS çıkarın ve oyuk başına geçirgenleştirme çözeltisi 500 ul ilave edin. Yavaşça oda sıcaklığında 25 dakika boyunca sallayın örnekleri. </li><li> Geçirgenleştirmeden sonra, 1x PBS ile numuneleri 3 kere yıkayın. </li><li> Bloke etme çözeltisi 500 ul içinde en az 30 dakika boyunca numune bloke eder. </li><li> Engelleme, e sonraçözümü engelleme seyreltilmiş primer antikor 500 ul engelleme çözüm Xchange ve 4 ° CO / N de örnekleri tutmak. NOT: Dondurulmuş ve parafin bölümlerde işe birincil antikor dilüsyonları genellikle Ancak genellikle bu protokolü (örneğin, BrdU) ile iyi çalışmaz nükleer antijen alımı gerektiren antikorlar, çok Vibratome bölümlerde iyi çalışır. </li><li> Ertesi gün, bloke etme çözeltisi ile 15 dakika boyunca numuneleri her 3 kez yıkayın. </li><li> Yıkamadan sonra, bloke etme çözeltisi içinde seyreltilmiş sekonder antikor, 500 ul de geçerlidir. Oda sıcaklığında 2 saat 45 dakika boyunca örnekleri ışıktan korumak ve kaya florofor için alüminyum folyo ile 24 oyuklu plaka sarın. </li><li> 15 dakika her biri için 1x PBS ile numuneleri 3 kez yıkayın ve Bölüm 2.4 &#39;de açıklandığı gibi slaytlar üzerinde vibratome bölümleri monte edin. </li></ol></li><li> Vibratome bölümleri Montaj <ol><li> Vibratome çift sti ince dilim keserek montaj için slaytlar hazırlayınck bant ve slaytlar uzun kenarları boyunca onları yerleştirerek. </li><li> Dikkatle 1x PBS 100 ul bir damla slaytlar üzerinde çift kaset sopa arası 5-10 vibratome bölümleri yerleştirin. </li><li> Tüm bölümlerini açın ve slaytta tek bir tabaka içine yayıldı. </li><li> Düz oturan bir coverslip engelleyecek herhangi bir aşırı agarozun veya düzensiz parçalarını çıkarın. </li><li> Dikkatle Vibratome bölümler etrafında aşırı 1x PBS kadar emmek için bir laboratuar doku kullanın. </li><li> Vibratome bölümlerin üstüne monte sulu ortam bir damla ekleme ve sonra da lamel. </li><li> Valap bir pamuklu çubukla uygulanan eritilmiş slaytlar üzerine lamelleri Seal. </li><li> Görüntü ya bir dik veya ters konfokal mikroskop vibratome bölümleri. Bir görüntüleme sisteminin seçimi konusunda ayrıntılı bilgi için tartışmaya bakınız. </li></ol></li></ol> Tüm Bağırsaklar 3. Canlı Görüntüleme Bağırsaklar fetuse hasatBağlı omentum sağlam sol s ile E12.5 sonra, başarıyla yukarıdaki sistemini kullanarak kültür içinde villus gelişir. Bu nedenle, bu ex vivo sistem zamanla morfolojilerinden bir üç boyutlu görünüm için yeniden olabilir gelişen bağırsak canlı z-bölüm görüntüler yakalama izin verir. <ol><li> Canlı görüntüleme ayarlama <ol><li> Ince örgülü elek için tek bir polikarbonat membran bağlı anında tutkal. Bu dik konfokal mikroskop daldırma lens aşağıda yerinde bağırsak tutmak için bir destek iskele sağlar. Canlı görüntüleme mikroskoplar seçimi hakkında ayrıntılı bilgi için tartışma bölümüne bakınız. </li><li> Merkezinde iyi bir kültür tepsisi içine elek yerleştirin. </li><li> Polikarbonat membran üzerine disseke bağırsak yerleştirin ve her iki ucunda anlık tutkal bir damla ekleyin. Bağırsak tutturmak için yalnızca yeterli tutkal kullanın, ama ceket o! </li><li> Polikarbonat altında fenol kırmızısının kültür ortamı ücretsiz eklentiMerkezi de kültür plakasında membran. </li><li> Nem temin etmek üzere kültür plakasının dış kenarı için su ilave edilir. </li><li> Görüntüleme sırasında bağırsak hareketini kontrol etmek için ortam 3 ml 1 x PBS&#39;de ksilazin 5 çözeltisi: fetal barsak kültürde peristaltik kasılma benzeri dalgalar maruz kaldığından, 1 100 ul ilave edin. Bu dozaj villus gelişimini ödün vermeden yaklaşık 8-10 saat bağırsak hareketlerini kontrol edecek. </li><li> Bağırsağın bir enine görünüm için, 3-4% agaroz çözeltisi içinde canlı bağırsakları gömmek ve bir vibratom kalın kesitler (150-500 mikron) kesilir. Kesilmiş olarak, bağırsak sertliğe sahip agaroz sertliğini maç ve ampirik olarak bir gelişme aşamasındaki belirlenir. Genellikle,% 3 agaroz çözüm E14.5 daha genç bağırsaklar için iyi çalışır ve% 4 çözelti bağırsaklar E15-E16 için iyi çalışır. </li><li> (Kısım 3.1.1 gibi) gözenekli bir elek tutturulmuş bir polikarbonat membran vibratome bölümleri ve TutkalKültür olarak Bölüm 3.1.2-3.1.6 tarif edilmiştir. </li><li> Ayarlanabilir bir tablo ile dik bir stand üzerine bir iki-foton konfokal floresan mikroskop (690-1,040 nm arasında uyarma lazer) kullanılarak canlı görüntüleme Davranış. </li><li> İki foton lazer GFP sinyalleri için en iyi çözünürlük ile derin penetrasyon sağlar nm 900 ayarlı. Görüntülenir iken bu dokulara zarar azaltır. Buna ek olarak, mümkün olan en düşük emisyon için lazerin gücünü ayarlayıcı, doku hasarı azaltır. Tipik olarak, GFP iyi bir uyarma elde iki foton lazer% 12 güç kullanmak. </li></ol></li></ol>

