Primary Acinar Cell Isolation: A Method to Isolate Acinar Cells from Mouse Pancreas

All procedures involving animal models have been reviewed by the local institutional animal care committee and the jove veterinary review board.

1. Acinar Cell Isolation

1. Sacrifice the mouse via CO₂ induction, and perform cervical dislocation. Immediately dissect the pancreas. To dissect the pancreas, first pin the paws of the mouse to the polystyrene lid, orient the mouse such that the tail is facing the researcher, and spray the abdomen with 70% ethanol.
   NOTE: The mouse used in the representative results was a 12-week old non-transgenic female with C57BL/6 background. Mouse selection is further discussed in the discussion section.
   1. Using a set of autoclaved scissors and forceps, lift the fur/skin with the forceps at the midline and use the scissors to make an incision through the fur and skin from the urethral opening to the diaphragm/ribcage area.
   2. Make additional incisions to the left and right such that the fur/skin is cut away to create 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, as was done with the fur/skin.
   3. Lift the intestines with the forceps and put it to the left side of the mouse creating space to see the pancreas, which is light pink in color, and attached to the spleen, which is a dark red oval. The pancreatic tissue is distinguished by its soft, spongy texture. Cut out the pancreas which will run along the stomach and intertwine with the intestines.
   4. Separate the spleen from the pancreas. Then, put the pancreas in a 50 mL tube containing 10 mL of HBSS with 1x penicillin-streptomycin and bring this to the laminar flow hood.
      NOTE: The remaining steps of the protocol should be done utilizing sterile technique in a laminar flow hood.
2. Pour the pancreas and HBSS (with penicillin-streptomycin) into an empty weigh boat and, using forceps, wash the pancreas by swirling it. Then, move the pancreas with the forceps to a second weigh boat containing HBSS (with penicillin-streptomycin). Again, wash the pancreas by swirling.
3. Move the pancreas to the third HBSS (with penicillin-streptomycin)-containing weigh boat and begin cutting the pancreas into small pieces of 5 mm or less. Next, pour the pancreas pieces and HBSS into an empty 50 mL tube.
   1. To do this, use the forceps to move the pancreas pieces into the liquid as the weigh boat is being tipped. Pour once all the pieces are no longer attached to the weigh boat. Pick up any remaining pieces with the forceps and wash the forceps in the 50 mL tube containing the pancreas in HBSS with penicillin-streptomycin.
4. Centrifuge at 931 x g for 2 min at 4 °C and then remove the HBSS (and any fat that is floating) by pipetting it off with a 5 mL pipette.
5. Add 5 mL of collagenase (Make 40 mL of HBSS + 5% FBS, 20 mL of HBSS + 30% FBS, and 5 mL of 2 mg/mL collagenase in HBSS. Sterilize the collagenase by filtration (0.22 µm pore) and keep at room temperature. Keep each of the HBSS solutions on ice) to the pancreas. Ensure a sealed lid by wrapping the 50 mL tube in plastic paraffin film and then place it in an incubator shaking at 220 rpm for 20 min at 37 °C.
   NOTE: The collagenase digestion time may vary, as noted in the discussion. At the end of incubation, no large pieces of tissue should remain.
6. To stop the dissociation, place the pancreas-containing tube on ice and add 5 mL of cold HBSS + 5% FBS. Centrifuge at 931 x g for 2 min at 4 °C and then pipet off the supernatant using a 5 mL pipet.
7. Resuspend in 10 mL of HBSS + 5% FBS and centrifuge at 931 x g for 2 min at 4 °C. Pipet off the supernatant using a 5 mL pipet and repeat this step with another 10 mL of HBSS + 5% FBS.
8. Resuspend in 5 mL of HBSS + 5% FBS and, using a P1000, transfer 1 mL at a time through a 500 µm mesh into a 50 mL tube. Add an additional 5 mL of HBSS + 5% FBS through the mesh with a P1000 to wash any remaining pancreatic cells through the mesh.
9. Put the cell suspension from the 500 µm mesh through the 105 µm mesh by pipetting 1 mL at a time using a P1000. Next, gently pipet the cell suspension into a tube containing HBSS + 30% FBS. A layer of cell suspension will form at the top; upon centrifugation, the acinar cells will sink to form a pellet.
10. Centrifuge at 233 x g for 2 min at 4 °C. Remove the supernatant and resuspend the pellet in 1x Waymouth's complete media.
    1. If proceeding directly to embedment of cells in collagen or basement membrane matrix, resuspend in a volume of 7 mL per pancreas. If proceeding to adenoviral or lentiviral infection, resuspend in 4 mL per pancreas.