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.

NOTA: Todos los ratones fueron manejados con humanidad utilizando los protocolos aprobados por la Universidad de Michigan Medical School Unidad de Laboratorio de Medicina Animal y de acuerdo con las directrices del Comité de la Universidad sobre el uso y cuidado de los animales. 1. System Organ Culture Whole <ol><li> Preparación de medios de comunicación y placas de cultivo <ol><li> En una campana de cultivo de tejidos, eliminar 5 ml de medio BGJb de la botella stock y añadir 5 ml de penicilina / estreptomicina. Prepare una solución de trabajo de los medios de comunicación en un tubo cónico de 50 ml, mediante la adición de 1 ml de 5 mg / ml de ácido ascórbico a 49 ml de medio BGJb (con Pen / Strep). </li><li> Preparar una placa de cultivo de 6 pocillos mediante la adición de 1 ml por debajo de cada uno de los cultivos polarizados. NOTA: Si no se utilizan los 6 transwells, mover transwells adicionales en una placa de 6 pocillos estéril fresco para su uso posterior. Añadir 3 ml de los medios de trabajo BGJb a cada una de tres placas de Petri de 10 cm estériles. </li></ol></li><li> Preparación para la disección <ol><li> Remoje las herramientas de disección en el 70% ethanol y asegúrese de mantener las herramientas limpias durante el trabajo. </li><li> Configurar la zona de disección con un gran cubo de hielo y colocar la placa de cultivo de 6 pocillos y 10 cm placas de Petri con medios BGJb sobre hielo. Trabajar en el hielo tanto como sea posible mientras que la disección y preparación de los intestinos para la cultura. </li><li> Anestesiar a los ratones hembra adulta en una cámara con isofluorano (100 l) antes de la eutanasia por dislocación cervical para la recolección de los intestinos fetales. </li><li> Después de cosechar los fetos de su madre, etapa cada feto cuidadosamente según la Theiler puesta en escena gráfica 13. No confíe en los días post-coito para determinar la edad de cada feto como variaciones de hasta 24 horas en la etapa de desarrollo se observan habitualmente en una sola camada. </li></ol></li><li> La disección de los intestinos fetales <ol><li> La eutanasia a los fetos por decapitación antes de la extracción de los intestinos. </li><li> Diseccionar cada intestino en 1x de Dulbecco tamponada con fosfato salino (DPBS) y luego place en una placa de Petri de 10 cm con el trabajo de medios BGJb. </li><li> Retire cuidadosamente el bazo desde el estómago y el páncreas desde el estómago y el duodeno superior. </li><li> Separar suavemente el epiplón teniendo cuidado de dejar el epiplón adjunta tanto como sea posible y la separación de las conexiones sólo lo suficiente para permitir que el intestino para ser enderezada. NOTA: Para intestinos mayores de E14.5 o los que están siendo cultivados más de 24 horas, separar suavemente el intestino en 3 segmentos iguales para permitir el contenido luminal fluyan a través del intestino y ser expulsados ​​de los extremos cortados. Sin embargo, para que las culturas se inició antes de E13.5, espere hasta después de 24 horas de cultivo antes de la separación de los 3 segmentos del intestino para permitir la serosa crezca adecuadamente en toda la longitud del intestino. </li><li> Usar una pipeta de boca para transferir las piezas intestinales en los cultivos polarizados de la placa de cultivo (1.1.2). </li><li> Reposicionar suavemente los intestinos a medida que sea necesario para que estén en línea recta la Transwell. NOTA: Una vez que los intestinos comienzan a moverse contenido luminal a través del tubo, cualquier torcedura en el intestino