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.

NOTE: Toutes les souris ont été traitées avec humanité en utilisant des protocoles approuvés par l&#39;Université du Michigan Medical School Unité de médecine de laboratoire et les animaux selon les directives du Comité de l&#39;Université sur l&#39;utilisation et l&#39;entretien des animaux. 1 Tout le Système de culture d&#39;organes <ol><li> Préparation des milieux et des plaques de culture <ol><li> Dans une culture de tissu capot, enlevez 5 ml de BGJb médias de la bouteille de et ajouter 5 ml de Pen / Strep. Préparer un stock de travail des médias dans un tube conique de 50 ml, en ajoutant 1 ml de 5 mg / ml d&#39;acide ascorbique à 49 ml de BGJb médias (avec Pen / Strep). </li><li> Préparer une plaque de culture à 6 puits en ajoutant 1 ml de chacune des sous transwell. NOTE: Si les 6 transwell ne sont pas utilisés, déplacer transwell supplémentaires dans une plaque à 6 puits stérile frais pour une utilisation ultérieure. Ajouter 3 ml de travailler BGJb médias à chacune des trois boîtes de Pétri de 10 cm stériles. </li></ol></li><li> Préparation pour la dissection <ol><li> Faire tremper les outils de dissection dans 70% ethanol et être sûr de garder les outils propres tout en travaillant. </li><li> Mettre en place la zone de dissection avec un grand seau à glace et placer la plaque de culture à 6 puits et 10 cm boîtes de Pétri avec BGJb médias sur la glace. Travailler sur la glace autant que possible, tout en disséquant et la préparation des intestins de la culture. </li><li> Anesthésier les souris femelles adultes dans une chambre avec isofluorane (100 pi) avant l&#39;euthanasie par dislocation cervicale pour la récolte de l&#39;intestin du foetus. </li><li> Après la récolte, les fœtus de leur mère, en scène chaque fœtus soigneusement selon le tableau 13 Theiler mise en scène. Ne vous fiez pas aux jours post-coït pour déterminer l&#39;âge de chaque fœtus comme des variations allant jusqu&#39;à 24 heures dans le stade de développement sont régulièrement observés dans une seule portée. </li></ol></li><li> Dissection de l&#39;intestin du foetus <ol><li> Euthanasier fœtus par décapitation avant le retrait de l&#39;intestin. </li><li> Disséquer chaque intestin en tampon phosphate salin (DPBS) de 1x Dulbecco puis plaCE dans une boîte de Pétri de 10 cm de travailler BGJb médias. </li><li> Retirez délicatement la rate de l&#39;estomac et le pancréas de l&#39;estomac et du duodénum supérieur. </li><li> Séparez délicatement l&#39;épiploon prenant soin de laisser l&#39;épiploon attachée autant que possible et en séparant seulement assez de connexions pour permettre à l&#39;intestin pour être redressé. NOTE: Pour les intestins plus que E14.5 ou ceux qui sont mis en culture pendant plus de 24 heures, séparer délicatement l&#39;intestin en 3 segments égaux pour permettre contenu luminal de couler à travers l&#39;intestin et être expulsés des extrémités coupées. Toutefois, pour les cultures commencé avant E13.5, attendre après 24 heures de culture avant de se séparer les 3 segments de l&#39;intestin pour permettre la séreuse de croître correctement sur toute la longueur de l&#39;intestin. </li><li> Utilisez une pipette de bouche pour transférer les morceaux intestinales sur les transwell de la plaque de culture (1.1.2). </li><li> Repositionner délicatement les intestins au besoin afin qu&#39;ils soient directement à travers la transwell. NOTE: Une fois que les intestins commencent à s&#39;installer contenu luminal dans le tube, les plier dans l&#39;intestin entraînera une sauvegarde et des dommages à l&#39;intestin. </li></ol></li><li> Culture et le traitement <ol><li> Retirez le support sous le transwell, ajouter 700 ul de milieu de traitement (médias travaillent avec des protéines recombinantes ajoutés ou des inhibiteurs pharmacologiques) dans le cadre du transwell. </li><li> Ajouter 300 ul de milieu de traitement goutte à goutte sur le dessus de l&#39;intestin et laisser tremper à travers le transwell. Repositionner les intestins comme nécessaire. </li><li> Changer agents de traitement au moins une fois par jour ou plus souvent fonction de la demi-vie du réactif de traitement. </li></ol></li><li> Préparation de protéine des billes d&#39;agarose imprégné d&#39;une source localisée de réactif de traitement <ol><li> Remettre en suspension les billes d&#39;agarose dans leur conteneur de stockage et transférer 100 pl de billes d&#39;agarose dans un tube à centrifuger de 1,5 ml. </li><li> Remplir le volume restant du tube avec 1x stérile Phosphate solution saline tamponnée (PBS) et mélanger les perles pour les laver. Placez le tube de centrifugeuse dans un rack pendant quelques minutes pour permettre aux billes de se déposent au fond et puis pipette au large de la PBS à partir au-dessus des perles. Remplir le tube