The overall goal of this procedure is to grow whole organ cultures of mouse embryonic intestines, ex vivo, thus allowing for the modulation of cell signaling pathways. This is accomplished by first harvesting mouse intestines from fetuses between embryonic day 12.5 to 18.5. The second step is to place the intestines in a transwell culture or live imaging culture plate.
Next, treat the intestines with media prepared with the pharmacological inhibitors of cell signaling pathways or place. The aro speeds soaked with recombinant proteins to alter cell signaling on top of the intestines. The final step is to image the cultured intestines either in thick viome sections or hole mount.
Ultimately, confocal imaging followed by three dimensional reconstruction, is used to visualize how modulation of cell signaling pathways has affected the development of the tissue layers during villus formation. Visual demonstration of this method is critical as the embryonic tissue is very delicate and requires great care during dissection and handling. To prepare for dissection, soak the dissecting tools in 70%ethanol.
Place the six well culture plate and 10 centimeter Petri dishes with BGJB media on ice. Then remove the intestines from the euthanized fetuses. Dissect them one by one in one X-D-P-B-S.
Carefully remove the spleen from the stomach, then the pancreas from the stomach and the upper duodenum. After that, gently separate the omentum, leaving it attached as much as possible, and separating the connections only enough to allow the intestine to be straightened. Then place the samples in a 10 centimeter Petri plate with working BGJB media.
Next, use a mouth pipette to transfer the intestinal pieces onto the transwells of the culture plate. Remove the media from under the transwell. Then add 700 microliters of treatment media under the trans.
Well subsequently, add 300 microliters of treatment media dropwise on the intestines, and allow it to soak through the transwell. Change the treatment media at least once daily or more often depending on the half-life of the treatment reagent. Alternatively, gently place the agro beads on the intestines using a mouth pipette with a pulled capillary needle.
Place the beads with extreme caution as rough handling will damage the intestine and prevent villus development in the injured area. In this procedure, embed the intestines in 7%aros in small plastic molds, and allow the aros blocks to cool and solidify. Afterward, remove the aros blocks from the plastic molds.
Glue the aros blocks to the Vibram collection stage with instant glue. Next, fill the collection stage with ice cold one X-D-P-B-S. Cut the sections at a thickness of 100 to 150 micrometers.
Then transfer the sections from the collection stage into a well of the six well plate for storage at four degrees Celsius. To prepare the slides for mounting place double-sided tape along the long slide of each slide. Place a drop of one XPBS on it.
Then carefully place five to 10 sections on the slide. Unfold all the sections and spread them out onto a single layer. Across the slide.
Remove any excess aros or uneven bits that would prevent the cover slip from lying flat. Carefully use a lab tissue to soak up excess one XPBS from around the sections. Next at a drop of aqueous mounting medium on top of the sections.
Then place the cover slip over it and seal it onto the slide with melted vap. Afterward, image the viome sections on either an upright or inverted confocal microscope. Now use instant glue to adhere an individual polycarbonate membrane to a fine mesh screen.
Place the mesh screen in the center well of a culture plate. Then place the dissected intestine onto the polycarbonate membrane and add a drop of instant glue to each of the ends. Next, add culture media free of phenol red below the polycarbonate membrane in the center well of the culture plate.
Add water to the outer rim of the culture plate to provide humidity. Since the fetal intestine undergoes peristaltic like waves of contraction in culture, add 100 microliters of xylazine solution to three milliliters of media in the center well of the plate to control movement of the intestine while imaging. Conduct the live imaging using a two photon confocal fluorescence microscope on an upright stand.
Shown here are the cross sections of freshly harvested E 14, E 15, and E 16 duodenal tissue stained with ecat herein and dpi. This intestinal segment was harvested at E 14 and cultured for two days. At E 14, the unmodeled epithelium was still pseudo stratified.
After two days in culture VII are visible, though they are slightly delayed compared to vii. At E 16, here is the three dimensional reconstruction of the optical sections of pcam stained vasculature in the E 14 duodenum, and this figure shows the three dimensional reconstructions of confocal images from E 15.5 viome sectioned intestines that were antibody immunofluorescence stained. After watching this video, you should have a good understanding of how to dissect the embryonic tissue, set the intestines up for whole organ culture, and prepare the tissue for high quality three-dimensional imaging.