provocarán una copia de seguridad y daños en el intestino. </li></ol></li><li> Cultura y tratamiento <ol><li> Retire el papel de debajo de la transwell, añadir 700 l de medio de tratamiento (los medios de comunicación que trabajan con proteínas recombinantes añadidos o inhibidores farmacológicos) bajo la transwell. </li><li> Añadir 300 l de medio de tratamiento gota a gota en la parte superior de los intestinos y deje que penetre a través de la transwell. Vuelva a colocar el intestino en caso necesario. </li><li> Cambie medio de tratamiento por lo menos una vez al día o más a menudo, dependiendo de la vida media del reactivo de tratamiento. </li></ol></li><li> Preparación de proteínas empapado perlas de agarosa para una fuente localizada de reactivo de tratamiento <ol><li> Resuspender las perlas de agarosa en su recipiente de almacenamiento y la transferencia de 100 l de perlas de agarosa en un tubo de microcentrífuga de 1,5 ml. </li><li> Llenar el volumen restante del tubo con 1x estéril Phosphate-Buffered Saline (PBS) y mezclar las perlas para lavarlos. Colocar el tubo de microcentrífuga en un bastidor por unos pocos minutos para permitir que las perlas se depositan en el fondo y luego pipetee de la PBS en la parte superior de la de las perlas. Vuelva a llenar el tubo de microcentrífuga con 1x PBS estéril y repetir el proceso de lavado dos veces más. </li><li> Preparar la proteína de interés a dos veces la concentración deseada para el tratamiento. </li><li> Mezclar 5 l de perlas de agarosa lavadas con 5 l de la proteína de stock 2x y agite suavemente las perlas en la proteína. Se coloca el tubo en hielo durante 1 hora, mezclando suavemente cada 10-15 min. </li><li> Enjuague las perlas brevemente en PBS 1x estéril antes de colocar las perlas en los intestinos. </li></ol></li><li> La colocación de perlas de agarosa <ol><li> Con suavidad, coloque las bolas de agarosa en la parte superior de los intestinos usando una pipeta de boca con agujas capilares tirados. Coloca las bolitas con extremo cuidado ya que el manejo brusco puede dañar el intestino y prevenir el desarrollo de vellosidades en el área lesionada. </li><li> Coloque el doble de cuentas que sean necesarios para el análisis, ya que los intestinos se someterán a los movimientos peristálticos y aproximadamente la mitad de los granos colocados rodará fuera de los intestinos durante el tiempo de cultivo. </li></ol></li><li> La fijación de los intestinos cultivadas <ol><li> Retire con cuidado los medios de tratamiento de debajo de la transwell y permitir que los intestinos se sequen al aire durante 3 min. </li><li> Añadir 1 ml de fijador bajo el transwell durante 2 min, luego cubrir la parte superior del intestino con 2 ml de fijador para el resto del tiempo de fijación. Fijar con paraformaldehído al 4% O / N a 4 ° C o durante 2 horas a temperatura ambiente. </li></ol></li></ol> 2. Imaging de intestinos fijos <ol><li> Imágenes Wholemount <ol><li> Coloque el conjunto de los intestinos (con fluorescencia endógena) en un portaobjetos con 100 l de 1x DPBS. El uso de fórceps para arreglar suavemente los intestinos. </li><li> Utilice un pañuelo de papel de laboratorio para retirar cuidadosamente los DPBS y añadir inmediatamente 100 l de 70% de glicerol para hacer el mo tejidovolver transparente y aumentar la posible profundidad de formación de imágenes. NOTA: Si se desea una mayor penetración del intestino, en lugar de montar el intestino en un 70% de glicerol, empape el intestino en unas gotas de Focus Claro solución para 10-30 min. Pipeta de fuera de la solución de enfoque claro y montar el