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.

NOTA: Tutti i topi sono stati trattati umanamente utilizzando protocolli approvati dalla University of Michigan Medical School Unità per la Medicina di Laboratorio animali e secondo le linee guida della Commissione di Ateneo per l&#39;uso e la cura degli animali. 1. intero sistema Cultura Organo <ol><li> Preparazione dei supporti e piastre di coltura <ol><li> In una cappa di coltura tissutale, rimuovere 5 ml di BGJb supporto dalla bottiglia stock e aggiungere 5 ml di Pen / Strep. Preparare uno stock di lavoro di media in un tubo conico da 50 ml, con l&#39;aggiunta di 1 ml di 5 mg / ml di acido ascorbico a 49 ml di BGJb media (con Pen / Strep). </li><li> Preparare una piastra di coltura ben 6 con l&#39;aggiunta di 1 ml di sotto ciascuna delle transwells. NOTA: Se non si utilizzano tutti i 6 transwells, spostare transwells in più in una sterile piatto 6 e fresco per un uso successivo. Aggiungere 3 ml di lavorare BGJb media per ciascuna delle tre piastre di Petri sterili 10 centimetri. </li></ol></li><li> Preparazione per la dissezione <ol><li> Immergere gli strumenti di dissezione nel 70% etanolo perNol ed essere sicuri di mantenere gli strumenti puliti durante il lavoro. </li><li> Impostare l&#39;area dissezione con un grande secchio di ghiaccio e posizionare la piastra di coltura ben 6 e 10 centimetri di Petri con BGJb multimediale su ghiaccio. Lavorare sul ghiaccio il più possibile mentre dissezione e preparare l&#39;intestino per la cultura. </li><li> Anestetizzare adulto topi femmina in una camera con isofluorane (100 ml) prima euthanization da dislocazione cervicale per la raccolta di intestino fetale. </li><li> Dopo la raccolta i feti dalla madre, in scena ogni feto con attenzione secondo la Theiler messa in scena tabella 13. Non fare affidamento nei giorni post-coito per determinare l&#39;età di ogni feto come variazioni fino a 24 ore in fase di sviluppo sono abitualmente osservati all&#39;interno di una singola cucciolata. </li></ol></li><li> Dissezione di intestini fetali <ol><li> Euthanize feti di decapitazione prima della rimozione di intestino. </li><li> Sezionare ogni intestino di 1x Dulbecco Phosphate-Buffered Saline (DPBS) e poi place in una piastra di Petri 10 centimetri con il lavoro BGJb media. </li><li> Rimuovere con cautela la milza dallo stomaco e il pancreas dallo stomaco e duodeno superiore. </li><li> Separare delicatamente il omento avendo cura di lasciare il omento divisoria per quanto possibile e separare connessioni solo sufficiente a permettere l&#39;intestino da raddrizzare. NOTA: Per intestini di età superiore a E14.5 o quelli in coltura più di 24 ore, separare delicatamente l&#39;intestino in 3 segmenti uguali per consentire contenuto luminale di fluire attraverso l&#39;intestino ed essere espulsi dalle estremità tagliate. Tuttavia, per le culture iniziato prima E13.5, attendere fino a dopo 24 ore di coltura prima di separare i tre segmenti di intestino per permettere la sierosa di crescere correttamente sulla lunghezza dell&#39;intestino. </li><li> Utilizzare una pipetta bocca per trasferire i pezzi intestinali sui transwells della piastra di coltura (1.1.2). </li><li> Riposizionare delicatamente l&#39;intestino, se necessario in modo che siano dritti alla transwell. NOTA: Una volta che gli intestini cominciano muoversi contenuto luminale attraverso il tubo, eventuali pieghe a livello intestinale causerà un backup e danni per l&#39;intestino. </li></ol></li><li> Cultura e trattamento <ol><li> Rimuovere il supporto da sotto il transwell, aggiungere 700 ml di mezzi di trattamento (dei media che lavorano con l&#39;aggiunta di proteine ​​ricombinanti o inibitori farmacologici) sotto il transwell. </li><li> Aggiungere 300 ml di supporti trattamento goccia a goccia sulla parte superiore dell&#39;intestino e lasciare in ammollo attraverso il transwell. Riposizionare l&#39;intestino, se necessario. </li><li> Cambiare mezzi trattamento almeno una volta al giorno o più spesso a seconda del tempo di dimezzamento del reagente trattamento. </li></ol></li><li> Preparazione di proteine ​​imbevuto agarosio perline per una fonte localizzata di reagente trattamento <ol><li> Risospendere le perline agarosio nel loro contenitore e trasferire 100 ml di perline agarosio in una provetta da 1,5 ml microcentrifuga. </li><li> Riempire il volume rimanente del tubo con sterile 1x Phosphate-Buffered Saline (PBS) e mescolare le perle di lavarli. Collocare la provetta da microcentrifuga in un rack per alcuni minuti per consentire le perle di depositarsi sul fondo e poi pipettare fuori dalla PBS in cima alle perline. Riempire la provetta