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Encyclopedia of Experiments: Cancer Research

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Pancreatic Islets Isolation through Collagenase Perfusion

 

Pancreatic Islets Isolation through Collagenase Perfusion: An Enzymatic Method to Isolate Pancreatic Islet Cells

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- Islet cells in the pancreas produce essential hormones involved in endocrine functions. To isolate islets, first, secure a euthanized mouse and surgically expose its abdominal cavity. Brush aside the liver and intestines to locate and clamp the common bile duct. Identify the ampulla of Vater, the duct formed by the union of pancreatic and common bile ducts.

Next, inject a collagenase enzyme solution into the common bile duct. The backflow from the duct successfully inflates the pancreas, facilitating collagenase-induced dissociation of pancreatic tissues. Excise the pancreas into a tube containing more collagenase solution. Mechanically chop the tissue and incubate to aid tissue digestion by the collagenase.

Once obtaining a homogeneous suspension, stop the enzymatic digestion by adding a solution containing proteins that inhibit collagenase activity. Centrifuge to gather a tissue pellet. Resuspend the pellet in an appropriate density gradient medium, layer a suitable buffer over it, and centrifuge to generate gradient layers.

Collect islets from the layer formed between the density gradient medium and the buffer. Finally, pick healthy islets for culturing. In the following protocol, we will demonstrate murine pancreatic islets' isolation by collagenase administration followed by density gradient centrifugation.

- Begin by taping the limbs of the mouse in the supine position and spraying the body with 70% ethanol. Using cover glass forceps and curved surgical scissors, make an approximately 3-centimeter horizontal abdominal skin incision, and pull open the skin to expose the abdominal wall. Make a 3 to 4 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 taut, insert a 30-gauge half-inch needle attached to a 3-milliliter syringe containing 3 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 3 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 3 milliliters of ice-cold collagenase P solution. To harvest the islets, first, use fine surgical scissors to chop the pancreas for 3 to 5 seconds before placing the tube in a 37 degrees 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 homogeneous, 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 digestive 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 5 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 5 milliliters of room temperature density gradient to the tube. Now use a 10-millimeter pipette to gently and slowly add 10 millimeters 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 5 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 3 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 4 milliliters of 37 degrees Celsius RPMI 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 5 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 transferring to a new Petri dish containing 10 milliliters of complete RPMI 1640 medium. When all of the islets have been collected, place the islets in the sterile incubator at 37 degrees Celsius and 5% carbon dioxide in humidified air overnight.

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