Name: Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development (Video) | JoVE Journal
Uploaded: 2014-09-04T14:00:00.000+00:00
Duration: 6 min 46 s
Description: Learn whole organ culture of mouse embryonic intestines for ex vivo signaling pathway modulation and live imaging of villus development. This method enables three-dimensional visualization of intestinal organogenesis.

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5 minutes

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: All mice were handled humanely using protocols approved by the University of Michigan Medical School Unit for Laboratory Animal Medicine and according to the guidelines of the University Committee on Use and Care of Animals.
1. Whole Organ Culture System
<ol>
	<li>Preparation of media and culture plates
	<ol>
		<li>In a tissue culture hood, remove 5 ml of BGJb media from the stock bottle and add 5 ml of Pen/Strep. Prepare a working stock of media in a 50 ml conical tube, by adding 1 ml of 5 mg/ml ascorbic acid to 49 ml of BGJb media (with Pen/Strep).</li>
		<li>Prepare a 6 well culture plate by adding 1 ml underneath each of the transwells. 
		NOTE: If all 6 transwells are not used, move extra transwells into a fresh sterile 6 well plate for later use. Add 3 ml of working BGJb media to each of three 10 cm sterile petri dishes.</li>
	</ol>
	</li>
	<li>Preparation for dissection
	<ol>
		<li>Soak dissecting tools in 70% ethanol and be sure to keep the tools clean while working.</li>
		<li>Set up the dissecting area with a large ice bucket and place the 6 well culture plate and 10 cm petri dishes with BGJb media on ice. Work on ice as much as possible while dissecting and preparing the intestines for culture.</li>
		<li>Anesthetize adult female mice in a chamber with isofluorane (100 &#181;l) prior to euthanization by cervical dislocation for the harvesting of fetal intestines.</li>
		<li>After harvesting the fetuses from their mother, stage each fetus carefully according to the Theiler staging chart13. Do not rely on days post-coitus to determine the age of each fetus as variations of up to 24 hr in developmental stage are routinely observed within a single litter.</li>
	</ol>
	</li>
	<li>Dissection of fetal intestines
	<ol>
		<li>Euthanize fetuses by decapitation prior to removal of intestines.</li>
		<li>Dissect each intestine in 1x Dulbecco&#39;s Phosphate-Buffered Saline (DPBS) and then place it into a 10 cm petri plate with working BGJb media.</li>
		<li>Carefully remove the spleen from the stomach and the pancreas from the stomach and upper duodenum.</li>
		<li>Gently separate the omentum taking care to leave the omentum attached as much as possible and separating connections only enough to allow the intestine to be straightened. 
		NOTE: For intestines older than E14.5 or those being cultured longer than 24 hr, gently separate the intestine into 3 equal segments to allow for luminal contents to flow through the intestine and be expelled from the cut ends. However, for cultures started prior to E13.5, wait until after 24 hr of culturing before separating the 3 segments of the intestine to allow the serosa to properly grow over the length of the intestine.</li>
		<li>Use a mouth pipet to transfer the intestinal pieces onto the transwells of the culture plate (1.1.2).</li>
		<li>Gently reposition the intestines as needed so that they are straight across the transwell. 
		NOTE: Once the intestines begin moving luminal contents through the tube, any kinks in the intestine will cause a backup and damage to the intestine.</li>
	</ol>
	</li>
	<li>Culture and treatment
	<ol>
		<li>Remove the media from under the transwell, add 700 &#956;l of treatment media (working media with added recombinant proteins or pharmacological inhibitors) under the transwell.</li>
		<li>Add 300 &#956;l of treatment media drop-wise on top of the intestines and allow it to soak through the transwell. Reposition the intestines as necessary.</li>
		<li>Change treatment media at least once daily or more often depending on the half-life of the treatment reagent.</li>
	</ol>
	</li>
	<li>Preparation of protein soaked agarose beads for a localized source of treatment reagent
	<ol>
		<li>Resuspend the agarose beads in their storage container and transfer 100 &#181;l of agarose beads into a 1.5 ml microfuge tube.</li>
		<li>Fill the remaining volume of the tube with sterile 1x Phosphate-Buffered Saline (PBS) and mix the beads to wash them. Place the microfuge tube in a rack for a few minutes to allow the beads to settle to the bottom and then pipet off the PBS from on top of the beads. Refill the microfuge tube with sterile 1x PBS and repeat the washing process twice more.</li>
		<li>Prepare the protein of interest at two times the concentration desired for treatment.</li>
		<li>Mix 5 &#181;l of washed agarose beads with 5 &#181;l of the 2x protein stock and gently agitate the beads in the protein. Place the tube on ice for 1 hr, mixing gently every 10-15 min.</li>
		<li>Rinse the beads briefly in sterile 1x PBS before placing the beads on the intestines.</li>
	</ol>
	</li>
	<li>Placement of agarose beads
	<ol>
		<li>Gently place the agarose beads on top of the intestines using a mouth pipet with pulled capillary needles. Place the beads with extreme caution as rough handling will damage the intestine and prevent villus development in the injured area.</li>
		<li>Place twice as many beads as are needed for analysis since the intestines will undergo peristaltic movements and about half of the beads placed will roll off of the intestines over the culturing time.</li>
	</ol>
	</li>
	<li>Fixation of cultured intestines
	<ol>
		<li>Carefully remove the treatment media from below the transwell and allow the intestines to air dry for 3 min.</li>
		<li>Add 1 ml of fixative under the transwell for 2 min, then cover the top of the intestine with 2 ml of fixative for the remainder of the fixation time. Fix with 4% paraformaldehyde O/N at 4&#176;C or for 2 hr at RT.</li>
	</ol>
	</li>
</ol>
2. Imaging of Fixed Intestines
<ol>
	<li>Wholemount imaging
	<ol>
		<li>Place the whole intestines (with endogenous fluorescence) on a slide with 100 &#181;l of 1x DPBS. Use forceps to gently arrange the intestines.</li>
		<li>Use a lab tissue to carefully remove the DPBS and immediately add 100 &#181;l of 70% glycerol to make the tissue more transparent and increase the possible depth of imaging. 
