Title: Normothermic preservation for pancreas transplantation
Funder: Department of Health (England)

Abstract: Research Plan A full freedom to operate search has not been undertaken but extensive IP searches were undertaken by both US and European patent offices prior to grant of the above patents. Aims Aims:The aim of this project is to develop a novel method of normothermic perfusion to preserve and resuscitate donor organs for pancreas transplantation. This will involve initial proof of principle experiments and then development of a prototype device that will be suitable for clinical trials. By the end of the project, we intend to have generated evidence that normothermic preservation can enable the preservation of the pancreas as measured by whole organ endocrine function and the quality of islets isolated following digestion. This will be the prelude to a clinical trial. Methodology:As an experimental model, we plan to use human organs that have been retrieved with the intention of transplantation, but discarded because of concerns about warm ischaemia or other donor factors (this occurs in more than 50% of livers and an even higher proportion of pancreases from non-heart-beating donors). This will be organisationally challenging and will require close working with the NHS Blood and Transplant (NHSBT) organ donation service to identify potentially suitable donors, seek consent from donor families and collaborate in other logistic aspects. As part of the National Organ Retrieval Service, the Oxford Transplant Centre regularly provides a team for multi-organ retrieval. We have carried out a pre-clinical liver perfusion study in recent months using organs from the same source and have established the necessary logistics with the organ donation network. Perfusion circuit:Experiments will first be performed using the OrganOx Metra perfusion device that has been developed for the liver. This is a sealed circuit in which blood is pumped out of the liver by a centrifugal pump and passes through a heat exchanger and oxygenator, splitting to the hepatic artery directly and, via a variable resistance, to a reservoir supplying the portal vein. The circuit receives infusions of heparin, prostacyclin, bile salt nutrition and, if necessary, insulin. The temperature is regulated to 37 C and on-line blood gas measurements are used to control the flow and proportions of oxygen/air to the oxygenator. The system is now fully automated with the exception of glucose monitoring (which is manual). The most recent version of this device is fully transportable, with sufficient battery capacity to run independently of mains electricity for several hours. Some modification of this design will be needed to allow an isolated pancreas to be perfused (see below). We will transport the perfusion device to the donor hospital in cases where the suitability of these donor organs for clinical transplantation is believed to be marginal, anticipating the possibility of the organs being declined for clinical use. At this point we will transfer the organ(s) to the perfusion device and commence perfusion with minimum delay after cessation of the circulation in the donor. The perfusion device will be transported to the perfusion laboratory in Oxford and the perfusion continued for a total of 24 hours. During this time the function of the perfused pancreas and markers of damage to the pancreas will be measured (see below). At the end of the procedure, the pancreas will be processed for islet isolation. Oxford is one of three designated national centres for pancreatic islet isolation for clinical transplantation; all necessary skills, facilities protocols are in place. Islet isolation and testing will be invaluable, first, as a sensitive marker of the quality of organ preservation and, second, because this preservation technology is equally applicable to islet transplantation as to solid organ pancreas transplantation. The yield and function of islets will be assessed using multiple parameters including islet cell mitochondrial integrity and in vitro function. Phase 1 experiments - pancreas and liver en bloc:In order to test the principle of normothermic perfusion without an immediate redesign of the perfusion device, we will retrieve the pancreas and liver en bloc (a well-recognised technique), in order to connect the organs to the existing device in a similar way to the liver alone: by cannulation of the combined superior mesenteric and coeliac arteries (instead of coeliac artery alone) and of the superior mesenteric vein (rather than portal vein). Both the bile duct and the duodenum will be drained. All other aspects of the perfusion will be as with liver-only perfusion. 15 perfusions are planned in this series of experiments. Phase 2 experiments - isolated pancreas:The most notable difference in the vascular supply to the pancreas compared to that of the liver, is that the liver has two vascular inflows, the low pressure, high flow portal vein and the high pressure lower flow hepatic artery. In the isolated pancreas perfusion, the arterial perfusion will require, as before, cannulation of coeliac and superior mesenteric arteries, but the reservoir will function simply as a bypass to maintain constant arterial pressure and drain into the inflow channel of the pump. This modification will allow the essential design of the circuit to be maintained, together with much of the control hardware and software, as well as the underlying concept of auto-regulation. 