Islet cell transplantation represents a potentially valuable therapeutic approach for the treatment of type 1 diabetes mellitus. As recent clinical trials carried out in Edmonton, our institute, and other centers have definitively shown, islet transplants carried out under the umbrella of steroid-free immunosuppression protocols can indeed result in a 100% success rate in type 1 diabetic patients, conferring insulin independence, provided that a sufficient number of cells are transplanted (1). The relative ease of the islet-transplantation procedure, performed by simple gravity infusion of the final islet preparation (similar to a blood transfusion) after percutaneous catheterization of the portal vein under local anesthesia, makes it a much more desirable alternative to the complex and higher-risk whole-organ pancreas transplantation procedure.
Yet, despite these new and promising results, only a fraction of the donor organs that become available each year are ever used for either clinical pancreas or islet cell transplantation. Of the 6,000 pancreata made available last year, only 1,500 were used for clinical transplantation (2). Thousands of organs were not used for either procedure because of long ischemia times postprocurement. Given the oxygen-rich environment of the native pancreas, it is conceivable that the harvested pancreata and especially the insulin-producing islet cells of the pancreata suffer irreversible damage following prolonged ischemia. Other factors contributing to the gross underuse of donor pancreata are the strict donor criteria currently in place, particularly regarding donor age and circulatory status (heart beating vs. non-heart beating). This is because of the fact that isolations from so-called marginal donors above the age of 50 or who are hemodynamically unstable consistently produce inadequate islet yields for clinical transplantation.
The use of perfluorocarbon (PFC), together with University of Wisconsin (UW) cold preservation solution, has been previously described by Matsumoto et al. (3–9) as a way of improving islet recovery from animal pancreata. The aim of this study was to determine whether the addition of PFC to the UW could result in improved use of human pancreata in the so-called marginal donor age group, a group that does not directly compete with the selection criteria of most whole-organ transplant programs, therefore maximizing use of donor pancreata that would be otherwise largely unused for clinical applications.
MATERIALS AND METHODS
Pancreas Preservation and Islet Isolation
Human pancreata were obtained from organ procurement organizations after they could not be allocated for organ transplantation. Thirty-three consecutive human islet isolations were performed from pancreata preserved with preoxygenated (30 min) PFC–UW (n=15) or with UW alone (n=18, controls). Donor age ranged from 51 to 69 years. Immediately following organ harvest, the procured pancreata were placed inside a sterile calibrated stainless steel grid (#CX-800C, Small Parts Inc., Miami, FL) that had been previously positioned inside a standard 1L nalgene jar containing 500 mL of UW solution and a bottom layer of 333 mL of preoxygenated (30 min by way of a sterile cannula submerged in PFC from filtered medical grade oxygen tank) perfluorodecalin (FluoroMed, Round Rock, TX). In this manner, the pancreata were partially submerged with 50% to 70% of the organ resting in the PFC layer. For three organs that were procured at remote sites, the PFC incubation was started after the pancreas arrived at the islet isolation facility. The mean cold-ischemia time (CIT) for the control UW group was 8.4±3.2 hours. The mean preservation time in PFC was 7.6±5.5 hours, and the mean total CIT of the PFC group was 15.3±9.4 hours. Following cold preservation, the islets were isolated using a modification of the Ricordi automated method (10).
Static Glucose Challenge
To determine the physiologic well-being of isolated islets, aliquots of 50 islet equivalents (IEQs) were subjected to static glucose challenge. Briefly, 10 aliquots of 50 IEQs were placed into 6 mL polypropylene tubes; 5 were labeled low glucose, 5 labeled high. Glucose solutions were prepared with RPMI 1640 without glucose and supplemented with HEPES, sodium bicarbonate, and 0.1% bovine serum albumin. Glucose was added to make 1.67 mM and 16.7 mM solutions for low and high incubation, respectively. The pH was adjusted to 7.2. Five hundred microliters of low solution and high solution were added to the five tubes with corresponding low and high labels. The tubes were placed in a standard 95%/5% incubator at 37°C for 1 hour. At the end of the hour, 200 μL were carefully removed from each tube after the islets had settled and were stored at −80°C in microcentrifuge tubes for later analysis of insulin.