de centrifugeuse stérile de PBS 1x et répéter deux fois plus le processus de lavage. </li><li> Préparation de la protéine d&#39;intérêt à deux fois la concentration désirée pour le traitement. </li><li> Mélanger 5 pi de billes d&#39;agarose lavés avec 5 pi de la protéine actions 2x et agiter doucement les perles dans la protéine. Placer le tube sur la glace pendant 1 heure, en mélangeant délicatement toutes les 10-15 min. </li><li> Rincer les perles brièvement stérile 1x PBS avant de placer les perles sur les intestins. </li></ol></li><li> Placement des billes d&#39;agarose <ol><li> Placez délicatement les billes d&#39;agarose sur le dessus de l&#39;intestin à l&#39;aide d&#39;une pipette de bouche avec des aiguilles capillaires tirés. Placez les perles avec une extrême prudence, car une manipulation brutale peut endommager l&#39;intestin et prévenir le développement des villosités dans la zone lésée. </li><li> Placez deux fois plus de billes que nécessaire pour l&#39;analyse depuis les intestins vont subir les mouvements péristaltiques et environ la moitié des billes placées roulera hors de l&#39;intestin au cours de la durée de la culture. </li></ol></li><li> Fixation des intestins culture <ol><li> Retirez délicatement les médias de traitement de dessous de la transwell et permettre aux intestins sécher à l&#39;air pendant 3 min. </li><li> Ajouter 1 ml de fixatif sous la Transwell pendant 2 min, puis recouvrir la partie supérieure de l&#39;intestin avec 2 ml de fixatif pour le reste de la période de fixation. Fixer avec 4% de paraformaldéhyde O / N à 4 ° C ou pendant 2 heures à température ambiante. </li></ol></li></ol> 2. imagerie de l&#39;intestin fixes <ol><li> Imagerie Wholemount <ol><li> Placez les intestins entiers (avec fluorescence endogène) sur une diapositive avec 100 pi de 1x DPBS. Utilisez une pince à organiser doucement les intestins. </li><li> Utilisez un tissu de laboratoire pour retirer soigneusement les DPBS et ajouter immédiatement 100 pi de 70% de glycérol à faire la mo tissure transparente et augmenter la profondeur possible de l&#39;imagerie. REMARQUE: Si une plus grande pénétration de l&#39;intestin est souhaité, au lieu de monter l&#39;intestin dans 70% de glycérol, tremper l&#39;intestin dans quelques gouttes d&#39;orientation claire solution pour 10-30 min. Pipeter au large de la solution mise au point claire et monter l&#39;intestin avec le Mont Effacer. </li><li> Tremper chaque des quatre coins d&#39;une lamelle en pâte à modeler et ramasser une petite quantité d&#39;argile à utiliser comme entretoises entre la lame et lamelle pour maintenir la lamelle de l&#39;aplatissement de l&#39;intestin. </li><li> Sceller la lamelle sur la lame avec valap fondu appliquée avec un coton-tige. </li><li> L&#39;image sur les intestins soit un microscope confocal debout ou inversé. S&#39;il vous plaît se référer à la discussion pour plus de détails. </li></ol></li><li> Vibratome sectionnement de l&#39;intestin fixes (pour une vue en coupe transversale de structures intestinales) <ol><li> Intégrer dans les intestins 7% d&#39;agarose dans de petits moules en plastique (10 x 10 x 15 mm) et permettre aux blocs d&#39;agarose àrefroidir et se solidifier. </li><li> Retirer des blocs d&#39;agarose à partir de moules en plastique et des blocs d&#39;agarose colle à l&#39;étape de collecte de vibratome avec de la colle instantanée. </li><li> Remplir le stade de la collecte de froid de glace 1x DPBS. </li><li> Couper des sections à une épaisseur comprise entre 100-150 um. Pour les sections plus épaisses utilisent les solutions de compensation (décrits dans la section 2.1.2) pour visualiser plus profondément dans la section. </li><li> Verser vibratome sections de l&#39;étape de la collecte dans un puits d&#39;une plaque à 6 puits pour le stockage à 4 ° C. </li></ol></li><li> Immunofluorescence, de tissus vibratome sectionnés fixes <ol><li> Placez 5-12 sections vibratome par puits dans une plaque de 24 puits avec 1x DPBS. </li><li> Retirer 1x DPBS et ajouter 500 ul de solution de perméabilisation par puits. Secouez délicatement les échantillons pendant 25 min à température ambiante. </li><li> Après perméabilisation, laver les échantillons 3 fois avec du PBS 1X. </li><li> Bloquer les échantillons pendant au moins 30 min dans 500 pi de solution de blocage. </li><li> Après blocage, eXCHANGE la solution de blocage avec 500 ul d&#39;anticorps primaire dilué dans la solution de blocage et conserver les échantillons à 4 ° CO / N. REMARQUE: Les dilutions d&#39;anticorps primaires qui fonctionnent bien sur des coupes congelées et paraffine fonctionnent généralement bien sur vibratome sections aussi, cependant anticorps nécessitant l&#39;extraction de l&#39;antigène nucléaire en général ne fonctionnent pas bien avec ce protocole (par exemple, BrdU). </li><li> Le