Impact of two-layer pancreas preservation on islet isolation and transplantation1 (2002)

Ischemic injury of the human cadaver donor pancreas during cold storage is conceivably the single most ignored impediment to successful islet cell transplantation. Protecting pancreatic islets from ischemic injury during cold storage of the donor pancreas is likely to result in markedly improved results and increased applicability of islet transplantation in the treatment of diabetes. Dr. Yoshikazu Kuroda and his group at Kobe University in Japan deserve a great deal of credit for having developed the two-layer (perfluorochemical preservation solution) pancreas preservation method in 1988 (1). Dr. Shinichi Matsumoto, one of his former co-workers, was the first to evaluate the two-layer preservation method in the clinical setting of vascularized pancreas transplantation while working at the University of Minnesota (2). The excellent review authored by Drs. Matsumoto and Kuroda and published in this forum of Transplantation makes apparent the wealth of research performed in the past 15 years on the two-layer method, culminating in the first clinical trial in 2000. Their review also emphasizes very clearly the possible future ramifications of the two-layer method for vascularized pancreas and pancreatic islet transplantation and beyond. The most significant impact of the two-layer preservation method will be in islet transplantation. It is becoming increasingly apparent that both the yield of islets that can be retrieved from a human cadaver donor pancreas and the ability of isolated islets to reverse diabetes after transplantation are profoundly reduced even by short periods (3–12 hr) of cold storage of the donor pancreas in University of Wisconsin (UW) preservation solution. This is an unexpected observation, given the consistent diabetes reversal after transplantation of cadaver human pancreases stored for 24 hr in UW solution (3,4). During the process of liberation from the pancreatic tissue and subsequent purification, culture, and transplantation, pancreatic islet cells encounter immense stress caused by, among other conditions, hypoxia, sudden and repeated changes in temperature, shear forces, disruption of cell matrix interactions, removal of critical growth factors, high concentrations of reactive oxygen intermediates, and proinflammatory cytokines. It is hypothesized that this stress is markedly compounded by any preceding cold ischemia of the donor pancreas and exceeds the cellular stress associated with reperfusion injury that islets encounter after vascularized pancreas transplantation. Consequently, pancreas preservation techniques that are adequate for prolonged cold storage before vascularized pancreas transplantation are presumably inadequate for even a short paired of cold pancreas storage before islet isolation and transplantation. How compelling is the available evidence that cold storage of the donor pancreas makes islets exquisitely susceptible to noxious events during islet isolation and, consequently, is detrimental to the yield and function of isolated islets? Hesse et al. were the first to show in a preclinical model that cold preservation of the pancreas in Collins solution and silica-gel–filtered plasma before islet preparation is associated with a reduced success rate after as little as 3 hr of storage, whereas whole vascularized segmental pancreas grafts function well even after 24 hr of storage (5). Two additional articles by the University of Minnesota group by Munn et al. (6) and Tanioka et al. (7) corroborated these findings by demonstrating a significant reduction in both canine islet yield and autograft success rate after vascular flush and brief periods of cold preservation in UW solution. In the study performed by Munn et al. (6), a 12-hr cold storage period in UW solution of the canine pancreas resulted in a significantly diminished number of islets compared with dog pancreases not subjected to cold storage (4,370 islets/kg body weight vs. 10,600 islets/kg body weight). Importantly, the canine’s islet autograft success rate dropped from 86% without cold storage to 20%. In the study by Tanioka et al. (7), cold storage of the donor pancreas in UW solution for only 3 hr did not result in reduced islet yields, but did reduce the islet autograft success rate from 83% to 33%. These findings are in contrast to a report by Zucker et al. (8) indicating that preservation of canine pancreas for 24 hr in UW solution did not negatively affect the islet yield or the autografts success rate. There are at least seven reports in the literature demonstrating that cold ischemia of the human cadaver donor pancreas is detrimental to islet yield (9–15). Some of these reports also indicate a reduced in vitro islet insulin secretory response to glucose after short periods of cold storage of the donor pancreas (15). Perhaps even more compelling is the lack of successful single-donor islet transplants after cold preservation of the donor pancreas for more than 10 hr. Of the 16 successful single-donor islet transplants performed in type 1 diabetic recipients through 1996, pancreas cold ischemia time was less than 8 hr in 15 cases and 9.2 hr in a single case (16). From 1997 on, additional reports of insulin independence after single-donor islet transplantation have been communicated, but again, the majority, if not all, transplanted islets prepared from donor pancreases were stored for less than 8 to 10 hr. Although it could be argued that islets retrieved from pancreases with longer cold storage time have rarely been transplanted, and therefore the success rate after prolonged cold storage is unknown, recent data published by the Edmonton group provide evidence of a detrimental impact of cold ischemia on posttransplant islet function (17). In this analysis, the ischemic index, which takes into account both the cold ischemia time and the number of transplanted islets, correlated with the insulin secretory response as expressed by insulin area under the curve derived from the intravenous glucose tolerance test. Does