15 perfusions are planned in this series of experiments. Control group - isolated pancreas:Five further organs will be retrieved and preserved for 12 hours using conventional static cold storage (UW solution) and then processed for islet isolation and testing using the same protocol. Outcome and whole organ monitoring:Monitoring of pancreas-liver en-bloc perfusions will comprise metabolic (acid-base homeostasis), synthetic (bile and pancreas exocrine production) and haemodynamic variables (blood flows and pressures). In all pancreas perfusions (including en bloc), markers of pancreatic damage and function will be measured: Pancreatic damage:Levels of pancreatic enzymes (amylase and/or lipase) in the perfusate will be measured. Micro-dialysis will be used to look for metabolic markers of ischaemia-reperfusion injury. The micro-circulation will be assessed by means of side-stream dark field (SDF) imaging (a validated tool for assessing in vivo micro-circulatory changes). The pancreas will be assessed macroscopically for evidence of pancreatititis (fat saponification). Islet yield and viability will be tested after islet isolation. Histological samples will be taken immediately prior to islet isolation (see below). Pancreatic function:Testing of beta cell function will be performed during perfusion by metabolic stimulation. Glucose will be measured in the perfusate; the metabolic capability of the pancreas will be studied by means of glucose-stimulated insulin secretion, with frequent measures of glucose, C-peptide and insulin levels after injection of a bolus of glucose. Islet isolation and testing:The pancreas will be taken off the warm-perfusion circuit, placed in University of Wisconsin solution at 4oC, and transferred to the Human Isolation Facility that is adjacent to the laboratory where the warm-perfusion is conducted. Once in the islet facility, any residual fat and vessels will be removed from the pancreas, and a 0.5 cm tissue block taken from the head, body, and tail of each pancreas for histochemistry. The pancreas will then be processed according to well-established protocols, using a modification of the semi-automated method described by Ricordi. Essentially, after the `biopsy' sites have been sealed with fibrin glue or with a surgical clip, the pancreatic duct will be cannulated, and the pancreas distended by syringe loading with Serva collagenase and neutral protease. The distended pancreas will be placed in a Ricordi chamber and digested. 3 ml samples will be taken from the digestion circuit every 3 minutes, stained with dithizone, and viewed under the light microscope in order to monitor digestion. Once liberated islets are seen, the digest will be collected, spun down, and the pooled digest placed in University of Wisconsin solution for 45 minutes. The digest will be then purified using a Continuous Ficoll-based density gradient loaded onto a COBE 2991 Cell Processor. The purified islets will then be placed in RPMI-based culture medium in culture flasks and cultured at 37oC. Each islet preparation will undergo the following in vitro tests that are well established in our laboratory, and that collectively provide an objective assessment of islet viability and function: viability testing using propidium iodide (PI)/ fluoroscein diacetate (FDA) staining; static incubation for insulin secretion using glucose concentations of 1mmol and 20mmol; an assessment of insulin and glucagon content; a measure of ATP content per islet; and an assessment of apoptosis using Annexin V-FITC. Data analysis and interpretation:This is a feasibility study of a new technology and not primarily a controlled study. The key end-points will be: markers of pancreatic injury and function and assessment of microcirculation. Islet yield, viability and function will be used as a surrogate for whole organ viability and will be compared with a small cohort of conventionally- preserved organs. The group size (15 in each normothermic group) is not based on statistical power, but is based on our experience in developing the OrganOx Metra perfusion device in normothermic perfusion of discarded human livers. Benefit This innovation will enable more patients to receive pancreas transplants at an appropriate time and at lower risk.The pancreases from more than 60% of donors within current pancreas donation criteria are currently turned down. The safe use of even a proportion of these donor organs would transform the outlook of the many patients who are currently waiting for a pancreas transplant. The reason that so many potential transplants do not take place is concern that the combination of organ damage and cold preservation will lead to a severe (and life-threatening) reperfusion injury. By avoiding cooling and ischaemia and by allowing viability (functional) testing during storage, it will be possible to transplant organs that would otherwise have been damaged excessively by preservation and also, select those that will not function and avoid the risks of transplanting non-viable organs.At present patients wait for many months or years, having been placed on the waiting list for transplantation. During this time, their condition deteriorates (the mortality of a diabetic patient requiring dialysis is 5-10% per year) and the risk of transplantation increases. Timely transplantation would bring great benefits in outcome and is also cost-effective - transplantation is substantially less costly than dialysis over the long-term.The logistic advantages of a longer preservation time are considerable. Patients travel considerable distances (up to 200 miles or more in some cases) to the nearest pancreas transplant centre and this compromises the rapidity with which the procedure can be completed. UK data show a clear detriment in outcome with every hour of cold ischaemia time. If normothermic preservation allows a longer storage time without detriment (24 hours is the target), then the patient could be brought into hospital once the organ has been assessed and deemed suitable, and the operation set up with less urgency probably as a day-time procedure.This technology will transform the patient experience, not only by increasing the number but also by changing the indications for a pancreatic transplant. With more organs becoming available, even without any changes in current donation practices, it should become possible to offer transplantation to patients suffering from severe diabetic complications at an earlier disease stage. This would be expected to result in significant improvements in patient quality-of-life, and considerable savings in healthcare from reduced treatment of diabetic complications.


Research activity codes: 5.4 SURGERY
Health categories: ORAL AND GASTROINTESTINAL