Surgical Perfusion and Isolation of the Porcine Pancreas for Islet Isolation
Summary
This protocol provides a step-by-step guide for the procurement of a porcine pancreas for islet isolation and purification.
Abstract
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.
Introduction
Type 1 diabetes is caused by the autoimmune destruction of pancreatic beta cells1. 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.
Protocol
Representative Results
Discussion
Pancreatic islet xenotransplantation using porcine donors is a promising strategy for the treatment of Type I diabetes. Islet isolation is challenging, and final islet viability and yield are highly susceptible to hypotension and tissue ischemia encountered during organ procurement16,17. To optimize pancreas procurement and preservation, this protocol presents a new method for aortic cannulation, visceral isolation, and perfusion during the procurement of the por…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This manuscript was supported by a clinical innovation grant from the Mid-America Transplant Foundation.
Materials
| 1 M NaOH, 100 mL | Millipore Sigma | 1310-73-2 | |
| 10x HBSS, 500 mL (15 to 30 °C) | Thermo Fisher | 14065056 | |
| 1x D-PBS, 500 mL | Thermo Fisher | 14200075 | |
| 1x HBSS, 1 L | Thermo Fisher | 14025076 | |
| 250 mL Conical Tubes | Millipore Sigma | CLS430236-6EA | |
| Amphotericin B [50 mg] | Millipore Sigma | PHR1662 | |
| Antiseptic Povidone Iodine 10% | Millipore Sigma | 25655-41-8 | |
| BioRad Gradient Former | BioRad | 395 | |
| Calcium Chloride dihydrate | Thermo Fisher | 10035-04-8 | |
| COBE Cell Processor | Ebay | 317690 | |
| Digestion circuit reservoir, Gibco bottle, 1 L | Thermo Fisher | 10341001 | |
| Dimethylsulfoxide (DMSO) | Thermo Fisher | 85190 | |
| Dithizone | Thermo Fisher | 60-10-6 | |
| Gradient Former Connection Kit | BioRad | 1652008 | |
| Heparin [1000 U/mL] | Emergency Medical Products | 0409-2720-01 | |
| HEPES Buffer (1 M), 100 mL | Thermo Fisher | 15630080 | |
| Human Serum Albumin 25%, 100 mL | Celprogen | HSA2001-25-1 | |
| Liberase (100 mg) | Millipore Sigma | 5401020001 | |
| LIVE/DEAD Viability/Cytotoxicity Kit | Thermo Fisher | L3224 | |
| Lympholyte 1.1 Media 500 mL | Cedarlane Labs | CL5020 | |
| Masterflex LS 16 Tubing | Masterfle | 96419-16 | |
| Masterflex Peristaltic Pump | Masterflex | 07522-30 | |
| PenStrep (2 mg/mL) | Celprogen | PS-30-002 | |
| Pulmozyme (1 mg/mL, 2.5 mL/vial) | Dornase Alfa | 8931278 | |
| Recovery Medium, PIM(R) | Prodo Laboratories, Inc. | PIM-R001GMP | |
| RPMI 1640 | Millipore Sigma | R8758-1L | |
| Standard Culture Medium, PIM(S) | Prodo Laboratories, Inc. | PIM-S001GMP | |
| University of Wisconsin (UW) Solution, 3 L | Global Transplant Solutions | 1000-0046-06 | |
| Waterbath | Sigma Alderich | CLS6783-1EA |
References
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