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 procedure^2,3. Islet xenotransplantation from porcine donors is a promising alternative as these animals are readily available. Optimization and scali....

Protocol

All procedures involving animals are approved by the Institutional Animal Care and Use Committee (IACUC) at the Washington University School of Medicine Division of Comparative Medicine. Adult Yorkshire pigs ranging from 35 to 35 kg are ideal for this procedure; however, the protocol can be adapted for pigs of different sizes depending on the experimental context. The entire procedure should be performed in a sterile fashion in an operating room setup.

1. Preoperative preparation and ane.......

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 procurement^16,17. 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.......

Materials

Name	Company	Catalog Number	Comments
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
Isoflurane	Covetrus	Item#:11695067772
Ketamine	Patterson	Item #:07-894-8462
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
Tzed™ (tiletamine and zolazepam) Injection C	Patterson	Item #:07-894-6240
University of Wisconsin (UW) Solution, 3 L	Global Transplant Solutions	1000-0046-06
Waterbath	Sigma Alderich	CLS6783-1EA
Xylazine	Patterson	Item #:07-891-6165