Chemical Oxygenation of Pancreatic Tissue Prior to Islet Isolation and Transplantation
Brandhorst H., Johnson P., Brandhorst D.
Islet transplantation has been established as a promising treatment for patients suffering from life-threatening hypoglycemic episodes. Apart from the achievement of insulin independence, islet transplantation has been shown to improve metabolic control and to ameliorate the progression of diabetic complications. However, this procedure is limited with respect to the number of suitable donor pancreata that are required to achieve long-term insulin independence in diabetic patients. Numerous studies indicate that prolonged ischemia is a major obstacle for the frequent success of islet isolation and subsequent islet transplantation. Since 10 – 12% of the normal metabolic activity is operative in tissue stored at 4°C, hypothermic organ perfusion and subsequent immersion in various preservation solutions do not completely prevent energy depletion during prolonged cold storage. For that reason an urgent need exists to optimize organ preservation techniques to benefit transplant recipients. We hypothesized that a substantial improvement of islet isolation success and posttransplant function in diabetic patients can be obtained by effective oxygenation of ischemic pancreatic tissue during cold storage and organ shipment resulting in maintenance of ATP generation. One option to prevent or even reverse ischemically induced damage from procured organs is continuous hypothermic perfusion with oxygenated organ preservation solutions such as UW solution, Custadiol or modifications of these solutions as demonstrated in different animal models. Nevertheless, the complex and expensive equipment that is required to perform continuous perfusion is available only for a maximum of 4% of transplanted organs. The simple incubation of organs in oxygen-precharged perfluorocarbons represents an approach that is easier to apply. One member of this chemical group, perfluorodecalin, has been extensively investigated in pancreata obtained from different species. In the proposed chapter we will provide first a short overview about the impact of ischemia on viability and quality of subsequently transplanted organs particularly focussing on the specific sensitivity of pancreatic islet tissue toward ischemically induced damage. Next, we will discuss our findings utilizing different techniques to oxygenate pig pancreata during prolonged ischemia prior to isolation and transplantation of pig islets. It was observed that islet tissue isolated after prolonged cold storage is characterized by a significantly higher potency in terms of insulin secretory capacity, membrane integrity, mitochondrial activity, ATP generation and posttransplant function in diabetic nude mice when the organ is completely immersed in the hyperoxygen carrier perfluorodecalin (one-layer method) and not oxygenated with the two-layer method representing the current state-of-the-art procedure for pancreas oxygenation. By increasing the storage temperature for oxygenated pig pancreata from 4 to 20°C it was revealed that a temperature-stimulated ATP production does neither reflect tissue viability nor predict posttransplant outcome particularly if the donor tissue is predamaged by warm ischemia. The relatively low efficiency of perfluorodecalin to prevent or reverse ischemically induced damage from pancreatic tissue was additionally verified in 200 human donor pancreata procured for subsequent islet transplantation into diabetic patients with established kidney grafts. Compared to storage in UW solution pancreas oxygenation in precharged perfluorodecalin was unsufficient to increase islet yield, insulin secretory capacity and posttransplant function in recipients. The higher efficiency of perfluorodecalin that had been demonstrated in ischemic rat, pig and canine pancreata indicated that the specific texture and firmness of human pancreatic tissue may be responsible for the heterogeneous results observed worldwide in human islet transplantation. We hypothesized that oxygenation of ischemic human pancreatic tissue can significantly be improved when lipophilic oxygen carriers are used for cold storage or shipment from the donor to the recipient center. In a series of experiments made in rats and pigs we demonstrated that lipophilic compounds, such as perfluorohexyloctan, are superior for the oxygen supply of pancreatic tissue compared to inert substances such as perfluorodecalin in particular if the partial oxygen pressure in the pancreatic core region is determined by means of Clark probes or optic fiber sensors. These results could be confirmed in human donor pancreata processed for subsequent islet isolation. Prior to islet isolation the organs were stored for at least 24 hours in either oxygen-precharged perfluorodecalin, the current standard oxygen carrier, or perfluorohexyloctan which was adjusted to a pancreas-specific density utilizing polydimethylsiloxane, another potential oxygen carrier. This strategy dispenses from the need to use custom-made transport equipment as required for perfluorodecalin. The pancreata were subjected to prolonged cold ischemia for at least 24 hours until islet isolation was performed. Utilizing a newly established technique that combines capillary gas chromatography and mass spectroscopy we could clearly demonstrate that our new strategy for pancreas oxygenation facilitates oxygen carrier penetration into the tissue. Since perfluorohexyloctane is already in clinical use for surgical ophthalmology and passed the required panel of toxicity tests a clinical study is in preparation to isolate human islets from perfluorohexyloctane-oxygenated pancreata for subsequent transplantation into diabetic patients.