		NOTE: If further penetration of the intestine is desired, instead of mounting the intestine in 70% glycerol, soak the intestine in a few drops of Focus Clear solution for 10-30 min. Pipet off the Focus Clear solution and mount the intestine with Mount Clear.</li>
		<li>Dip each of the four corners of a coverslip into modeling clay and pick up a small amount of clay to use as spacers between the slide and coverslip to keep the coverslip from flattening the intestine.</li>
		<li>Seal the coverslip onto the slide with melted VALAP applied with a cotton swab.</li>
		<li>Image the intestines on either an upright or inverted confocal microscope. Please refer to the discussion for further details.</li>
	</ol>
	</li>
	<li>Vibratome sectioning of fixed intestines (for cross-sectional view of intestinal structures)
	<ol>
		<li>Embed intestines in 7% agarose in small plastic molds (10 x 10 x 15 mm) and allow the agarose blocks to cool and solidify.</li>
		<li>Remove agarose blocks from the plastic molds and glue agarose blocks to the vibratome collection stage with instant glue.</li>
		<li>Fill the collection stage with ice cold 1x DPBS.</li>
		<li>Cut sections at a thickness between 100-150 &#181;m. For thicker sections use clearing solutions (described in section 2.1.2) to visualize deeper into the cross-section.</li>
		<li>Pour vibratome sections from the collection stage into a well of a 6-well plate for storage at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Immunofluorescence staining of fixed, vibratome sectioned tissues
	<ol>
		<li>Place 5-12 vibratome sections per well into a 24-well plate with 1x DPBS.</li>
		<li>Remove 1x DPBS and add 500 &#181;l of permeabilization solution per well. Gently rock the samples for 25 min at RT.</li>
		<li>After permeabilization, wash the samples 3 times with 1x PBS.</li>
		<li>Block the samples for at least 30 min in 500 &#181;l of Blocking Solution.</li>
		<li>After blocking, exchange the blocking solution with 500 &#181;l of primary antibodies diluted in blocking solution and keep the samples at 4 &#176;C O/N. 
		NOTE: The primary antibody dilutions that work well on frozen and paraffin sections generally work well on vibratome sections too, however antibodies requiring nuclear antigen retrieval generally do not work well with this protocol (e.g., BrdU).</li>
		<li>The following day, wash the samples 3 times for 15 min each time with blocking solution.</li>
		<li>After washing, apply 500 &#181;l of the secondary antibody diluted in blocking solution. Wrap the 24-well plate in aluminum foil to protect the fluorophores from light and rock the samples for 45 min to 2 hr at RT.</li>
		<li>Wash the samples 3 times with 1x PBS for 15 min each and mount the vibratome sections on slides as described in Section 2.4.</li>
	</ol>
	</li>
	<li>Mounting vibratome sections
	<ol>
		<li>Prepare slides for vibratome mounting by cutting thin slices of double-stick tape and placing them along the long sides of the slides.</li>
		<li>Carefully place 5-10 vibratome sections between the double-stick tape on the slides in a drop of 100 &#181;l of 1x PBS.</li>
		<li>Unfold all sections and spread them out into a single layer across the slide.</li>
		<li>Remove any excess agarose or uneven bits that would prevent a coverslip from sitting flat.</li>
		<li>Carefully use a lab tissue to soak up excess 1x PBS from around the vibratome sections.</li>
		<li>Add a drop of aqueous mounting medium on top of the vibratome sections and then coverslip.</li>
		<li>Seal the coverslips onto the slides with melted VALAP applied with a cotton swab.</li>
		<li>Image the vibratome sections on either an upright or inverted confocal microscope. Please refer to the discussion for further details on selection of an imaging system.</li>
	</ol>
	</li>
</ol>
3. Live Imaging of Whole Intestines
Intestines harvested from fetuses after E12.5, with the connected omentum left intact, successfully develop villi in culture using the above system. Therefore, this ex vivo system permits the capture of live z-section images of the developing intestines which can be reconstructed for a three-dimensional view of morphogenesis over time.
<ol>
	<li>Setting up live imaging
	<ol>
		<li>Use instant glue to adhere an individual polycarbonate membrane to a fine mesh screen. This provides a support scaffold to hold the intestine in place below the dipping lens of the upright confocal microscope. Please refer to the discussion section for further details on selection of live imaging microscopes.</li>
		<li>Place the mesh screen in the center well of a culture plate.</li>
		<li>Place the dissected intestine onto the polycarbonate membrane and add a drop of instant glue at either end. Use only enough glue to affix the intestine, but not coat it!</li>
		<li>Add culture media free of phenol red below the polycarbonate membrane in the center well of the culture plate.</li>
		<li>Add water to the outer rim of the culture plate to provide humidity.</li>
		<li>Since the fetal intestine undergoes peristaltic-like waves of contraction in culture, add 100 &#956;l of a 1:5 solution of xylazine in 1x PBS to 3 ml of media to control movement of the intestine while imaging. This dosage will control intestinal movements for about 8-10 hr without compromising villus development.</li>
		<li>For a transverse view of the intestine, embed the live intestines in a 3-4% agarose solution and cut thick sections (150-500 &#181;m) on a vibratome. Match the rigidity of the agarose with the stiffness of the intestine being cut and empirically determined the developmental stage. Generally, a 3% agarose solution works well for intestines younger than E14.5 and a 4% solution works well for intestines E15-E16.</li>
		<li>Glue the vibratome sections to a polycarbonate membrane affixed to a mesh screen (as in Section 3.1.1) and culture as described in Section 3.1.2-3.1.6.</li>
		<li>Conduct the live imaging using a two-photon confocal fluorescence microscope (excitation laser between 690-1,040 nm) on an upright stand with a tunable table.</li>
		<li>Two-photon laser tuned to 900 nm provides deep penetration with the best resolution for GFP signals. It reduces damage to tissues while imaging. In addition, setting the power of the laser to the lowest possible emission reduces tissue damage. Typically to obtain a good excitation of GFP, use 12% power on the two-photon laser.</li>
	</ol>
	</li>
</ol>