per microcentrifuga con 1x PBS sterile e ripetere il processo di lavaggio altre due volte. </li><li> Preparare la proteina di interesse a due volte la concentrazione desiderata per il trattamento. </li><li> Mescolare 5 ml di perline agarosio lavato con 5 ml di proteina magazzino 2x e agitare delicatamente le perline nella proteina. Porre la provetta in ghiaccio per 1 ora, mescolando delicatamente ogni 10-15 min. </li><li> Sciacquare le perline brevemente sterile 1x PBS prima di mettere le perline su intestini. </li></ol></li><li> Posizionamento di perline agarosio <ol><li> Posizionare delicatamente le perline agarosio sulla parte superiore dell&#39;intestino utilizzando una pipetta bocca con aghi capillari tirato. Posizionare le perle con estrema cautela in quanto un trasporto sicuro danneggia l&#39;intestino e prevenire lo sviluppo dei villi nella zona lesa. </li><li> Posizionare il doppio delle perle come sono necessari per l&#39;analisi in quanto l&#39;intestino saranno sottoposti a movimenti peristaltici e circa la metà delle perline poste rotolerà fuori degli intestini oltre il tempo di coltura. </li></ol></li><li> Fissazione di intestino in coltura <ol><li> Rimuovere con cautela il supporto di trattamento da sotto la transwell e permettere l&#39;intestino di aria secca per 3 min. </li><li> Aggiungere 1 ml di fissativo sotto la transwell per 2 minuti, quindi coprire la parte superiore dell&#39;intestino con 2 ml di fissativo per il resto del tempo di fissazione. Fissare con 4% paraformaldeide O / N a 4 ° C o per 2 ore a temperatura ambiente. </li></ol></li></ol> 2 Imaging di Intestino fisse <ol><li> Immagini Wholemount <ol><li> Posizionare l&#39;intero intestino (con fluorescenza endogena) su un vetrino con 100 ml di 1x DPBS. Usare pinze per organizzare delicatamente l&#39;intestino. </li><li> Utilizzare un tessuto laboratorio per rimuovere con attenzione i DPBS e immediatamente aggiungere 100 ml di glicerolo 70% per rendere il mo tessutiri trasparente e aumentare la possibile profondità di imaging. NOTA: Se si desidera ulteriore penetrazione dell&#39;intestino, invece di montare l&#39;intestino in 70% glicerolo, immergere l&#39;intestino in poche gocce di soluzione di messa a fuoco libero per 10-30 min. Pipettare fuori la soluzione messa a fuoco chiara e montare l&#39;intestino con il Monte Chiaro. </li><li> Immergere ciascuno dei quattro angoli di un coprioggetto in creta e raccogliere una piccola quantità di argilla da utilizzare come distanziatori tra il vetrino e coprioggetto per mantenere il coprioggetto da appiattimento dell&#39;intestino. </li><li> Sigillare il coprioggetto sul vetrino con VALAP fusa applicato con un batuffolo di cotone. </li><li> Immagine l&#39;intestino su entrambi un microscopio confocale in posizione verticale o invertita. Si prega di fare riferimento alla discussione per ulteriori dettagli. </li></ol></li><li> Vibratome sezionamento di intestino fissi (per la vista in sezione trasversale di strutture intestinali) <ol><li> Intestini incorporare nel 7% di agarosio in piccoli stampi di plastica (10 x 10 x 15 mm) e consentire i blocchi di agarosio araffreddare e solidificare. </li><li> Rimuovere i blocchi di agarosio dagli stampi di plastica e blocchi di colla di agarosio alla fase di raccolta vibratome con colla istantanea. </li><li> Riempire la fase della raccolta con il freddo ghiaccio 1x DPBS. </li><li> Tagliare sezioni ad uno spessore compreso tra 100-150 micron. Per le sezioni più spesse utilizzano le soluzioni di compensazione (descritti nella sezione 2.1.2) per visualizzare più in profondità la sezione trasversale. </li><li> Versare vibratome sezioni dalla fase di raccolta in un pozzetto di una piastra da 6 pozzetti per la conservazione a 4 ° C. </li></ol></li><li> Immunofluorescenza colorazione dei tessuti, vibratome sezionati fissi <ol><li> Posizionare 5-12 sezioni vibratome per bene in un 24-pozzetti con 1x DPBS. </li><li> Rimuovere 1x DPBS e aggiungere 500 ml di soluzione di permeabilizzazione per pozzetto. Agitare delicatamente i campioni per 25 minuti a temperatura ambiente. </li><li> Dopo permeabilizzazione, lavare i campioni 3 volte con PBS 1x. </li><li> Bloccare i campioni per almeno 30 minuti in 500 ml di Blocking Solution. </li><li> Dopo il blocco, exchange la soluzione di saturazione con 500 ml di anticorpi primari diluiti in soluzione bloccante e mantenere i campioni a 4 ° CO / N. NOTA: Le diluizioni degli anticorpi primari che funzionano bene su sezioni congelate e paraffina generalmente funzionano bene su vibratome sezioni troppo, ma gli anticorpi che richiede il recupero dell&#39;antigene nucleare in genere non funzionano bene con