intestino con el Monte Claro. </li><li> Sumerja cada una de las cuatro esquinas de un cubreobjetos en plastilina y recoger una pequeña cantidad de arcilla para usar como separadores entre el portaobjetos y cubreobjetos para mantener el cubreobjetos de aplanamiento del intestino. </li><li> Sellar el cubreobjetos sobre el portaobjetos con valap derretida aplicada con un bastoncillo de algodón. </li><li> Imagen de los intestinos a ambos un microscopio confocal en posición vertical o invertida. Por favor refiérase a la discusión para más detalles. </li></ol></li><li> Seccionamiento Vibratome de intestinos fijos (para vista en sección transversal de las estructuras intestinales) <ol><li> Intestinos Insertar en el 7% de agarosa en pequeños moldes de plástico (10 x 10 x 15 mm) y permitir que los bloques de agarosa aenfriar y solidificar. </li><li> Retire los bloques de agarosa de los moldes de plástico y pegamento bloques de agarosa a la etapa de recolección vibratome con pegamento instantáneo. </li><li> Llenar la etapa de recolección de frío hielo 1x DPBS. </li><li> Cortar secciones con un espesor de entre 100-150 micras. Por secciones más gruesas utilizan las soluciones de compensación (que se describen en la sección 2.1.2) para visualizar más en la sección transversal. </li><li> Vierta vibratome secciones de la etapa de recolección en un pocillo de una placa de 6 pocillos para el almacenamiento a 4 ° C. </li></ol></li><li> La tinción de inmunofluorescencia de tejidos, vibratome seccionadas fijos <ol><li> Coloque 5-12 secciones vibratome por pocillo en una placa de 24 pocillos con 1x DPBS. </li><li> Retire 1x DPBS y añadir 500 l de solución de permeabilización por pocillo. Agite suavemente las muestras durante 25 minutos a temperatura ambiente. </li><li> Después de permeabilización, lavar las muestras 3 veces con 1 × PBS. </li><li> Bloquee las muestras durante al menos 30 min en 500 l de disolución de bloqueo. </li><li> Después del bloqueo, eXchange la solución de bloqueo con 500 l de anticuerpos primarios diluidos en solución de bloqueo y mantener las muestras a 4 ° CO / N. NOTA: Las diluciones de anticuerpos primarios que funcionan bien en las secciones congeladas y parafina generalmente funcionan bien en vibratome secciones también, sin embargo, los anticuerpos que requiere la recuperación de antígeno nuclear generalmente no funcionan bien con este protocolo (por ejemplo, BrdU). </li><li> El día siguiente, se lavan las muestras 3 veces durante 15 min cada vez con solución de bloqueo. </li><li> Después del lavado, se aplican 500 l del anticuerpo secundario diluido en solución de bloqueo. Envolver la placa de 24 pocillos en papel de aluminio para proteger los fluoróforos de la luz y el rock las muestras durante 45 min a 2 horas a RT. </li><li> Lávese las muestras 3 veces con 1 × PBS durante 15 minutos cada uno y montar las secciones vibratome en las diapositivas como se describe en la Sección 2.4. </li></ol></li><li> Montaje vibratome secciones <ol><li> Preparar en otro portaobjetos para vibratome montaje cortando lonchas finas de doble stick y la colocación de la cinta a lo largo de los lados largos de las diapositivas. </li><li> Coloque cuidadosamente 5-10 secciones vibratome entre la cinta de doble cara en las diapositivas en una gota de 100 l de PBS 1x. </li><li> Desplegar todas las secciones y se extendió a cabo en una sola capa en toda la diapositiva. </li><li> Elimine cualquier exceso de agarosa o trozos irregulares que impida un cubreobjetos de sentarse plana. </li><li> Con cuidado, usar un pañuelo desechable de laboratorio para absorber el