Insulin values were determined using conventional enzyme-linked immunosorbent assay (ELISA) kits, ultrasensitive for human insulin (ALPCO Windham, NH), and values were determined using a spectrophotometric plate reader.
Fluorescein Diacetate–Propidium Iodide Viability Staining
Briefly, 45 μL of each postisolation islet pellet was added to a 10×35 mm counting Petri dish. Four hundred fifty-eight microliters of DPBS and 10 μL each of a 24-μM fluorescein diacetate and a 750-μM propidium iodide stock solution were added to a Petri dish, resulting in final concentrations of 0.67 μM and 75, μM respectively. Through use of fluorescent microscopy, 50 islets were then quantified for cell viability by estimating the percentage of viable cells (green) versus the percentage of nonviable cells (red) within each islet. The mean and standard deviation were then calculated for each fifty-islet measurement.
In the PFC–UW group, an average of 438,381±182,226 IEQs per pancreas were procured (prepurification), whereas in the control group (UW alone), 219,050±95,849 islets were obtained (P <0.001). Postpurification, the PFC group yielded 305,790±159,654 compared with 148,854±80,449 in the control group (P =0.005). Of the 18 islet preparations from the control-group pancreata, only 2 were considered suitable and used for transplantation, whereas 8 of 15 from the PFC–UW group produced clinically acceptable yields and were used for transplant. Four patients with type 1 insulin-dependent diabetes mellitus have now been insulin independent following transplantation at our institution with islets obtained from these PFC preserved organs.
More convincingly, there were no significant differences between the two groups in other critical factors that might translate into differences in islet yield (Table 1). The mean donor age of the control group was 56.7±5.4 years, whereas that of the PFC–UW group was 55.9±4.2 years (P =0.63). The mean weight of the harvested pancreata from the control group was 107.5±20.8 g, whereas that of the PFC-UW group was 119.2±36.2 g (P =0.28). Postpurification viability and functionality tests were indicative of a pattern but not conclusive as to the physiologic benefits of the PFC. The mean viability of the PFC–UW group was 94±3.6% and of the UW group 88±5.8% (P =0.016). The mean stimulation index of the PFC–UW group was 1.92±0.97 (n=8) and of the UW group 2.08±1.26 (n=7, P =not significant). The insulin output per IEQ for each preparation in both groups is outlined in Table 2.
The addition of PFC to UW solution for cold preservation of organs before transplantation has been proposed for several years (3–9,11–22). PFCs have an innate ability for the maintenance of high-oxygen partial pressures for extended periods of time and the ability to offload oxygen to areas of lower-oxygen partial pressure. Their unique chemical characteristics allow them to serve as oxygen “reservoirs” for harvested organs in transport to processing and transplant centers. A one-time 30 minute saturation of a PFC two-layer with 100% oxygen is sufficient to maintain oxygen levels significantly above the partial pressure of room air (20.9%) for more than 48 hours. Most of the results regarding the use of the two-layer method have been obtained in animal models in which the effect of PFC following immersion of the donor organs into the oxygenated solution could have been related to small volume of the organ and the relative thinness of parenchyma being treated in the absence of perfusion of the preservation solution. As early as 1994, there was movement toward applying this model to the procurement of human organs, specifically pancreata (7,11,21). However, several groups, including our own, had been skeptical about the real potential of this solution when applied to the preservation of larger organs such as the human pancreas. Specifically, no significant improvement could be observed when using a direct comparison of human-donor pancreata that were divided into two portions (head and body-tail segments) and processed in parallel (Bernhard Hering, personal communication, July 2001). Despite this lack of clear advantage, the Minneapolis group was the first to introduce this pancreas preservation method in the clinical practice with very encouraging preliminary results (4 of 6 recipients were made insulin independent following islet transplantation; Bernhard Hering, personal communication, 2002). Although other factors, such as optimal donor age and CIT along with low recipient weight and body mass index, may have contributed to these results, it could not be excluded that the incubation of the donor pancreas in PFC–UW before islet isolation may have also contributed. We thought that a direct comparison between ideal donors would represent a more challenging model system in which to define potentially significant differences, and in which a beneficial, clinically relevant effect could be better defined in pancreata from marginal donors.