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The authors have no financial conflicts to disclose.

Dinamik yapısı ve gelişen bağırsak karmaşık doku etkileşimleri bu morfogenetik olayların tam bir takdir 3D görselleştirme gerektirir. Görüntüleme Gelişen teknolojiye, yetenek / gelişmekte iyileştirilmesi ve onunla, organogenezis sırasında mekansal iletişim ve etkileşim anlayışı oldukça gelişmiş edilir detaylı olarak villus morfogenetiğine incelemek. Bütün bağırsakları kültürü için alternatif yöntemleri de test edilmiştir, ancak Transwell sistem uzun k?...

Each intestinal villus is composed of two main tissue compartments: an epithelial surface layer and a mesenchymal core. The mouse small intestine is formed at embryonic day 10 when a sheet of endoderm closes and seals to form a tube of epithelium surrounded by mesenchymal cells3. This flat tube of epithelium undergoes rapid proliferation, growing both in length and girth and undergoes dramatic rearrangements involving dynamic cell shape changes1. At the same time, the surrounding mesenchyme also undergoes many developmental processes including the formation of the vascular plexus, differentiation of smooth muscle and recruitment of enteric neurons4. In the proximal small intestine at embryonic day 14.5, condensations (clusters) of Hedgehog- and PDGF-responsive cells begin to form adjacent to the epithelium2,5. Formation of mesenchymal clusters continues to spread along the length of the intestine so that they cover the entirety of the small intestine by embryonic day 16.52. As mesenchymal clusters form, the epithelial cells closest to the clusters begin to withdraw from the cell cycle, while the other epithelial cells continue to proliferate. Those cells directly above the mesenchymal cluster that have withdrawn from the cell cycle begin to change shape as the emerging villus buckles into the lumen. Further growth of the villus is driven in part by the continued proliferation of the epithelium between the emerging villi. The mesenchymal clusters remain tightly adhered to the epithelium of the growing villus and continue to express a variety of signaling molecules. The wave of villus emergence propagates along the length of the small intestine following the formation of mesenchymal clusters. As the intestine continues to grow and the intervillus region extends between emerging villi, new mesenchymal clusters form adjacent to the intervillus epithelium and further rounds of villus emergence and growth ensue6.
Synchronized development of the epithelium and mesenchyme is essential for villus morphogenesis. Signaling molecules are secreted from one layer to the other where receptors receive and transduce the signal message in order to coordinate development between the epithelium and mesenchyme. Mesenchymal clusters act as signaling centers and express a variety of developmental morphogens7-10. Disruption of cluster formation or pattern results in loss of villus emergence and pattern. Inhibition of PDGF signaling results in fewer clusters and fewer villi and those villi that do form are misshapen following the abnormal clusters11. Loss of Hedgehog signaling results in complete loss of cluster formation and failure of villus emergence2,12. Together, these data demonstrate that clusters coordinate development of the villus epithelium with its mesenchymal core.
Using this whole organ culture system, we are able to alter signaling involved in epithelial-mesenchymal cluster cross-talk to determine the role of those signals in villus morphogenesis. Two-photon confocal optical sectioning and reconstruction afford the ability to visualize cluster formation and villus emergence in three-dimensions and better understand the spatial relationships between the mesenchymal clusters and their overlying epithelium. Extending the culture system to four dimensions, we can capture z-stacks of developing clusters and villi over time and observe these interactions. Ultimately, the ability to follow villus development in this manner and observe changes that occur with altered signaling will revolutionize the understanding of epithelial mesenchymal interactions in villus morphogenesis.