jour suivant, laver les échantillons 3 fois pendant 15 min à chaque fois avec une solution de blocage. </li><li> Après lavage, 500 ul d&#39;appliquer l&#39;anticorps secondaire dilué dans une solution de blocage. Enveloppez la plaque de 24 puits dans du papier d&#39;aluminium pour protéger les fluorophores de la lumière et de la roche les échantillons pendant 45 minutes à 2 heures à température ambiante. </li><li> Laver les échantillons 3 fois avec du PBS 1X pendant 15 minutes chacun et monter les sections de vibratome sur des lames comme décrit à la section 2.4. </li></ol></li><li> Montage sections vibratome <ol><li> Préparer des lames pour vibratome de montage par couper de fines tranches de double-stick bande et en les plaçant sur les côtés longs des lamelles. </li><li> Placez délicatement 5-10 sections vibratome entre le ruban adhésif double-bâton sur les lames dans une goutte de 100 pi de PBS 1x. </li><li> Déplier toutes les sections et les étaler en une seule couche sur la diapositive. </li><li> Retirez tout excès d&#39;agarose ou des morceaux inégaux qui empêcherait une lamelle de siéger plat. </li><li> Soigneusement utiliser un tissu de laboratoire pour absorber l&#39;excès de PBS 1x autour des sections de vibratome. </li><li> Ajouter une goutte de milieu de montage aqueux sur des sections de vibratome et lamelle. </li><li> Sceller les lamelles sur les lames avec fondu valap appliquée avec un coton-tige. </li><li> Image, les sections de vibratome sur soit un microscope confocal droit ou inversé. S&#39;il vous plaît se référer à la discussion pour plus de détails sur la sélection d&#39;un système d&#39;imagerie. </li></ol></li></ol> 3. imagerie en direct de l&#39;intestin entiers Intestins récolté de fetuses après E12.5, avec l&#39;épiploon connecté laissée intacte, développer avec succès villosités en culture en utilisant le système ci-dessus. Par conséquent, ce système ex vivo permet la capture d&#39;images en direct z-section de l&#39;intestin de développement qui peut être reconstituée pour une vue en trois dimensions de la morphogenèse au cours du temps. <ol><li> Mise en place d&#39;imagerie en temps réel <ol><li> Utilisez de la colle instantanée à respecter une membrane de polycarbonate individu à un tamis à mailles fines. Cela fournit un échafaudage de support pour maintenir l&#39;intestin en place sous l&#39;objectif de trempage du microscope confocal verticale. S&#39;il vous plaît se référer à la section de discussion pour plus de détails sur la sélection de microscopes d&#39;imagerie en direct. </li><li> Placer le tamis dans le puits central d&#39;une plaque de culture. </li><li> Placer l&#39;intestin disséqué sur la membrane de polycarbonate et on ajoute une goutte de colle instantanée à chaque extrémité. Utilisez seulement assez de colle pour fixer l&#39;intestin, mais pas l&#39;enduire! </li><li> Ajouter culture libre de médias de phénol rouge ci-dessous le polycarbonatemembrane dans le puits central de la plaque de culture. </li><li> Ajouter de l&#39;eau à la bordure extérieure de la plaque de culture à fournir de l&#39;humidité. </li><li> Depuis l&#39;intestin foetal subit ondes péristaltiques analogues de contraction dans la culture, ajouter 100 ul d&#39;une solution 1: 5 de xylazine dans du PBS 1x à 3 ml de milieu à contrôler les mouvements de l&#39;intestin, tandis que l&#39;imagerie. Cette posologie sera de contrôler les mouvements intestinaux pour environ 8-10 heures sans compromettre le développement des villosités. </li><li> Pour une vue transversale de l&#39;intestin, intégrer les intestins vivants dans une solution d&#39;agarose à 3-4% et couper des sections épaisses (150-500 de um) sur un vibratome. Correspondre à la rigidité de l&#39;agarose avec la rigidité de l&#39;intestin étant déterminée empiriquement et couper le stade de développement. En général, une solution d&#39;agarose à 3% fonctionne bien pour les intestins de moins de E14.5 et une solution à 4% fonctionne bien pour les intestins E15-E16. </li><li> Coller les sections de vibratome à une membrane de polycarbonate, apposée sur un tamis à mailles (comme dans la section 3.1.1) etculture comme décrit dans la Section 3.1.2-3.1.6. </li><li> Effectuer l&#39;imagerie en temps réel en utilisant un microscope confocal à fluorescence à deux photons (laser d&#39;excitation entre 690-1,040 nm) sur un support vertical avec une table réglable. </li><li> Laser à deux photons réglé à 900 nm permet une pénétration profonde avec la meilleure résolution pour les signaux de GFP. Il réduit les dommages aux tissus en imagerie. En outre, le réglage de la puissance du laser pour l&#39;émission le plus bas possible pour réduire les dommages des tissus. En général pour obtenir une bonne excitation de la GFP, en utilisant 12% de la puissance du laser à deux photons. </li></ol></li></ol>

