continuous gradient method for porcine islet purification

Purification of Porcine Islets by Surgical Perfusion of Pig Pancreas

Type 1 diabetes is caused by the autoimmune destruction of pancreatic beta cells1.&#160;Consequently, patients are dependent on exogenous insulin, placing them at high-risk glycemic fluctuations with episodes of hyper- and hypoglycemia2. Islet allotransplantation is a potential cure for type I diabetes; however, the limited availability of pancreatic organ donors remains a major barrier to widespread adoption of the procedure2,3. Islet xenotransplantation from porcine donors is a promising alternative as these animals are readily available. Optimization and scaling of porcine pancreatic islet isolation will be critical for progressing islet xenotransplantation.
Although several porcine pancreas procurement techniques have been previously published, many of these procedures describe pancreatectomy after induction of cardiac death or exsanguination4,5,6,7,8,9,10. One major disadvantage of these techniques is the variable warm ischemic time (WIT) occurring between the time of cardiac death and the initiation of intraarterial and/or intraductal infusion of preservation solution. Even 10 min of WIT will negatively impact islet yield and viability5. Minimizing WIT requires prompt perfusion of the pancreas with a preservation solution. During abdominal organ procurement, aortic cannulation followed by intracorporeal perfusion with University of Wisconsin (UW) solution is crucial for removing blood, preventing intravascular thrombosis, protecting against ischemic damage, and minimizing cellular injury11,12.
Previous studies have demonstrated that during porcine pancreas procurement, low-pressure flushing of the celiac trunk and superior mesenteric artery with UW solution improves islet yield and purity4. However, previously published methods for porcine pancreas procurement include open aortic cannulation, which can be technically challenging, particularly for smaller pigs13,14,15. In this manuscript, we present a detailed, step-by-step protocol with accompanying visual aids for the surgical perfusion and procurement of the porcine pancreas, followed by islet isolation and purification. This new technique of aortic cannulation during pancreatic isolation was developed specifically to minimize WIT and allow the pancreatic procurement to be performed in a bloodless field as a means of maximizing islet isolation yield and viability.

Author Spotlight: Techniques and Challenges in Islet Purification for Xenotransplantation

Surgical Perfusion and Isolation of the Porcine Pancreas for Islet Isolation

Our lab focuses on microencapsulation for pancreatic islet xenotransplantation. This requires reliable islet isolation and purification. We tackle this challenge by isolating the pancreas without damaging it from warm ischemia or mechanical injury.And this offers strategies for effective digestion and a high islet yield. Isolating pancreatic islets is very challenging. Both the yield and purity depend on every phase of the process being executed effectively and efficiently from the organ procurement and digestion to the islet purification.Furthermore, ethical and humane considerations are important when planning animal procedures that will facilitate optimal outcomes while reducing any animal suffering. So here we demonstrate that we can percutaneously cannulate the aorta to selectively perfuse the pancreas. This technique allows us to efficiently preserve the pancreas and then isolate the islets.So a major advantage of our protocol is the use of Seldinger technique for aortic cannulation. This helps maintain a bloodless operative field and preserves the dissection planes. This technique can also be easily adapted for smaller pigs simply by using a smaller sheath.Our lab is currently investigating methods for creating an on-demand supply of pancreatic islets. Specifically, we're interested in seeing if encapsulating islets in a hydrogel can prolong their lifespan and improve their function in vitro and in culture. The ultimate goal is to reliably isolate porcine islets to use it in transplantation research, or even for xenotransplantation.

Continuous Gradient Method for Porcine Islet Purification

To begin, set a pump to a flow rate of 200 milliliters per minute and load 125 milliliters of high-density gradient solution into the continuous gradient former, and then into the cell processor. Start the cell processor centrifuge function at 1, 000 RPM. Release air from the system and rep-rime it with a high-density gradient solution.Then add 125 milliliters of high-density gradient solution and 130 milliliters of low-density solution to the gradient former. Load 100 milliliters of digested tissue suspension. Increase the cell processor speed to 2, 000 RPM for three minutes.To prepare 16 50 milliliter conical tubes for collecting purified islet fraction, add 25 milliliters of RPMI medium to each tube and arrange them in ascending order. Collect the purified islet fractions in the prepared conical tubes. To stain the islets, collect 100 to 200 microliter aliquots from each purified islet fraction, and assess islet content using DTZ staining in a 24-well tissue culture plate.Inspect each fraction using light microscopy at 4X where the islets will appear dark red to black. Using this protocol, pancreatic islet isolation was performed on three pigs. Brightfield images showed purified pancreatic islets, which were characterized for viability using live/dead staining.Representative islet size distribution after the islet isolation showed size distribution around 50 to 100 microns. Further, yield and purity were evaluated using an islet cell counter, indicating efficient porcine pancreatic isolations.

continuous-gradient-method-for-porcine-islet-purification

Research

JoVE Journal

Medicine

55 Views. To begin, set a pump to a flow rate of 200 milliliters per minute and load 125 milliliters of high-density gradient solution into the continuous gradient former, and then into the cell processor. Start the cell processor centrifuge function at 1, 000 RPM. Release air from the system and rep-rime it with a high-density gradient solution.Then add 125 milliliters of high-density gradient solution and 130 milliliters of low-density solution to the gradient former. Load 100 milliliters of d...