<table><tbody><tr><td>Fine dissecting forceps&amp;nbsp;</td><td>Fine Science Tools</td><td>&amp;nbsp;11254-20</td><td>2 pairs</td></tr><tr><td>70% Ethanol</td><td></td><td></td><td></td></tr><tr><td>1x sterile Dulbecco&#039;s Phosphate-Buffered Saline (DPBS)</td><td>Gibco&amp;nbsp;</td><td>14040-133</td><td>500 ml</td></tr><tr><td>6 well plates</td><td>Costar</td><td>3516</td><td></td></tr><tr><td>24 well plates</td><td>Costar&amp;nbsp;</td><td>3524</td><td></td></tr><tr><td>60 x 15 mm petri dishes</td><td>Falcon</td><td>&amp;nbsp;451007</td><td></td></tr><tr><td>Transwell plates, 24 mm inserts, 8.0 mm polycarbonate membranes</td><td>Corning Costar&amp;nbsp;</td><td>3428</td><td>6 inserts per plate</td></tr><tr><td>BGJb media</td><td>Invitrogen&amp;nbsp;</td><td>12591-038</td><td>500 ml</td></tr><tr><td>PenStrep (10,000U/ml Penicillin; 10,000 mg/ml Streptomycin)</td><td>Gibco</td><td>&amp;nbsp;15140</td><td></td></tr><tr><td>Ascorbic Acid</td><td>Sigma&amp;nbsp;</td><td>A0278</td><td>make 5 mg/ml stock, filter, aliquot and store at -20 &amp;deg;C</td></tr><tr><td>Mouth pipet (Drummond 1-15 inch aspirator tube assembly)</td><td>Fisher</td><td>&amp;nbsp;21-180-10</td><td>remove the aspirator assembly and replace it with a 1,000 &amp;micro;l pipet tip which acts as an adaptor to plug in a 6 inch glass Pasteur pipet.</td></tr><tr><td>6 inch glass pasteur pipets</td><td></td><td></td><td></td></tr><tr><td>Capillary Tubes</td><td>World Precision Instruments&amp;nbsp;</td><td>TW100F-4</td><td>pull to needles</td></tr><tr><td>4% Paraformaldehyde</td><td></td><td></td><td>made in 1 x PBS, pH to 7.3</td></tr><tr><td>Culture plates</td><td>Falcon&amp;nbsp;</td><td>353037</td><td></td></tr><tr><td>Fine mesh stainless steel screen</td><td>purchase at hardware store</td><td></td><td></td></tr><tr><td>Polycarbonate membranes</td><td>Thomas scientific&amp;nbsp;</td><td>4663H25</td><td>alternatively, cut Corning Costar 3428 membranes off of transwell supports</td></tr><tr><td>Instant glue</td><td>purchase at hardware store</td><td>gel based preferrably</td><td></td></tr><tr><td>35 x 10 mm plates</td><td>Falcon&amp;nbsp;</td><td>351008</td><td></td></tr><tr><td>7% agarose</td><td>Sigma</td><td>&amp;nbsp;A9414</td><td>prepare w/v in 1x DPBS, heating to dissolve in a waterbath</td></tr><tr><td>minutien pins</td><td>Fine Science Tools&amp;nbsp;</td><td>26002-20</td><td></td></tr><tr><td>Phenol red free media (DMEM)</td><td>Gibco&amp;nbsp;</td><td>21063-029</td><td></td></tr><tr><td>Xylazine (100 mg/ml)</td><td>AnaSed&amp;nbsp;</td><td>139-236</td><td></td></tr><tr><td>Matrigel</td><td>BD</td><td>356231</td><td>basement