A limitation of this study was that the two groups were not randomized. Nevertheless, the isolations were alternated between the two groups, depending on whether the additional PFC preservation step was available or not. All isolations were performed with similar reagents and the same standard operating procedures. One variable that could also interfere with the interpretation of the results is the variability between enzyme blends used for pancreatic digestion. Although we were unable to use the exact same enzyme lot for all isolations, we have used lots with similar activities in all isolations, and, more importantly, 16 pancreata were processed with exactly the same lot (12 PFC–UW and 4 UW-alone controls). These isolations yielded results that were comparable with the results reported in Table 1 for all isolations (n=33). In fact, a partial analysis of only the 16 pancreata processed using the same enzyme lot revealed that islet yields in these groups were comparable with the overall results (Table 1), with a mean of 431,381 IEQs for the PFC–UW group and an average 219,051 IEQs in the UW-alone group.
Our results clearly indicate that the use of PFC–UW cold-storage preservation can indeed improve islet isolation outcome in donors older than 50 years of age, providing indirect evidence that this intervention could also be of value in improving the quality of islets retrieved from any other age-group donor pancreata. In fact, we are now routinely using this preservation method with donor pancreata from all ages, incubating them in PFC–UW for a period of 2 to 8 hours before islet processing. The preliminary results across all age groups are very encouraging (data not shown) and may allow for an extension of donor-selection criteria not only to older donors but also to pancreata with more prolonged CITs, an important step toward the definition of an optimization strategy that will maximize human pancreas use for treatment of patients with type 1 diabetes.
It is now apparent that the use of the two-layer method can significantly impact the use of harvested organs for clinical transplantation. The oxygenating characteristics of the perfluorochemical layer serve to resuscitate damaged tissue, despite extended CITs. The task, now, is to identify the pathways through which PFCs preserve and revitalize damaged organs. Much research has been done in this area, but to date, no one has conclusively determined the mechanism by which these inert liquids function to preserve ischemic tissue. Some groups, such as Kuroda et al. (8,9), believe it is the maintenance of tissue ATP levels that keep tissue viable. Others, such as Fujino et al. (23–25), believe that PFCs may attenuate free-radical damage and may up-regulate cytoprotective genes. Regardless of the means through which the two-layer method exerts its observed effects (and most likely through multiple mechanisms), the outcome of PFC preservation is irrefutable. The future applications of this method to islet research and transplantation are numerous because it may also have a significant role in areas such as postisolation culture and tissue engineering. Optimistically, as more long-term investigation is completed in the physiologic function of PFC-preserved islets, the yields of marginal pancreata procured with the two-layer method may even provide a sufficient number of cells to reproducibly transplant one recipient with one donor, despite strict recipient-size criteria.
1. Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 2000; 343 ( 4): 230.
2. United Network of Organ Sharing. 2001 annual report of the United States Scientific Registry of Transplant Recipients and the Organ Procurement and Transplantation Network: transplant data 1991 to 2000. Available at: http://www.unos.org/Data/anrpt_main.htm
. Accessed June 2002.
3. Kuroda Y, Tanioka Y, Matsumoto S, et al. Difference in energy metabolism between fresh and warm ischemic canine pancreases during preservation by the two-layer method. Transpl Int 1994; 7 (suppl 1): S441.
4. Kuroda Y, Hiraoka K, Tanioka Y, et al. Metabolic intervention to affect canine pancreas recovery following ischemia during preservation by the two-layer method. Transpl Int 1994; 7 (suppl 1): S436.
5. Matsumoto S, Fujino Y, Suzuki Y, et al. Evidence of protein synthesis during resuscitation of ischemically damaged canine pancreas by the two-layer method. Pancreas 2000; 20 ( 4): 411.
6. Kim Y, Kuroda Y, Tanioka Y, et al. Recovery of pancreatic tissue perfusion and ATP tissue level after reperfusion in canine pancreas grafts preserved by the two-layer method. Pancreas 1997; 14 ( 3): 285.
