Our protocol offers direct visualization of Acinar-to-Ductal Metaplasia and allows the study of the contribution of ectopically administered factors or protein modulation to this process. The main advantage of this technique is that Acinar-to-Ductal Metaplasia can be viewed in real time and cell signaling studies can be performed. To dissect the pancreas from a euthanized mouse, first pin the paws of the mouse to the polystyrene lid, orienting it so that the tail is facing the researcher and spray the abdomen with 70%ethanol.
Use autoclaved forceps to lift the fur and skin at the midline, and with autoclaved scissors, make an incision through the fur and skin from the urethral opening to the diaphragm rib cage area. Make additional incisions to the left and right to cut away the fur and skin, creating a clear view of the abdominal cavity. Then, cut into the peritoneal lining, down the middle and to the right and left and pull it away from the organs.
Use the forceps to lift the intestines and move them to the left side of the mouse, creating space to see the light pink pancreas attached to the dark, red, oval spleen. The pancreatic tissue will have soft, spongy texture. Cut out the pancreas, which runs along the stomach, intertwining with the intestines.
Separate the spleen from the pancreas and place the pancreas in a 50 milliliter tube containing 10 milliliters of HBSS, with one times penicillin streptomycin and transfer it to the laminar flow hood. Pour the contents of the tube into an empty weigh boat and use forceps to wash the pancreas by swirling it. Then, use the forceps to move the pancreas to a second weigh boat containing HBSS with penicillin streptomycin and wash again by swirling.
Move the pancreas to the third weigh boat containing HBSS with penicillin streptomycin and begin cutting the pancreas into five millimeter or smaller pieces. To pour the contents of the weigh boat into a fresh 50 milliliter tube, first use the forceps to move the pancreas pieces into the liquid as the boat is being tipped. Once the pieces are no longer attached to the boat, pour its contents into the tube.
Pick up any remaining pieces with the forceps and wash the forceps in the tube. After centrifuging the tube at 931 times gravity at four degrees Celsius for two minutes, use a five milliliter pipette to remove the HBSS and any floating fat. Add five milliliters of collagenase diluted in HBSS.
Close the tube and ensure a sealed lid by wrapping the tube in plastic paraffin film. Place it in an incubator, shaking at 220 RPM at 37 degrees Celsius for 20 minutes. Which will yield the turbid solution, and few remaining tissue pieces.
To stop the dissociation, place the tube on ice and add five milliliters of cold HBSS with 5%FBS. After centrifuging at 931 times gravity at four degrees Celsius for two minutes, remove the supernatant using a five milliliter pipette. Re-suspend the pellet in 10 milliliters of HBSS with 5%FBS, and centrifuge again at 931 times gravity for two minutes at four degrees Celsius.
Remove the supernatant using a five milliliter pipette and repeat this step with another 10 milliliters of HBSS with 5%FBS. Then re-suspend the pellet in five milliliters of HBSS with 5%FBS. Use a P1000 pipette to filter the cell suspension one milliliter at a time through a 500 micrometer mesh into a 50 milliliter tube.
To wash any remaining pancreatic cells through the mesh, add an additional five milliliters of HBSS with 5%FBS. Then use a P1000 pipette to pass this filtrate one milliliter at a time through the 105 micrometer mesh into a 50 milliliter tube. Gently pipette this cell suspension into a 50 milliliter tube containing HBSS with 30%FBS, which will form a layer of cell suspension at the top.
Centrifuge this cell suspension at 233 times gravity at four degrees Celsius for two minutes and remove the supernatant. Re-suspend the pellet containing the Acinar cells in one times Waymouth's complete medium. Since one pancreas is sufficient for two viral infections, split the cell suspension between the lids of two 35 millimeter plates, which allows for infection in suspension.
For adenoviral infection, add the virus at a 1/1000 dilution to the lid of each plate and swirl. Place the plates in a cell culture incubator at 37 degrees Celsius and 5%CO2. Swirl every 15 minutes for the first hour and continue the incubation for an additional two hours.
To make a collagen gel, combine seven milliliters of type one rat tail collagen, 700 microliters of 10 times Waymouth's medium and 466.6 microliters of 0.34 molar sodium hydroxide in a 50 milliliter tube on ice to make collagen gel. In a 50 milliliter tube, combine equal volumes of cell suspension and collagen gel after three hours incubation and gently mix. Plate 2000 microliters, one well at a time in a six well plate while keeping the plate on ice and swirl to evenly distribute the collagen cell layer.
To solidify the medium, incubate the plate in a cell culture incubator at 37 degrees Celsius and 5%Carbon dioxide for 30 minutes. Then add 1.5 milliliters per well of one times Waymouth's complete medium with desired stimulus or inhibitors. Change the medium the following day and then every other day.
Depending on the stimulus used, Acinar-to-Ductal Metaplasia or ADM can be observed between day three and day five. 24 hours post infection with GFP adenovirus, images of cells embedded in collagen or basement membrane matrix, showed that infection was efficient. At five days post infection, cells embedded in collagen formed duct-like structures when stimulated with 50 nanograms per milliliter of TGF alpha.
Cells embedded in basement membrane matrix formed duct-like structures in the absence of a stimulus at five days post infection. Whereas larger ducts were formed upon stimulation with 50 nanograms per milliliter of TGF alpha. One of the most critical points in the protocol is the length of collagenase digestion.
Proper digestion is indicated by a turbid solution and few remaining tissue pieces.