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The authors have no financial conflicts to disclose.

La nature dynamique et les interactions de tissus complexes de l&#39;intestin en développement exige une visualisation 3D d&#39;avoir une appréciation complète de ces événements morphogénétiques. Avec l&#39;évolution des technologies d&#39;imagerie, la capacité d&#39;examiner en détail la morphogenèse des villosités se développe / amélioration et, avec elle, la compréhension de la communication spatiale et interaction pendant l&#39;organogenèse est grandement améliorée. Des...

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 plupart des processus morphogénétiques dans l&#39;intestin du foetus ont été déduites à partir des coupes minces de tissus fixés, présenter des instantanés de changements au cours des stades de développement. Des informations en trois dimensions à partir de coupes sériées minces peut être difficile à interpréter en raison de la difficulté de reconstituer des coupes en série et parfaitement maintenir l&#39;orientation correcte du tissu sur coupes sériées. Les études récentes de Grosse et al., 2011 soulignent l&#39;importance des trois dimensions informations pour comprendre la morphogenèse des villosités de l&#39;intestin en développement 1. Reconstruction tridimensionnelle de cellules intestinales individuellement marqués ont démontré que la majorité des cellules épithéliales de l&#39;intestin contact avec les deux surfaces apicales et basales. En outre, la reconstruction en trois dimensions du cytosquelette d&#39;actine à la surface apicale de l&#39;épithélium a démontré que la lumière intestinale est continue et que les lumières secondaires sont un artefact de sectioning. Ces deux points, ainsi que la démonstration de la migration nucléaire interkinetic dans l&#39;épithélium intestinal, définis l&#39;épithélium intestinal en développement un épithélium pseudo et non stratifiées comme le pensait auparavant 1. La capacité d&#39;observer l&#39;épithélium en trois dimensions a été décisif pour démontrer ce point et la redéfinition de la morphogenèse épithéliale dans l&#39;intestin du foetus. Avec l&#39;évolution des technologies d&#39;imagerie multi-photonique et logiciel de reconstruction tridimensionnelle, la capacité de visualiser intact, le développement des organes s&#39;améliore rapidement. Excitation à deux photons permet une pénétration moins dommageable plus profondément dans les tissus à haute résolution. Imagerie à deux photons et la reconstruction 3D de l&#39;ensemble des intestins de souris fœtales à Walton et al., 2012 ont contribué à définir le modèle de villosités excroissance 2. Nous décrivons ici un ensemble de système de culture d&#39;organe qui permet le développement ex vivo de villosités et extensions de ce système de culture pour permettreles intestins pour être en trois dimensions imagé au cours de leur développement.