experimental evidence indicate that two-layer pancreas preservation before islet isolation increases the potency of isolated islets to reverse diabetes? An increasing number of institutions record that improved islet yields can be obtained after prolonged cold storage if the two-layer method is used for pancreas preservation (S. Matsumoto, Ricordi, and Lakey, personal communication). Equally important is the question of whether the two-layer method is superior to standard UW solution for short-term human pancreas preservation with respect to the ability of islets to reverse diabetes. The University of Minnesota group has demonstrated in the preclinical canine islet autotransplant model the exceptional ability of the two-layer pancreas preservation method to protect islets from ischemic damage before islet isolation and transplantation (7). The functional success rate of canine islet autografts was 89% without pancreas preservation, dropped to 33% after only 3 hr of cold preservation in UW solution, but was 83% after 3 hr of preservation with the two-layer method. When pancreases were stored for 24 hr before islet isolation, the functional success rate with two-layer and standard UW preservation was 56%. A recent study in the authors’ laboratory compared the efficacy of pancreas preservation techniques before islet isolation in a Lewis rat–to–diabetic nude mouse islet transplant model (18). The functional success rate of islet transplants in this model after 6 hr of pancreas preservation was 100% with the two-layer method compared with 50% with standard UW pancreas preservation. Tissue adenosine triphosphate contents were significantly higher with the two-layer method than with UW (20.71 vs. 5.80 μmol/g protein;P <0.01). The available information suggests the potential of a significant impact of improved pancreas preservation on posttransplant islet function. The data are consistent with the assumption that the failure of single-donor human islet transplants to consistently reverse diabetes is largely because of ischemic injury encountered during even brief periods of cold storage in UW solution. What are likely implications of significant progress in the area of human pancreas preservation before islet isolation and transplantation? Is research on ischemic injury of human islets and on approaches to protect islets from ischemic injury a high priority? Immunosuppressive protocols have now been developed that prevent both alloimmune rejection and autoimmune destruction of transplanted islets. The availability of suitable collagenase preparations and improvement in pancreas preservation methods are likely to have the most significant impact on the success and applicability of islet cell transplantation. Provided that suitable collagenase lots can consistently be made available, improved pancreas preservation techniques could (1) increase the success rate of single-donor islet transplants, (2) extend the duration of acceptable cold ischemia to up to 24 hr, (3) facilitate the use of donor pancreases from expanded donors, and (4) reduce the immunogenicity of transplanted islets. Increasingly successful single-donor islet transplantation will affect donor pancreas allocation policies, substantially reduce the costs, facilitate insurance coverage, and therefore boost the applicability of islet transplantation. The University of Minnesota was the first to apply the two-layer pancreas preservation method in the clinical setting of islet transplantation (19). Preliminary results are consistent with the notion that the application of the two-layer pancreas preservation method allows diabetes reversal after single-donor islet transplantation on a more consistent basis. Although other factors such as refined immunosuppression may have contributed to the outcome, 12 of 14 type 1 diabetic patients in the recent Minnesota series who received single-donor islet transplants from two-layer preserved donor pancreases have achieved insulin independence. It will be important to determine whether these findings can be confirmed in a larger series of patients and at additional institutions. Islet transplantation will only become a sound treatment option for diabetes if protocols can be developed and implemented that allow diabetes reversal after single-donor transplantation. As long as two to three donor pancreases are required for diabetes reversal, the applicability of this procedure will remain extremely limited, especially given the high success rate of single-donor vascularized pancreas transplantations. Thus, diabetes reversal after single-donor islet transplantation will not simply double or triple the applicability of islet transplantation, rather, it will be a prerequisite for the implementation of islet transplantation as a reimbursable treatment option for type 1 diabetes. The preliminary results obtained at the University of Minnesota suggest that tools are available to achieve insulin independence with islets prepared from one donor pancreas. Whether the two-layer preservation method is a critical factor in the current protocols is unknown but clearly is testable in prospective clinical trials. Logistical obstacles also limit widespread implementation of islet transplantation. The promising data obtained recently at the University of Minnesota only included the use of donor pancreases preserved for up to 8 hr using the two-layer method. Diabetes reversal after transplantation of islets prepared from single-donor pancreases stored for 12 to 24 hr has not been documented. The implications would be far reaching—prolonged pancreas preservation before islet isolation would allow the exchange of donor pancreases between remote institutions, as currently practiced in whole-organ vascularized pancreas transplantation. Of the 150 pancreases transplanted annually at the University of Minnesota, approximately 100 are imported from distant organ procurement organizations (OPO), indicating that exchange of donor pancreases contributes substantially to the pancreas transplant activity at active programs. If maximization of donor