membrane matrix, growth factor reduced, phenol red-free</td></tr><tr><td>3-4% agarose</td><td>Sigma&amp;nbsp;</td><td>A9414</td><td>prepare w/v in 1x DPBS, heating to dissolve in a waterbath</td></tr><tr><td>Imaging of fixed intestines</td><td></td><td></td><td></td></tr><tr><td>Name of Material/Equipment</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>vaseline</td><td>purchase at pharmacy</td><td></td><td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td></tr><tr><td>lanolin</td><td>Sigma&amp;nbsp;</td><td>L7387</td><td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td></tr><tr><td>paraffin</td><td>Surgipath</td><td>39601006</td><td>used to make VALAP: equal parts vaseline, lanolin, paraffin</td></tr><tr><td>70% glycerol in 1 x PBS</td><td></td><td></td><td></td></tr><tr><td>Focus clear and Mount Clear</td><td>CelExplorer Labs Co.</td><td>&amp;nbsp;F101-KIT</td><td></td></tr><tr><td>Modeling clay</td><td>purchase at art supply store</td><td></td><td></td></tr><tr><td>double stick tape</td><td></td><td></td><td></td></tr><tr><td>cotton applicator swabs</td><td></td><td></td><td></td></tr><tr><td>plastic molds, 10mm x 10mm x 5 mm)</td><td>Tissue Tek&amp;nbsp;</td><td>4565</td><td></td></tr><tr><td>slides</td><td></td><td></td><td></td></tr><tr><td>coverslips</td><td></td><td></td><td></td></tr><tr><td>lab wipe</td><td>Kimberly Clark</td><td>34155</td><td>lint free delicate task wipe</td></tr><tr><td>Theiler staging chart&amp;nbsp;</td><td></td><td></td><td>&lt;a href=&quot;http://www.emouseatlas.org/emap/ema/theiler_stages/%20downloads/theiler2.pdf&quot;&gt;http://www.emouseatlas.org/emap/ema/theiler_stages/ downloads/theiler2.pdf&lt;/a&gt;</td></tr><tr><td>Leica SP5X confocal microscope&amp;nbsp;</td><td>Leica</td><td></td><td>Used to conduct the live imaging&amp;nbsp;</td></tr><tr><td>Leica DMI 6000 stand&amp;nbsp;</td><td>Leica</td><td></td><td>Used to conduct the live imaging&amp;nbsp;</td></tr><tr><td>Aqueous mounting medium (Prolong Gold)</td><td>Molecular Probes&amp;nbsp;</td><td>P36930</td><td></td></tr><tr><td>Name of Material/Equipment</td><td>Company</td><td>Catalog Number</td><td>Comments/Description</td></tr><tr><td>24 well plate</td><td>Costar</td><td>&amp;nbsp;3524</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma&amp;nbsp;</td><td>T-8787</td><td>used to make Permeabilization solution: 0.5% Triton X-100 in 1 x PBS</td></tr><tr><td>Goat serum</td><td></td><td></td><td>used to make Blocking Solution: 4% Goat serum, 0.1% Tween20 in 1x PBS</td></tr><tr><td>Tween20&amp;nbsp;</td><td>Sigma</td><td>&amp;nbsp;P9416</td><td></td></tr></tbody></table>

