Isolation and Decellularization of a Whole Porcine Pancreas

Tissue engineering of the whole pancreas is a challenge because of its exocrine and endocrine functions. We show a method for the dissection of an intact porcine pancreas and the process of successful decellularization by perfusion of detergents Triton X-100, sodium deoxycholate, and deoxyribonuclease.

Transcript

This method can be used for the dissection of porcine pancreas containing connection, duodenal, and splenic lobes and eventual decellularization via the perfusion of detergents at cold temperatures. Though this method of decellularization provides insight into the porcine pancreas, it may also be applied to pancreata from other large animals. The main advantage of this technique is that it facilitates the intact retrieval of a complicated organ, such as the pancreas, for further tissue engineering applications.

Generally, individuals new to this method, they will struggle because dissection of the intact pancreas while identifying and ligating all of the surrounding blood vessels requires a lot of practice to master. Before beginning the procedure, use one three-by-five-millimeter silicone tube to connect the detergent container to the peristaltic pump and a second three-by-five-millimeter silicone tube to connect the peristaltic pump to the organ chamber via the degasser. Connect a male Luer to the free end of the second tube, and use a third three-by-five-millimeter silicone tube to connect the organ chamber to the detergent collection container via the peristaltic pump.

Finally, connect a two-millimeter unlabeled pipette to the free ends of the tubes in the detergent container and the detergent collection container. Then, place the decellularization setup at four degrees Celsius. To harvest the pancreas, place a heparinized, 45-kilogram, female pig onto a dissection table in the supine position, and use a scalpel to make a 40-centimeter midline incision from the xiphoid process to the pubic bone to expose the abdominal organs.

After identifying the splenic, duodenal, and connection lobes, locate the major duodenal papilla, and use two individual sutures to orally ligate the duodenum from the site. Ligate the inferior esophagus with additional sutures, and cut between the ligatures with scissors to facilitate removal of the stomach. Separate the connective tissues from the colon to reach the small intestine, and separate the connective tissue of the colon that attaches to the splenic lobe of the pancreas.

Small intestines is attached to the pancreas by connective tissue at the splenic lobe, but also surrounds the pancreas. You have to carefully follow the intestinal path to detangle and separate the connective tissue between the intestine and the pancreas. After ligating the arteries, remove the colon from the small intestine.

Identify the inferior mesenteric vein, the inferior mesenteric artery tree, and ligate the vessels with one suture caudal to the pancreas and cut with scissors. Ligate the splenic artery and vein together in the hilum, close to the spleen, and cut distally with scissors to remove the spleen. Follow the duodenum until the duodenal and connection lobes are cleared, and ligate the duodenum at the end with two separate sutures.

The part of duodenum that is in close contact with the connection and duodenal lobes is removed with the pancreas, as the ligation of all the small blood vessels between the two organs is too difficult. After dissecting the portal vein, ligate the vessel with one suture to prevent any blood leakage from the liver, and dissect and ligate the bile duct and hepatic artery with two sutures. Then, cut portal vein, hepatic artery, and bile duct with scissors.

Locate the aorta under the renal vein, and dissect the aorta in the cranial direction from the muscle and connective tissue until the vessel reaches the pancreas. Now, gently flip the pancreas over, and dissect the aorta, keeping the superior mesenteric artery and the celiac trunk intact. Cut the aorta cranial to the celiac trunk, and blunt dissect the remaining surrounding tissue to allow harvest of the pancreas.

Then, use a 50-milliliter syringe connected to a four-millimeter arteriotomy cannula to flush the organ through the aorta with solution two until the whole organ is perfused or until the whole organ becomes cold. Place the solution two perfused pancreas on a metal tray on ice, with aorta facing up, and use scissors to cut the sutures from the duodenum. Use a 25-milliliter pipette to flush the duodenum with 50 to 150 milliliters of ultrapure water, and religate the tissue with fresh sutures.

Ligate one end of the aorta and all the branches, excluding the superior mesenteric artery and the celiac trunk, to prevent leakage, and insert a four-millimeter arteriotomy cannula into the other end of the aorta. Next, place the pancreas into the organ chamber, and connect the cannula in the pancreas to a male Luer. Then, perfuse the pancreas with solution three for one hour at 20 milliliters per minute, and freeze the double-perfused pancreas at minus 20 degrees Celsius in solution four until the start of the decellularization.

On the day of starting decellularization, thaw the pancreas at four degrees Celsius, and place the thawed pancreas in the decellularization organ chamber. Connect the detergent inlet tube to the aorta of the pancreas, and wash the pancreas with solution three at 20 milliliters per minute overnight at four degrees Celsius. The next morning, decant any solution remaining in the organ chamber, and perfuse the pancreas for 30 minutes at 20 milliliters per minute in solution five at four degrees Celsius.

At the end of the solution five perfusion, perfuse the pancreas for eight hours at 20 milliliters per minute in solution six and four degrees Celsius, followed by a 96-hour wash in fresh solution five at 20 milliliters per minute and four degrees Celsius. Replace the wash with 500 milliliters of DPBS supplemented with calcium and magnesium for 30 minutes at 37 degrees Celsius, followed by perfusion with 250 milliliters of solution seven for four hours at 20 milliliters per minute and 37 degrees Celsius. Then, wash the organ for another 120 hours with fresh solution five at 20 milliliters per minute and four degrees Celsius, and store the organ in solution eight.

Here, the gross morphology of a normal pancreas, which appears light pink and contains splenic, connection, and duodenal lobes, can be observed. After decellularization, the pink color is lost, and the processed pancreas appears pale white in color. In a normal pancreas, many blue nuclei are apparent by H and E staining, while in a decellularized pancreas the nuclei can no longer be visualized, confirming their loss.

The isolated intact porcine pancreas, following this procedure, it will allow a complete perfusion and successful decellularization while preserving the structure and also allowing some extracellular matrix proteins to be obtained. The decellularized pancreas can be used for developing whole-organ recellularization strategies in bioreactor systems with relevant cell types. Following recellularization with recipient stem cells, the tissue engineered pancreas may be used for clinical applications and drug testing.

Don't forget that working with Triton X-100 and SDC can be extremely hazardous and that precautions, such as wearing gloves and preparing the solutions under fume hood, should always be taken while performing this procedure.

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Journal (Bioengineering)

Journal (Bioengineering)

Isolation and Decellularization of a Whole Porcine Pancreas

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