This protocol requires the use of a single density gradient making it significantly less labor intensive and more cost effective compared to more complex conventional islet isolation methods. The main advantage of this protocol is that the enzyme is delivered directly to the pancreas resulting in an enhanced tissue digestion and increased islet yield. Begin by taping the limbs of the mouse in the supine position and spraying the body with 70%ethanol.
Using coverglass forceps and curved surgical scissors, make an approximately three centimeter horizontal abdominal skin incision and pull open the skin to expose the abdominal wall. Make a three to four centimeter vertical incision on the abdominal peritoneum to fully expose the pancreas and place the mouse under a dissecting microscope. Push the liver lobe superiorly to expose the bile duct which appears as a pale pink tube and gently move the intestines from the right lumbar and iliac region to the right of the abdominal cavity to expose the bile duct and hepatic artery.
Use Schwartz micro serrefines to carefully clamp the common bile duct as close to the liver as possible and identify the ampulla of Vater which is located at the duodenal papilla and formed by the union of the pancreatic duct and the common bile duct. Using micro Adson forceps to pull the common bile duct taught, insert a 30 gauge half-inch needle attached to a three milliliter syringe containing three milliliters of freshly prepared collagenase P solution into the ampulla of Vater pushing the needle into the duct parallel to the vessel for about a quarter of the length of the vessel. Once the needle is in place, stabilize the needle with micro Adson forceps and slowly and steadily inject three milliliters of collagenase P solution into the duct.
The injection is considered successful if the head, neck, body, and tail regions of the pancreas are fully inflated. Proper entry into the ampulla and cannulation of the common bile duct are critical for a successful digestion harvest. Piercing the ductal walls reduces the efficacy of the isolation.
Using curved and micro Adson forceps, carefully pull out the inflated pancreas starting at the spleen and continuing toward the stomach and along the duodenum. Then place the pancreas in a 50 milliliter digestion tube containing three milliliters of ice cold collagenase P solution. To harvest the islets, first use fine surgical scissors to chop the pancreas for three to five seconds before placing the tube in a 37 degree Celsius water bath at 100 to 120 rotations per minute for 12 to 13 minutes.
At the end of the incubation, gently shake the tube for about 30 seconds to disrupt the tissue until the solution is homogenous as confirmed by fine sand-like pancreatic tissue particles. As soon as the tissue is digested, place the tube on ice and add 40 milliliters of ice cold stop solution to terminate the enzymatic reaction. After gently disrupting the pellet, spin down the digested tissue in a swinging bucket centrifuge followed by two centrifugations with 20 milliliters of fresh stop solution per wash.
After the last wash, resuspend the pellet in 40 milliliters of ice cold HBSS for three additional washes, resuspending the pellet in five milliliters of room temperature density gradient solution after the last wash. Vortex the tube briefly at low speed until the solution is homogenized before adding another five milliliters of room temperature density gradient to the tube. Now use a 10 milliliter pipette to gently and slowly add 10 milliliters of room temperature HBSS to the tube in a drop-wise manner to allow the formation of a gradient and use a swinging bucket centrifuge to isolate the cell populations by density gradient separation.
At the end of the centrifugation, use a 10 milliliter pipette pre-wet with cold HBSS to collect five to 10 milliliters of the islet layer between the density gradient and the HBSS. Transfer the islets into a new 50 milliliter tube containing 20 milliliters of fresh ice cold HBSS and collect the cells by centrifugation in a swinging bucket centrifuge. Carefully aspirate all but the last three milliliters of the supernatant without disturbing the pellet and wash the islets at least three more times with 20 milliliters of fresh HBSS per wash.
After the last wash, replace the supernatant with four milliliters of 37 degree Celsius RMPI 1640 medium and gently swirl the tube to dislodge the pellet. Immediately pour the islets into a 100 millimeter Petri dish and wash the tube with five milliliters of fresh RPMI 1640 medium to collect any remaining islets pooling the wash with the other islets. Then use a dissection microscope and a 20 microliter pipette tip to pick healthy spherical or oblong golden brown islets with a smooth surface from the Petri dish for transfer into a new Petri dish containing 10 milliliters of complete RPMI 1640 medium.
When all of the islets have been collected, place the islets in a sterile incubator at 37 degrees Celsius and 5%carbon dioxide in humidified air overnight. Good healthy islets appear to have a smooth round shape while bad damaged islets exhibit rough edges. Undigested exocrine tissue is translucent in appearance and demonstrates an irregular shape.
A proper cannulation and distribution of the collagenase P enzyme can be confirmed by an inflation of the splenic portion of the pancreas. Overnight incubation in low glucose media can prime the islets for a glucose-stimulated insulin secretion assay at the following day allowing insight into the insulin secretory capacity of the islets.