7. Kuroda Y, Tanioka Y, Morita A, et al. The possibility of restoration of human pancreas function during preservation by the two-layer (University of Wisconsin solution/perfluorochemical) method following normothermic ischemia. Transplantation 1994; 57 ( 2): 282.
8. Hiraoka K, Kuroda Y, Tanioka Y, et al. Adenosine is a key component in preservation of ischemically damaged canine pancreas by the two-layer cold storage method. Transplant Proc 1994; 26 ( 2): 953.
9. Kuroda Y, Hiraoka K, Tanioka Y, et al. Role of adenosine in preservation by the two-layer method of ischemically damaged canine pancreas. Transplantation 1994; 57 ( 7): 1017.
10. Ricordi C, Lacy PE, Finke EH, et al. Automated method for isolation of human pancreatic islets. Diabetes 1988; 37 ( 4): 413.
11. Segel L, Minten J, Schweighardt FK. Fluorochemical emulsion APE-LM substantially improves cardiac preservation. Am J Physiol 1992; 32: H730.
12. Forman MB, Ingram DA, Murray JJ. Role of perfluorochemical emulsions in the treatment of myocardial reperfusion injury. Am Heart J 1992; 124 ( 5): 1347.
13. Breuninger HG, Rubenstein SD, Wolfson M, et al. Effect of exchange transfusion with a red blood cell substitute on neonatal hemodynamics and organ blood flows. J Pediatr Surg 1993; 28 ( 2): 144.
14. Jacobs HC, Mecrurio MR. Perfluorocarbons in the treatment of respiratory distress syndrome. Semin Perinatol 1993; 17 ( 4): 295.
15. Holman WL, McGiffin DC, Vicente WV, et al. Use of current generation perfluorocarbon emulsions in cardiac surgery. Artif Cells Blood Substit Immobil Biotechnol 1994; 22 ( 4): 979.
16. Spruell RD, Ferguson ER, Clymer JJ, et al. Perfluorocarbons are effective oxygen carriers in cardiopulmonary bypass. ASAIO J 1995; 41 ( 3): M636.
17. Wada S, Kajihara H, Murakami H, et al. Effects of FC43 emulsion against hyperacute rejection in rodent discordant xenotransplantation. J Heart Lung Transplant 1995; 14: 968.
18. Houmes RJ, Verbrugge SJ, Hendrik ER, et al. Hemodynamic effects of partial liquid ventilation with perfluorocarbon in acute lung injury. Intensive Care Med 1995; 21 ( 12): 966.
19. Tutuncu AS, Houmes RJ, Bos JA, et al. Evaluation of lung function after intratracheal perfluorocarbon administration in healthy animals. Crit Care Med 1996; 24 ( 2): 274.
20. Clark MC, Weiman DS, Pate JW, et al. Perfluorocarbons: future clinical possibilities. J Invest Surg 1997; 10 ( 6): 357.
21. Chiba T, Harrison MR, Ohkubo T, et al. Transabdominal oxygenation using perfluorocarbons. J Pediatr Surg 1999; 34(5): 895; discussion 900.
22. Matsumoto S, Kandaswamy R, Sutherland DE, et al. Clinical application of the two-layer (University of Wisconsin solution/perfluorochemical plus O2) method of pancreas preservation before transplantation. Transplantation 2000; 70 ( 5): 771.
23. Fujino Y, Suzuki Y, Tsujimura T, et al. Possible role of heat shock protein 60 in reducing ischemic-reperfusion injury in canine pancreas grafts after preservation by the two-layer method. Pancreas 2001; 23 ( 4): 393.
24. Heard SO, Puyana JC. The anti-inflammatory effects of perfluorocarbons: let’s get physical. Crit Care Med 2000; 28 ( 4): 1241.
25. Bekyarova G, Yankova T, Kozarev I. Increased antioxidant capacity, suppression of free radical damage and erythrocyte aggregrability after combined application of alpha-tocopherol and FC-43 perfluorocarbon emulsion in early postburn period in rats. Artif Cells Blood Substit Immobil Biotechnol 1996; 24 ( 6): 629.