<ol><li>Grosse, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+dynamics+in+fetal+intestinal+epithelium%3A+implications+for+intestinal+growth+and+morphogenesis%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Cell dynamics in fetal intestinal epithelium: implications for intestinal growth and morphogenesis.</a> Development. 138, 4423-4432 (2011).</li>
<li>Walton, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hedgehog-responsive+mesenchymal+clusters+direct+patterning+and+emergence+of+intestinal+villi%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Hedgehog-responsive mesenchymal clusters direct patterning and emergence of intestinal villi.</a> Proc Natl Acad Sci U S A. 109, 15817-15822 (2012).</li>
<li>Wells, J. M., Melton, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vertebrate+endoderm+development%5BTitle%5D)+AND+%22Annu+Rev+Cell+Dev+Biol%22%5BJournal%5D)">Vertebrate endoderm development.</a> Annu Rev Cell Dev Biol. 15, 393-410 (1999).</li>
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<li>Mathan, M., Moxey, P. C., Trier, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphogenesis+of+fetal+rat+duodenal+villi%5BTitle%5D)+AND+%22Am+J+Anat%22%5BJournal%5D)">Morphogenesis of fetal rat duodenal villi.</a> Am J Anat. 146, 73-92 (1976).</li>
<li>Burns, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Requirement+for+fibroblast+growth+factor+10+or+fibroblast+growth+factor+receptor+2-IIIb+signaling+for+cecal+development+in+mouse%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Requirement for fibroblast growth factor 10 or fibroblast growth factor receptor 2-IIIb signaling for cecal development in mouse.</a> Dev Biol. 265, 61-74 (2004).</li>
<li>Pabst, O., Schneider, A., Brand, T., Arnold, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mouse+Nkx2-3+homeodomain+gene+is+expressed+in+gut+mesenchyme+during+pre-+and+postnatal+mouse+development%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">The mouse Nkx2-3 homeodomain gene is expressed in gut mesenchyme during pre- and postnatal mouse development.</a> Dev Dyn. 209, 29-35 (1997).</li>
<li>Pabst, O., Zweigerdt, R., Arnold, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+disruption+of+the+homeobox+transcription+factor+Nkx2-3+in+mice+results+in+postnatal+lethality+and+abnormal+development+of+small+intestine%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Targeted disruption of the homeobox transcription factor Nkx2-3 in mice results in postnatal lethality and abnormal development of small intestine.</a> Development. 126, 2215-2225 (1999).</li>
<li>Kaestner, K. H., Silberg, D. G., Traber, P. G., Schütz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mesenchymal+winged+helix+transcription+factor+Fkh6+is+required+for+the+control+of+gastrointestinal+proliferation+and+differentiation%5BTitle%5D)+AND+%22Genes+%26+Development%22%5BJournal%5D)">The mesenchymal winged helix transcription factor Fkh6 is required for the control of gastrointestinal proliferation and differentiation.</a> Genes & Development. 11, 1583-1595 (1997).</li>
<li>Madison, B. B., McKenna, L. B., Dolson, D., Epstein, D. J., Kaestner, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FoxF1+and+FoxL1+link+hedgehog+signaling+and+the+control+of+epithelial+proliferation+in+the+developing+stomach+and+intestine%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">FoxF1 and FoxL1 link hedgehog signaling and the control of epithelial proliferation in the developing stomach and intestine.</a> J Biol Chem. 284, 5936-5944 (2009).</li>
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<li>Mao, J., Kim, B., Rajurkar, M., Shivdasani, R., Mcmahon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hedgehog+signaling+controls+mesenchymal+growth+in+the+developing+mammalian+digestive+tract%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Hedgehog signaling controls mesenchymal growth in the developing mammalian digestive tract.</a> Development. 137, 1721-1729 (2010).</li>
<li>Theiler, K. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aTheiler%2C+K+%22The+house+mouse.+Development+and+normal+stages+from+fertilization+to+4+weeks+of+age%22">The house mouse. Development and normal stages from fertilization to 4 weeks of age</a>. , 2, Springer-Verlag. (1989).</li>
<li>Hamilton, T. G., Klinghoffer, R. A., Corrin, P. D., Soriano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evolutionary+divergence+of+platelet-derived+growth+factor+alpha+receptor+signaling+mechanisms%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms.</a> Mol Cell Biol. 23, 4013-4025 (2003).</li>
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</ol>