exceso de 1x PBS desde alrededor de las secciones vibratome. </li><li> Añadir una gota de medio de montaje acuoso en la parte superior de las secciones de vibratome y luego cubreobjetos. </li><li> Sellar el cubreobjetos sobre los portaobjetos con derretida valap aplica con un algodón. </li><li> Imagen secciones vibratome a ambos un microscopio confocal vertical o invertida. Por favor refiérase a la discusión para más detalles sobre la selección de un sistema de imágenes. </li></ol></li></ol> 3. imágenes en vivo de los intestinos enteros Intestinos cosechado de fetuses después de E12.5, con el epiplón conectado deja intacto, desarrollar con éxito las vellosidades en la cultura con el sistema anterior. Por lo tanto, este sistema ex vivo permite la captura de imágenes de sección z vivo de los intestinos en desarrollo que pueden ser reconstruidas para una vista tridimensional de la morfogénesis en el tiempo. <ol><li> La creación de imágenes en directo <ol><li> Utilice pegamento instantáneo para adherir una membrana de policarbonato individuo a una pantalla de malla fina. Esto proporciona un andamio de apoyo para mantener el intestino en su lugar debajo de la lente de inmersión de microscopio confocal vertical. Por favor refiérase a la sección de discusión para más detalles sobre la selección de los microscopios de imágenes en directo. </li><li> Coloque la pantalla de malla en el centro de los pocillos de una placa de cultivo. </li><li> Coloque el intestino diseccionado sobre la membrana de policarbonato y añadir una gota de pegamento instantáneo en cada extremo. Utilice sólo el pegamento suficiente para colocar el intestino, pero no abrigo! </li><li> Añadir cultura libre medios de rojo fenol por debajo del policarbonatomembrana en el centro pocillo de la placa de cultivo. </li><li> Añadir agua hasta el borde exterior de la placa de cultivo para proporcionar humedad. </li><li> Desde el intestino fetal se somete a ondas peristálticas-como de contracción en cultivo, añadir 100 ml de una solución 1: 5 de xilazina en 1x PBS a 3 ml de medio para controlar el movimiento del intestino mientras que las imágenes. Esta dosis será controlar los movimientos intestinales por alrededor de 8-10 horas sin comprometer el desarrollo de las vellosidades. </li><li> Para una vista transversal del intestino, incrustar los intestinos vivos en una solución de agarosa al 3-4% y cortar secciones gruesas (150-500 M) en un vibratome. Coincidir con la rigidez de la agarosa con la rigidez del intestino de ser cortado y determinado empíricamente la etapa de desarrollo. Por lo general, una solución de agarosa al 3% funciona bien para los intestinos menores de E14.5 y una solución al 4% funciona bien para los intestinos E15-E16. </li><li> Pega las secciones vibratome a una membrana de policarbonato colocada en un tamiz de malla (como en la Sección 3.1.1) yla cultura como se describe en la Sección 3.1.2-3.1.6. </li><li> Llevar a cabo la formación de imágenes en vivo usando un microscopio confocal de fluorescencia de dos fotones (láser de excitación entre 690-1,040 nm) en un soporte vertical con una tabla ajustable. </li><li> Láser de dos fotones sintonizado a 900 nm proporciona una penetración profunda con la mejor resolución para las señales de las buenas prácticas agrarias. Reduce el daño a los tejidos, mientras que la formación de imágenes. Además, el establecimiento de la potencia del láser para la emisión más bajo posible reduce el daño del tejido. Típicamente para obtener una buena excitación de GFP, utilizar la energía 12% en el láser de dos fotones. </li></ol></li></ol>