Video: Continuous Gradient Method for Porcine Islet Purification

Pancreatic islet transplantation is an emerging treatment for type I diabetes; however, it is limited by donor matching and availability. Porcine islet xenotransplantation offers a promising alternative to allotransplantation, with the potential for large-scale production of on-demand, functional islets. The yield and viability of isolated islets is highly susceptible to the quality of the donor pancreas and the method of procurement, particularly the duration of warm-ischemia time. To improve organ preservation and subsequent islet yield and viability, we have developed a protocol for surgical perfusion and resection of the porcine pancreas. This protocol employs direct infrarenal aortic cannulation and organ perfusion to both minimize warm-ischemia time and simplify the procedure for operators who do not have extensive surgical expertise. Subsequent arterial perfusion of the pancreas via the aorta flushes stagnant blood from the microvasculature, thereby reducing thrombosis and oxidative damage to the tissue. This manuscript provides a detailed protocol for surgical perfusion and resection of the porcine pancreas, followed by islet isolation and purification.

This protocol provides a step-by-step guide for the procurement of a porcine pancreas for islet isolation and purification.

Pancreatic islet transplantation is an emerging treatment for type I diabetes; however, it is limited by donor matching and availability. Porcine islet ...

Dissection and Surgical Perfusion of Porcine Pancreas for Islet Isolation

Begin by preparing for the surgery. To perform retroperitoneal dissection, incise the peritoneum to the right of the midline from the level of the right kidney to the aortic bifurcation using electrocautery. Sharply expose the right kidney, right renal vein, right renal artery, and inferior vena cava, as well as the aorta.Using a large vessel loop, obtain circumferential control of the distal infrarenal aorta. Then, obtain circumferential control of the mid infrarenal IVC. And ligate the IVC using a 2-0 silk tie.Next, obtain circumferential control of the right and left renal arteries and veins. Ligate these structures using 2-0 silk ties. Proceed with intrathoracic dissection by retracting the liver inferiorly to expose the right hemidiaphragm.Apply two Allis clamps to the membranous portion of the right hemidiaphragm, and incise the diaphragm in between the two clamps using electrocautery. Extend the diaphragm myotome horizontally to approximately seven centimeters. Next, obtain circumferential control of the intrathoracic IVC.Place a 2-0 silk tie around the intrathoracic IVC and snap the ends of the suture with a small hemostat without occluding the IVC. Then, use two Allis clamps to retract the edge of the diaphragmotomy inferiorly. And extend it to the left hemidiaphragm.Administer systemic heparin to the pig for anticoagulation via peripheral venous access, and wait for at least three minutes before proceeding. Using an 18-gauge micropuncture needle, access the infrarenal aorta. Then, pass a J wire through the needle into the aorta and remove the needle over the wire.Place a 10 French sheath over the wire into the aorta and remove the wire. Next, connect IV tubing to the 10 French sheath, and infuse sterile UW solution through the sheath at a rate of 40 to 60 drops per minute in a sterile fashion. Then, using an aortic clamp, clamp the infrarenal aorta and the descending thoracic aorta.Tie the intrathoracic IVC with the previously placed 2-0 tie, and administer potassium chloride via peripheral venous access for euthanasia. Then, isolate the pancreatic duct by locating it at the entry of the duodenum. Ligate the duct using two 3-0 silk ties and transect the duct between the ties.Complete the pancreatectomy. To infuse the pancreas with the enzyme solution, pass 60-milliliter boluses into the pancreatic duct until the entire enzyme solution is infused. Placed the pancreas in the Ricordi chamber along with the mesh filter and rubber gasket.And secure the lid onto the Ricordi chamber to seal it. Then, place the heating coil in a hot water bath set to 47 degrees Celsius. Insert the inflow and outflow cannulas into the digestion circuit reservoir containing 500 milliliters of 1X HBSS.Initiate the pump at a flow rate of 450 milliliters per minute until the entire chamber is filled with the digestion solution. Then, decrease the flow rate to 100 milliliters per minute as the fluid circulates within the circuit.