The authors have no financial conflicts to disclose.

The dynamic nature and complex tissue interactions of the developing intestine requires 3D visualization to have a full appreciation of these morphogenetic events. With evolving imaging technology, the ability to examine villus morphogenesis in detail is developing/improving and with it, the understanding of the spatial communication and interaction during organogenesis is greatly enhanced.
Alternative methods for culturing whole intestines have also been tested, but the transwell system remai...

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

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

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Culture of whole explants of fetal intestines allows for the analyses of the location, distribution, and duration of the signaling molecules that coordinate intestinal development, as this system enables the manipulation of the signaling with pharmacological reagents or recombinant proteins. The transwell culture system (Figure 1A, reproduced from Walton et al., 2012)2 provides an air-liquid interface, which makes it possible to place drug- or protein soaked agarose beads onto the tis...

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.

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

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.

A System for ex vivo Culturing of Embryonic Pancreas

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

Tissue Engineering of the Intestine in a Murine Model

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The ex vivo upright droplet culture is an alternative to current in vitro and in vivo 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.

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

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.

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

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

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

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

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

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

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.

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

Here we describe constructing a basic three-dimensional (3D) intestinal cell line model system and a paraffin embedding protocol for light microscopic evaluation of fixed intestinal equivalents. Staining of selected proteins permits the analysis of multiple visual parameters from a single experiment for potential use in preclinical drug screening studies.

Basic Three-Dimensional (3D) Intestinal Model System with an Immune Component

Learn whole organ culture of mouse embryonic intestines for ex vivo signaling pathway modulation and live imaging of villus development. This method enables three-dimensional visualization of intestinal organogenesis.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development (Video) | JoVE Journal

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

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