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The authors have no financial conflicts to disclose.

La naturaleza dinámica e interacciones de tejidos complejos del intestino en desarrollo requiere la visualización en 3D para tener una apreciación más completa de estos eventos morfogenéticos. Con la evolución de la tecnología de imagen, la capacidad de examinar las vellosidades morfogénesis en detalle está desarrollando / mejora y con ello, la comprensión de la comunicación espacial y la interacción durante la organogénesis es mucho mayor. Los métodos alternativos para el cult...

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 mayoría de los procesos morfogenéticos en el intestino fetal se han deducido a partir de secciones finas de tejidos fijados, proporcionando instantáneas de los cambios más etapas de desarrollo. Información tridimensional a partir de secciones delgadas de serie puede ser difícil de interpretar debido a la dificultad de la reconstrucción de las secciones de serie perfectamente y mantener la orientación adecuada de los tejidos en las secciones de serie. Las investigaciones recientes de Grosse et al., 2011 ponen de manifiesto la importancia de la información dimensional tres en la comprensión de la morfogénesis de las vellosidades del intestino en desarrollo 1. Reconstrucción tridimensional de las células intestinales individualmente etiquetados demostró que la mayoría de las células epiteliales intestinales en contacto con ambas superficies apicales y basales. Además, la reconstrucción tridimensional del citoesqueleto de actina en la superficie apical del epitelio demostró que el lumen intestinal es continua y que los lúmenes secundarios son un artefacto de sectioning. Esos dos puntos, junto con la demostración de la migración nuclear interkinetic en el epitelio intestinal, definen el epitelio intestinal como el desarrollo de un epitelio pseudoestratificado y no estratificados como se pensaba anteriormente 1. La capacidad de observar el epitelio tridimensionalmente era seminal a demostrar este punto y la redefinición de la morfogénesis epitelial en el intestino fetal. Con la evolución de la tecnología de imágenes multi-fotón y software de reconstrucción en tres dimensiones, la capacidad de visualizar intacta, el desarrollo de órganos está mejorando rápidamente. Excitación de dos fotones permite la penetración más profunda menos perjudiciales en los tejidos con alta resolución. Dos fotones de imágenes y reconstrucción 3D de todo el intestino del feto de ratón en Walton et al., 2012 ayudaron a definir el patrón de vellosidades consecuencia 2. Aquí se describe un sistema de cultivo de órgano entero que permite ex vivo el desarrollo de las vellosidades y las extensiones de ese sistema de cultivo para permitirlos intestinos sean tridimensionalmente fotografiado durante su desarrollo.

Cultura de explantes enteros de intestinos fetales permite el análisis de la ubicación, la distribución, y la duración de las moléculas de señalización que coordinan el desarrollo intestinal, ya que este sistema permite la manipulación de la señalización con reactivos farmacológicos o proteínas recombinantes. El sistema de cultivo transwell (Figura 1A, reproducida de Walton et al., 2012) 2 proporciona una interfaz aire-líquido, lo que hace posible colocar de drogas o de ...

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.

Sistema de Cultura ratón fetal Whole Intestino para<em&gt; Ex Vivo</em&gt; Manipulación de vías de señalización y tridimensional de imágenes en vivo del desarrollo de vellosidades

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 vistas.  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.

Usando<em&gt; Ex Vivo</em&gt; Culturas Upright Gotita de enteros fetales Órganos para estudiar los procesos de desarrollo durante Ratón Organogénesis

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

Investigación

Biología del Desarrollo

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.

Los protocolos para analizar el papel de las células de Paneth en Regeneración del Murino Intestino utilizando condicional<em&gt; Cre-lox</em&gt; Ratón Modelos

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.

Ingeniería de tejidos del intestino en un modelo murino

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

Bioingeniería

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.

Cultivo tridimensional de criptas colónicas murinas para estudiar la función de las células madre 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 ...

Investigación sobre el cáncer

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-cultivo de estar con microbioma intestinal humana microconstruidos Villi en una Gut-on-a-Chip dispositivo de microfluidos

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.

Transición recapitulante de lechón a destete In Vitro usando organoides intestinales fetales

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.

los<em&gt; Ex Vivo</em&gt; Cultura y Reconocimiento de Patrones receptor de estimulación de ratón intestinal organoides

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

Inmunología e Infección

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 para la<em&gt; Ex vivo</em&gt; Cultivo de páncreas embrionario

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

Biología

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.

Mezcla de cultivos primarios de murino intestino delgado previstos para el Estudio de Gut Hormone secreción y Imágenes de células vivas de células enteroendocrinas

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

Aislamiento de criptas intestinales de ratas y generación de organoides intestinales

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.

Autor destacado: Generación y manipulación de organoides intestinales de rata  

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.

Sistema de Cultura ratón fetal Whole Intestino para

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Los protocolos para analizar el papel de las células de Paneth en Regeneración del Murino Intestino utilizando condicional

Ingeniería de tejidos del intestino en un modelo murino

Cultivo tridimensional de criptas colónicas murinas para estudiar la función de las células madre intestinales ex vivo

Co-cultivo de estar con microbioma intestinal humana microconstruidos Villi en una Gut-on-a-Chip dispositivo de microfluidos

Transición recapitulante de lechón a destete In Vitro usando organoides intestinales fetales

los

Un sistema para la

Mezcla de cultivos primarios de murino intestino delgado previstos para el Estudio de Gut Hormone secreción y Imágenes de células vivas de células enteroendocrinas

Generación y manipulación de organoides intestinales de rata