questo protocollo (ad esempio, BrdU). </li><li> Il giorno seguente, lavare i campioni 3 volte per 15 minuti ogni volta con soluzione bloccante. </li><li> Dopo il lavaggio, applicare 500 ml di anticorpo secondario diluito in soluzione bloccante. Avvolgere la piastra di 24 pozzetti in un foglio di alluminio per proteggere i fluorofori dalla luce e rock i campioni per 45 minuti a 2 ore a temperatura ambiente. </li><li> Lavare i campioni 3 volte con PBS 1x per 15 minuti ciascuno e montare le sezioni vibratome su vetrini come descritto nella Sezione 2.4. </li></ol></li><li> Montaggio vibratome sezioni <ol><li> Preparare i vetrini per vibratome il montaggio tagliando fette sottili di double-stinastro ck e metterli lungo i lati lunghi delle diapositive. </li><li> Posizionare con cura 5-10 sezioni vibratome tra il nastro biadesivo sulle diapositive in una goccia di 100 microlitri di PBS 1x. </li><li> Aprire tutte le sezioni e diffondere fuori in un unico livello in tutta la diapositiva. </li><li> Rimuovere eventuali agarosio eccesso o pezzi irregolari che potrebbero impedire un coprioggetto da seduta piatta. </li><li> Utilizzare con cautela un tessuto laboratorio per assorbire l&#39;eccesso di PBS 1x attorno alle sezioni vibratome. </li><li> Aggiungere una goccia di mezzo di montaggio acquoso sulla cima delle sezioni vibratome e poi coprioggetto. </li><li> Sigillare i coprioggetti sui vetrini con fuso VALAP applicato con un batuffolo di cotone. </li><li> Immagine sezioni vibratome su entrambi un microscopio confocale diritta o capovolta. Si prega di fare riferimento alla discussione per ulteriori dettagli sulla selezione di un sistema di imaging. </li></ol></li></ol> 3 Immagini dal vivo di Intestino interi Intestino raccolte da fetuses dopo E12.5, con l&#39;omento collegato lasciato intatto, sviluppare con successo villi in coltura utilizzando il sistema di cui sopra. Pertanto, questo sistema ex vivo permette la cattura di immagini z-sezione live dell&#39;intestino sviluppo che possono essere ricostruite per una vista tridimensionale della morfogenesi nel tempo. <ol><li> Impostazione di immagini dal vivo <ol><li> Utilizzare colla istantanea di aderire una membrana di policarbonato individuo a uno schermo a maglia fine. Questo fornisce un&#39;impalcatura di sostegno per tenere l&#39;intestino in posizione sotto la lente immersione del microscopio confocale montante. Si prega di consultare la sezione di discussione per ulteriori dettagli sulla selezione di microscopi di imaging dal vivo. </li><li> Posizionare lo schermo a rete al centro e di una piastra di coltura. </li><li> Posizionare l&#39;intestino sezionato sulla membrana di policarbonato e aggiungere una goccia di colla istantanea alle due estremità. Utilizzare solo abbastanza colla per apporre l&#39;intestino, ma non ricoprirlo! </li><li> Aggiungi terreni di coltura privo di fenolo rosso sotto il policarbonatomembrana nel centro pozzetto della piastra di coltura. </li><li> Aggiungere acqua al bordo esterno della piastra di coltura per fornire umidità. </li><li> Dal momento che l&#39;intestino fetale subisce onde peristaltiche simili di contrazione nella cultura, aggiungere 100 ml di una soluzione 1: 5 di xilazina in 1x PBS a 3 ml di media per controllare il movimento dell&#39;intestino durante l&#39;imaging. Questo dosaggio controllerà i movimenti intestinali per circa 8-10 ore senza compromettere lo sviluppo dei villi. </li><li> Per una vista trasversale dell&#39;intestino, incorporare gli intestini vivi in ​​una soluzione di agarosio 3-4% e tagliare sezioni spesse (150-500 micron) su un vibratome. Abbinare la rigidità del agarosio con la rigidità dell&#39;intestino essere tagliati ed empiricamente determinata fase di sviluppo. In generale, una soluzione di agarosio al 3% funziona bene per l&#39;intestino di età inferiore E14.5 e una soluzione al 4% funziona bene per l&#39;intestino E15-E16. </li><li> Incollare le sezioni vibratome ad una membrana di policarbonato, apposto su un schermo di maglia (come al punto 3.1.1) ecultura come descritto nella sezione 3.1.2-3.1.6. </li><li> Condurre l&#39;imaging dal vivo utilizzando un microscopio confocale a due fotoni (laser di eccitazione tra 690-1,040 nm) su un supporto verticale con un tavolo sintonizzabile. </li><li> Laser a due fotoni sintonizzato a 900 nm fornisce penetrazione profonda con la migliore risoluzione per i segnali GFP. Riduce i danni ai tessuti durante l&#39;imaging. Inoltre, impostando la potenza del laser per l&#39;emissione più bassa riduce il danno tissutale. Tipicamente per ottenere una buona eccitazione di GFP, utilizzare potenza 12% sul laser a due fotoni. </li></ol></li></ol>