Culture des explants entiers de l&#39;intestin fœtal permet l&#39;analyse de la localisation, la distribution, et la durée des molécules de signalisation qui coordonnent le développement intestinal, comme ce système permet la manipulation de la signalisation avec des réactifs pharmacologiques ou des protéines recombinantes. Le système de culture Transwell (Figure 1A, reproduit à partir de Walton et al., 2012) 2 fournit une interface air-liquide, ce qui permet de placer la dr...

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.

Souris foetal entier Intestin Système de culture pour<em&gt; Ex Vivo</em&gt; Manipulation des voies de signalisation et d&#39;imagerie en direct en trois dimensions du développement des villosités

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

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

14.9K vues.  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. 

Vidéo: 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.

Utilisation<em&gt; Ex Vivo</em&gt; Cultures Upright gouttelettes de toute fœtus organes pour étudier les processus de développement au cours de la souris Organogenèse

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

Recherche

Biologie du développement

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.

Protocoles pour analyser le rôle des cellules de Paneth dans la régénération de l&#39;intestin murin utilisant conditionnelle<em&gt; Cre-lox</em&gt; Modèles de souris

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.

Génie tissulaire de l&#39;intestin dans un modèle murin

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

Bio-ingénierie

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.

Culture tridimensionnelle de cryptes coliques murines pour étudier la fonction des cellules souches intestinales 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 ...

Recherche en cancérologie

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-culture de la vie microbiome avec Microengineered Human Intestinal Villi dans un Gut-on-a-Chip microfluidique périphérique

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.

Récapituler la transition de lassuck au soudement in Vitro à l'aide d'organoïdes intestinaux fœtaux

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.

le<em&gt; Ex Vivo</em&gt; Culture et Pattern Recognition Receptor Stimulation de la souris Intestinal organites

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

Immunologie et infection

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 système d&#39;<em&gt; Ex vivo</emLa culture&gt; de pancréas embryonnaire

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

Biologie

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.

Cultures primaires mixtes de murine Petit Intestin destiné à l&#39;étude de la Gut hormone Sécrétion et imagerie de cellules vivantes de cellules entéroendocrines

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

Isolement des cryptes intestinales de rat et génération d’organoïdes intestinaux

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.

Pleins feux sur l’auteur : Génération et manipulation d’organoïdes intestinaux de rats  

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.

Souris foetal entier Intestin Système de culture pour

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Utilisation

Protocoles pour analyser le rôle des cellules de Paneth dans la régénération de l'intestin murin utilisant conditionnelle

Génie tissulaire de l'intestin dans un modèle murin

Culture tridimensionnelle de cryptes coliques murines pour étudier la fonction des cellules souches intestinales ex vivo

Co-culture de la vie microbiome avec Microengineered Human Intestinal Villi dans un Gut-on-a-Chip microfluidique périphérique

Récapituler la transition de lassuck au soudement in Vitro à l'aide d'organoïdes intestinaux fœtaux

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Un système d'

Cultures primaires mixtes de murine Petit Intestin destiné à l'étude de la Gut hormone Sécrétion et imagerie de cellules vivantes de cellules entéroendocrines

Génération et manipulation d’organoïdes intestinaux de rat