pancreas use for islet transplantation is to be achieved, the development and implementation of suitable methods for pancreas preservation before islet transplantation will be of paramount importance. The two-layer preservation method holds promise in that regard, but widespread implementation of this or any other new preservation method will be contingent; on unequivocal demonstration of efficacy in type 1 diabetic islet transplant recipients. A different approach would not require the presence of the islet team at the pancreas recovery site or the training of OPOs across the country. Results reported by the Seattle, Edmonton, and Miami groups suggest that donor pancreases preserved in UW solution for prolonged periods can be rescued by an additional period of preservation with the two-layer method (20) (J.R. Lakey, personal communication; C. Ricordi, personal communication). This approach, if proven effective, would markedly reduce islet team transplantation costs for air charters and expedite the broad application of the two-layer method. The applicability of islet transplantation could further be increased if methods could be developed that would allow the successful use of donor pancreases from expanded donors. From the 6,096 cadaver donors reported in the United States in 2001, 1,405 pancreases were recovered for vascularized whole-organ transplantation and 476 for research (21). Poor donor quality has been the single most prevalent reason for turning down a pancreas offer for whole-organ transplantation in the 15- to 50-year age group. Pancreases from donors with cardiac arrests, prolonged hypotensive episodes, a history of high-dose vasopressor administration, or evidence of kidney or liver dysfunction are frequently rejected as pancreas or islet donors. Demonstration that exposure of a cadaver pancreas from an expanded donor to two-layer pretreatment (rescue) before initiation of islet isolation renders otherwise unsuitable pancreases suitable would markedly increase the number of islet transplants performed. Preliminary evidence presented by Ricordi et al. from Miami (22) suggests that this can be accomplished. The Miami group showed significant improvement in human islet isolation from marginal (older) donors when pancreases were preserved using the two-layer method. If methods were available to reverse the effects of warm ischemia on subsequent islet isolation and transplantation, then pancreases obtained from non–heart-beating donors could also be used more frequently. Preliminary data obtained in clinical pancreas transplantation suggest a reduced incidence of rejection in the two-layer group compared with the UW control group (2). This unexpected and particularly interesting finding awaits confirmation in adequate experimental models. If confirmed, additional studies addressing the mechanism by which two-layer pancreas preservation reduces pancreas and islet immunogenicity would be of great value, and less toxic immunosuppressive protocols would further increase the applicability of islet transplantation. Taken together, a method that protects pancreatic islets from ischemic injury would markedly expand the use of cadaver pancreases for islet transplantation. The experience with islet autotransplantation after total pancreatectomy suggests that fewer islets are required to maintain normoglycemia and insulin independence if ischemia and immunity are circumvented (23). Given the constraints related to ischemic injury encountered by islets during cold preservation as currently practiced, the number of type 1 diabetes patients in the United States in whom sustained diabetes reversal can be accomplished by cadaver donor islet transplantation could be limited to 500 per year. The two-layer pancreas preservation method, however, could increase that number up to 10-fold, to 5,000 per year. As discussed above, this profound increase in availability of islet transplantation secondary to improved pancreas preservation would result from a higher proportion of successful single-donor islet transplants, exchange of pancreases between distant centers, and use of pancreases from expanded and non–heart-beating donors. Thus, the availability of a suitable pancreas preservation method tailored to the specific requirements posed by islet isolation and transplantation after cold storage appears to be the single most important determinant of the applicability of islet transplantation. Compelling evidence that the two-layer method overcomes the problems associated with human pancreas preservation before islet isolation is lacking and difficult to obtain. Preliminary results suggest that more islets can be isolated from human donor pancreases preserved with the two-layer method as opposed to standard UW preservation, but a carefully designed prospective study has not yet been done. An analysis of the effects of two-layer preservation on the yield and function of isolated islets, moreover, is confounded by the immense variability between human donor pancreases. Consequently, a substantial number of organs needs to be prospectively processed using identical methods in both the two-layer and standard UW preservation groups. Such a study should ideally incorporate predictive islet potency assays, which have yet to be identified. Future research on pancreas preservation will likely incorporate sophisticated molecular and genetic assays of the integrity of acinar, ductal, and islet endocrine cells, these increasing understanding of the critical events that determine posttransplant islet function. As a consequence, refinement of the two-layer method or other preservation methods will continue. Ultimately, a prospective, randomized clinical trial will be required to assess the efficacy. The primary outcome measure in such a clinical trial should be sustained insulin independence at 1 year, and preferably at 2 years, after islet transplantation. Demonstration of efficacy in such a clinical trial would provide a strong rationale for universal implementation of the two-layer or any other improved method for pancreas preservation.