The overall goal of this procedure is to dissect and culture mouse embryonic pancreatic buds, ex vivo, allowing direct visualization of the developing pancreas. This is accomplished by first dissecting embryonic day 11.5 or 12.5 mouse embryos and removing the upper body and tail region to isolate the mid body of the embryo. The second step is to expose the duodenum region and the stomach for location, and then dissection of the dorsal pancreatic bud.
Next, the bud is transferred into the micro well of a MacTech dish and cultured for up to one week. Finally, the explan can be analyzed for live cell imaging, or the explan can be fixed, whole mount stained and analyzed by immunofluorescence imaging. Ultimately pancreatic cell differentiation, cytoskeleton organization and cell polarity and branching morphogenesis can be evaluated.
Ex vivo culturing of pancreas explants can help to answer key questions in pancreas organogenesis, such as branching morphogenesis. How does it take place in the developing pancreas and how this event is connected to growth and differentiation. On the day before the experiment, coat the 20 millimeter diameter glass bottom microwells of 35 millimeter MacTech Petri dishes with about 150 microliters of fibronectin covering the whole glass surface.
Then place the dishes in a four degree Celsius refrigerator for overnight incubation. On the day of the dissection, aspirate the fibronectin. Rinse the micro wells with cell culture grade water, and add a minimum volume of 150 microliters of dissection medium to each well.
To ensure success of the dissection, it is essential to know the anatomical structure of the embryo well and to be able to locate the dorsal pancreatic bud Under a stereo microscope with illumination from above. Use dumont forceps to separate the uterus into individual embryo segments. Then gently peel away the muscle of the uterus and remove the individual embryos which are surrounded by the decidua.
Using standard embryo dissection techniques, expose the embryos and remove the yolk sack and amnion. Subsequently, remove the tail region and the upper body of the embryo above the liver. Now using only trans illumination from below, use forceps to gently make a lateral incision to open the mid body of the embryo, facilitating exposure of the inner organs.
Locate the stomach and the spleen. The dorsal pancreatic bud is attached laterally to the posterior region of the stomach. Using dumont forceps, dissect the dorsal pancreatic bud away from the stomach and spleen, but leaves some mesenchyme around the epithelium as for the pancreatic bud shown in this figure.
Finally, use a glass pasture pipette to transfer the isolated bud into a 35 millimeter Petri dish containing cold culture medium. After pre warming the culture medium, replace the dissection medium in the fiber nin coated matte tech dishes with about 150 microliters of the culture medium. In a sterile laminar flow hood, It is important to place the pancreatic exponent carefully in the center of the Matt plate to ensure attachment and spreading of the cells.
Then use a glass pasture pipette to carefully transfer one pancreatic explan into each of the coated matte tech dishes. To ensure spreading during culture, partially rip the mesenchymes surrounding the explan with a fine needle. Now gently place the explan into a tissue culture incubator for few hours to let them attach to the glass bottom of the micro wells.
Once the explan have attached, fill the mat tech dishes with 1.5 to two milliliters of prewarm culture medium. Change the culture medium every other day for five days up to one week. Begin by aspirating the culture medium and wash the explan once with two milliliters of PBS.
Then remove the PBS and fix the X implants with two milliliters of ice cold, 4%para formaldehyde, and place the matte tech dish on ice for 20 minutes, swirling the plate gently from time to time. Next, remove the paraform aldehyde and wash the explan three times with two milliliters of PBS for 10 minutes each at room temperature After the wash block with two milliliters of blocking solution for at least 30 minutes at room temperature. Now transfer the mat tech plates to the cold room and place them inside a humid chamber.
To prevent evaporation, add a minimum of 150 microliters of the primary antibody diluted in blocking solution directly into the center of the 20 millimeter microwell. Depression of the mat tech plates covering the whole explan surface for overnight incubation at four degrees Celsius. The next day, remove the antibody solution and wash away any unbound antibody three times with two milliliters PBS plus tween 20 for 30 minutes each at room temperature.
After the wash, remove PBT and cover the whole explan with a minimum of 150 microliters of the secondary antibody delusion and incubate the explan for one to two hours at room temperature in the dark after staining with the secondary antibody. Wash the explan three times with two milliliters of PBS plus tween. 20 for 30 minutes each at room temperature and then once with PBS only to remove the tween 20.
Finally remove PBS and add aqueous mounting medium with anti fading dropwise into the 20 millimeter micro well of the mat tech plates. And then gently cover the explan with a glass cover slip avoiding air bubbles After the pancreatic explan has firmly attached to the glass bottom dish, assemble and turn on the heater box at least one hour before imaging while the equipment is warming up, aspirate and replace the explan medium with fresh culture medium inside a laminar flow hood. Then gently transfer the implants to the microscope room and place the mat tech dish into the specimen holder of the confocal microscope.
Add water medium to the objective. Close the environmental chamber and then focus on the region of interest in the explan. After two days of culture, pancreatic explants undergo significant growth and start to organize themselves into branched epithelial structures.
Note the area where branching has been initiated. Demarcated in the right picture by the red dotted line, this representative 3D reconstruction of whole mount immuno staining on day three of pancreatic culture demonstrates how the explants exhibit tubules of PDX one positive cells that undergo extensive branching by 48 hours of culture explants on day four of culture undergo typical tip and trunk segregation of the epithelium, displaying staining for EC Ahern in red and phospho histone in blue carboxy peptidase one or CPA one in green appears in progenitor cells at the tips of the epithelial branches, which are also demarcated by the dashed lines. In this representative day three whole mount immuno staining for endocrine lineage markers in pancreatic explan insulin staining is represented in green glucagon appears in blue and eca herrin is in red with the yellow arrows indicating clusters of insulin and glucagon positive cells.
In the inset, one of the endocrine clusters is magnified. In addition, pancreatic epithelium in ex vivo cultures shows proper cytoskeleton organization and cell polarity with apical localization of F actin, which appears here in green and beta one integrin at the basal membranes appearing here in red. Note, the mesenchymal cells indicated by the asterisk interspersed among P pdx one positive epithelial cells.
In this figure representatives still frames taken every 12 minutes for a total of 15 hours from a time-lapse movie of membrane, tomato, pancreatic explants grown in culture are shown. The dashed lines indicate the tip of the branches and the border between the epithelium and mesenchyme. Note that after a 12 hour time lapse experiment, the membrane tomato fluorescent signal remains viable and detectable even though its intensity is reduced.
After watching this video, you should have a good understanding of how to dissect and culture do pancreatic buds, and this will help you to study various aspects in pancreatic development, including morphogenesis growth and differentiation.