<ol>
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The authors have no financial conflicts to disclose.

La natura dinamica e le interazioni complesse dei tessuti dell&#39;intestino in via di sviluppo richiede la visualizzazione 3D per avere una comprensione completa di questi eventi morfogenetici. Con la continua evoluzione tecnologia di imaging, la capacità di esaminare villi morfogenesi in dettaglio si sta sviluppando / miglioramento e con esso, la comprensione della comunicazione spaziale e l&#39;interazione durante l&#39;organogenesi è notevolmente migliorata. Metodi di coltura intestini...

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

La maggior parte dei processi morfogenetici nell&#39;intestino fetale sono stati dedotto dalle sezioni sottili di tessuti fissati, fornendo istantanee di cambiamenti nel corso stadi di sviluppo. Informazioni tridimensionale da sezioni seriali sottili può essere difficile da interpretare a causa della difficoltà di ricostruire sezioni seriali perfettamente e mantenere il corretto orientamento del tessuto su sezioni seriali. Recenti scoperte di Grosse et al. 2011 sottolineano l&#39;importanza di tre informazioni dimensionali nella comprensione morfogenesi dei villi sviluppo dell&#39;intestino 1. Ricostruzione tridimensionale delle cellule intestinali singolarmente etichettati dimostrato che la maggior parte delle cellule epiteliali intestinali contattare sia le superfici apicali e basali. Inoltre, ricostruzione tridimensionale del citoscheletro actina alla superficie apicale dell&#39;epitelio dimostrato che lume intestinale è continua e che lumen secondarie sono un artefatto di sectioning. Questi due punti, insieme con la dimostrazione della migrazione nucleare interkinetic nell&#39;epitelio intestinale, definita l&#39;epitelio intestinale in via di sviluppo come un epitelio pseudostratificato e non stratificati come si pensava 1. La capacità di osservare l&#39;epitelio tridimensionale è stato seminale per dimostrare questo punto e ridefinendo morfogenesi epiteliale nell&#39;intestino del feto. Con l&#39;evoluzione della tecnologia di imaging multi-fotone e software di ricostruzione tridimensionale, la capacità di visualizzare intatto, lo sviluppo di organi sta rapidamente migliorando. Eccitazione a due fotoni permette la penetrazione meno dannoso più in profondità i tessuti con alta risoluzione. Due fotoni di imaging e la ricostruzione in 3D di tutto il fetali intestini del mouse in Walton et al., 2012 hanno contribuito a definire il modello di villi escrescenza 2. Qui si descrive un intero sistema di coltura d&#39;organo che permette ex vivo sviluppo dei villi e le estensioni di quel sistema di coltura per consentirel&#39;intestino ad essere tridimensionalmente ripreso durante il loro sviluppo.