mouse fetal whole intestine culture system for ex vivo manipulation of signaling pathways and three-dimensional live imaging of villus development

Fetal bağırsakta en morfogenetik süreçler gelişim aşamaları üzerinde değişikliklerin anlık sağlayarak, sabit dokuların ince kesitlerinin olayla edilmiştir. İnce Seri kesitlerde üç boyutlu bilgiler, çünkü mükemmel seri kesitler yeniden ve seri kesitler üzerinde dokusunun uygun yönünü sürdürmenin zorluğu yorumlamak zor olabilir. Tarafından Grosse ve ark. Son bulgular, 2011 bağırsak 1 gelişmekte villuslarda morfogenetiğine anlamada üç boyutlu bilgilerin önemini vurgulamak. Tek başına etiketli bağırsak hücreleri üç boyutlu yeniden yapılandırma bağırsak epitel hücrelerinin çoğunluğu her iki en üst ve taban yüzeylerine temas gösterdi. Ayrıca, epitel apikal yüzeyinde aktin hücre iskeletinin üç boyutlu rekonstrüksiyon bağırsak lümen sürekli olduğunu ve ikincil lümen s bir eserdir olduğunu gösterdiectioning. Bu iki nokta, bağırsak epitelinde interkinetic nükleer göç gösteri ile birlikte, bir yalancı epiteli olarak gelişen bağırsak epiteli tanımlanmış ve önceden 1 düşünce olarak tabakalı değil. Epitel üç boyutlu görebilme bu noktayı gösteren ve fetal bağırsakta epitel morfogenetiğine yeniden tanımlıyor ufuk açıcı oldu. Çoklu foton görüntüleme teknolojisi ve üç boyutlu rekonstrüksiyon yazılım evrimi ile, yeteneği hızla iyileştirilmesi organ geliştirmeyi, sağlam görselleştirmek için. İki foton uyarma yüksek çözünürlüklü dokulara daha az zarar penetrasyon derin verir. İki foton görüntüleme ve Walton vd. Tüm fetal fare bağırsak 3D rekonstrüksiyon 2012 villus akıbet 2 deseni tanımlamak için yardımcı oldu. Burada izin vermek için bu kültür sisteminin villus ve uzantıları ex vivo gelişimi sağlayan bir bütün organ kültür sistemi açıklanmaktadırbağırsaklar üç boyutlu gelişim sırasında görüntülenmiş olması.

Bu sistem farmakolojik reaktifler veya rekombinant proteinleri ile sinyalizasyon manipülasyon sağlayan fetal Bağırsakların tüm eksplantlarının Kültür, bağırsak gelişimini koordine sinyal moleküllerinin konumu, dağılımı ve süresi analizler için izin verir. (Walton ve ark yeniden Şekil 1A., 2012) Transwell kültür sistemi 2 mümkün doku üzerine ilaç veya proteini batırılmış agaroz boncuklar yerleştirmek ve böylece lokal sinyal kaynaklarının bir...

Watch this Scientific Journal Video about Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development at JoVE.com

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Için Fare Fetal Tüm Bağırsak Kültür Sistemi<em&gt; Ex vivo</em&gt; Sinyal facebook ve villus Kalkınma Üç boyutlu Canlı Görüntüleme Manipülasyon

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

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Biology

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

14.9K Görüntüleme.  University of Michigan. Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine. 

Video: Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

Kullanımı<em&gt; Ex Vivo</em&gt; Tüm Fetal Organları Dik Damlacık Kültürleri Fare Organogenesis sırasında Gelişim Süreçleri Eğitim için

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

Gelişim Biyolojisi

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a population of rapidly cycling intestinal stem cells (ISCs) characterized by their expression of the Lgr5 gene. These are supported by a quiescent stem cell population, marked by Bmi-1 expression, capable of replacing them in the event of injury. Investigating the interactions between these populations is crucial to understanding their roles in disease and cancer. The ISCs exist within crypts on the intestinal surface, these niches support the ISC in replenishing the epithelia. The interaction between active and quiescent ISCs likely involves other differentiated cells within the niche, as it has previously been demonstrated that the &#8216;&#8216;stemness&#8217;&#8217; of the Lgr5 ISC is closely tied to the presence of their neighboring Paneth cells. Using conditional cre-lox mouse models we tested the effect of deleting the majority of active ISCs in the presence or absence of the Paneth cells. Here we describe the techniques and analysis undertaken to characterize the intestine and demonstrate that the Paneth cells play a crucial role within the ISC niche in aiding recovery following substantial insult.

Protocols for Analyzing the Role of Paneth Cells in Regenerating the Murine Intestine using Conditional Cre-lox Mouse Models

Intestinal epithelial stem cells (ISCs) are intermingled with Paneth cells. These cells are differentiated progeny of the ISC, which support the ISCs and provide antibacterial protection. Here we demonstrate how we used transgenic conditional mouse models to establish that Paneth cells play a crucial role in maintaining the intestinal epithelia.

Şartlı kullanarak Fare İnce Bağırsağında Yenileyici içinde Paneth Hücrelerinin Rolü analiz için protokoller<em&gt; Cre-lox</em&gt; Fare Modelleri

The epithelial surface of the mammalian intestine is a dynamic tissue that renews every 3 - 7 days. Understanding this renewal process identified a ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

Bir fare modelinde Bağırsak Doku Mühendisliği

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

Biyomühendislik

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The present protocol describes establishing a murine colonic organoid system to study the activity and functioning of colonic stem cells in a claudin-7 knockout model.

Bağırsak Kök Hücre Fonksiyonunu Incelemek için Murin Kolonik Kriptlerinin Üç Boyutlu Kültürü Ex Vivo

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of ...

Kanser Araştırmaları

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

içinde Microengineered İnsan Bağırsak Villi Oturma mikrobiyomu Co-kültür, bir Gut-on-a-Chip mikroakışkan Aygıt

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

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 ...

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

<em&gt; Ex Vivo</emFare Bağırsak Organoids ve&gt; Kültür ve Desen Tanıma Reseptör Uyarım

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

İmmünoloji ve Enfeksiyon

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Için bir sistem<em&gt; Ex vivo</emEmbriyonik Pankreas&gt; ekimi

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Biyoloji

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

Enteroendokrin Hücre Gut Hormon salgısını Çalışması ve Canlı Hücre Görüntüleme için Amaçlanan Kemirgen Küçük Bağırsak Karışık İlköğretim Kültürler

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

Sıçan Bağırsak Kriptleri İzolasyonu ve Bağırsak Organoidleri Üretimi

Generation and Manipulation of Rat Intestinal Organoids

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. Accordingly, patient-derived organoids are used for disease modeling, drug discovery, and personalized treatment screening. Mouse intestinal organoids are commonly utilized to understand aspects of both intestinal function/physiology and stem cell dynamics/fate decisions. However, in many disease contexts, rats are often preferred over mice as a model due to their greater physiological similarity to humans in terms of disease pathophysiology. The rat model has been limited by a lack of genetic tools available in vivo, and rat intestinal organoids have proven fragile and difficult to culture long-term. Here, we build upon previously published protocols to robustly generate rat intestinal organoids from the duodenum and jejunum. We provide an overview of several downstream applications utilizing rat intestinal organoids, including functional swelling assays, whole mount staining, the generation of 2D enteroid monolayers, and lentiviral transduction. The rat organoid model provides a practical solution to the need of the field for an in vitro model which retains physiological relevance to humans, can be quickly genetically manipulated, and is easily obtained without the barriers involved in procuring human intestinal organoids.

Yazar Spotlight: Sıçan Bağırsak Organoidlerinin Üretimi ve Manipülasyonu  

Here, we present a protocol to generate rat intestinal organoids and use them in several downstream applications. Rats are often a preferred preclinical model, and the robust intestinal organoid system fills the need for an in vitro system to accompany in vivo studies.

Için Fare Fetal Tüm Bağırsak Kültür Sistemi

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Kullanımı

Şartlı kullanarak Fare İnce Bağırsağında Yenileyici içinde Paneth Hücrelerinin Rolü analiz için protokoller

Bir fare modelinde Bağırsak Doku Mühendisliği

Bağırsak Kök Hücre Fonksiyonunu Incelemek için Murin Kolonik Kriptlerinin Üç Boyutlu Kültürü Ex Vivo

içinde Microengineered İnsan Bağırsak Villi Oturma mikrobiyomu Co-kültür, bir Gut-on-a-Chip mikroakışkan Aygıt

Fetal Intestinal Organoidler Kullanılarak Tüp Bebek Teyidi--Weaning Geçiş

Için bir sistem

Enteroendokrin Hücre Gut Hormon salgısını Çalışması ve Canlı Hücre Görüntüleme için Amaçlanan Kemirgen Küçük Bağırsak Karışık İlköğretim Kültürler

Sıçan Bağırsak Organoidlerinin Üretimi ve Manipülasyonu