Cultura di espianti interi di intestini fetali consente per le analisi del luogo, la distribuzione, e la durata delle molecole di segnalazione che coordinano lo sviluppo intestinale, in quanto questo sistema consente la manipolazione della segnalazione con reagenti farmacologici o proteine ​​ricombinanti. Il sistema di coltura transwell (Figura 1A, riprodotto da Walton et al., 2012) 2 fornisce una interfaccia aria-liquido, che permette di posizionare droga o proteine ​​imbevu...

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.

Mouse fetale Whole Intestino Sistema Cultura per<em&gt; Ex Vivo</em&gt; La manipolazione delle vie di segnalazione e tridimensionale Imaging in diretta di Sviluppo del villo

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

mouse-fetal-whole-intestine-culture-system-for-ex-vivo-manipulation

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

14.8K Views.  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.

Utilizzando<em&gt; Ex Vivo</em&gt; Upright Droplet Culture di interi organi fetali per studiare i processi evolutivi durante mouse Organogenesi

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

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Biologia dello sviluppo

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.

Protocolli per l&#39;analisi del ruolo delle cellule Paneth in Rigenerazione della murino Intestino con condizionale<em&gt; Cre-lox</em&gt; Modelli mouse

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.

L&#39;ingegneria dei tessuti del colon in un modello murino

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

Bioingegneria

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.

Coltura tridimensionale di cripte coloniche murine per studiare ex vivo la funzione delle cellule staminali intestinali

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

Ricerca sul cancro

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.

Co-cultura del vivere Microbiome con Microengineered umana intestinale Villi in un Gut-on-a-chip microfluidico dispositivo

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.

Recapitulare la transizione Suckling-to-Weaning In Vitro utilizzando organoidi intestinali fetali

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.

Il<em&gt; Ex Vivo</em&gt; Cultura e Pattern Recognition recettore stimolazione del mouse intestinale organoidi

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

Immunologia e infezioni

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.

Un sistema per<em&gt; Ex vivo</emColtura&gt; del pancreas embrionale

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

Biologia

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.

Mixed colture primarie di murino tenue finalizzate allo studio di Gut secrezione dell&#39;ormone e imaging cellulare dal vivo di celle enteroendocrine

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

Isolamento delle cripte intestinali di ratto e generazione di organoidi intestinali

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.

Autore in primo piano: Generazione e manipolazione di organoidi intestinali di ratto  

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.

Mouse fetale Whole Intestino Sistema Cultura per

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Utilizzando

Protocolli per l'analisi del ruolo delle cellule Paneth in Rigenerazione della murino Intestino con condizionale

L'ingegneria dei tessuti del colon in un modello murino

Coltura tridimensionale di cripte coloniche murine per studiare ex vivo la funzione delle cellule staminali intestinali

Co-cultura del vivere Microbiome con Microengineered umana intestinale Villi in un Gut-on-a-chip microfluidico dispositivo

Recapitulare la transizione Suckling-to-Weaning In Vitro utilizzando organoidi intestinali fetali

Il

Un sistema per

Mixed colture primarie di murino tenue finalizzate allo studio di Gut secrezione dell'ormone e imaging cellulare dal vivo di celle enteroendocrine

Generazione e manipolazione di organoidi intestinali di ratto