Artificial Cells, Blood Substitutes, and Biotechnology, 33: 7582, 2005 Copyright Q Taylor & Francis, Inc.

ISSN: 1073-1199 print/1532-4184 online DOI: 10.1081/BIO-200046698

Clinical Application of Perfluorocarbons for Organ Preservation

Shinichi Matsumoto
Kyoto University Hospital Transplantation Unit, Kyoto, Japan

Abstract: Perfluorocarbons (PFC), which store and release high levels of oxygen, have been examined as oxygen carriers. PFC was first used in organ preservation as a component of the two-layer method (TLM). The TLM is comprised of University of Wisconsin solution [UW] and oxygenated PFC for pancreas preservation. Pancreata preserved in the TLM are oxygenated through the PFC and substrates are supplied by the UW. TLM has been shown to prolong the preservation period and repair pancreatic injury caused by warm ischemia. Currently the TLM was used for pancreas preservation prior to clinical whole organ transplant. In this first clinical trial, the morphologic quality of the human pancreas graft after reperfusion was excellent compared with the pancreas stored in UW. In addition, there was no acute rejection episode of pancreata preserved by the TLM. TLM preservation of human pancreata, without initial cold UW storage, prior to islet isolation, resulted in better isolation results and improved the success rate of islet transplantation. Thus preservation of human pancreata by the TLM has become an important process for successful islet transplantation. Nowadays, PFC is routinely used for pancreas preservation prior to islet isolation, which has had a significant impact on curing type 1 diabetes. INTRODUCTION Perfluorocarbons (PFC), which store and release high levels of oxygen, have been examined as oxygen carriers [1]. PFC was first used for organ preservation as a component of the two-layer method (TLM) of pancreas preservation that was developed by Dr. Kuroda in 1988 at Kobe University
The author thanks Lisa Upshaw, Jondell Clever-Hendrix and Yoshiko Tamura for their critical reading and editorial assistance. Address correspondence to Shinichi Matsumoto, Kyoto University Hospital Transplantation Unit, 54 Kawara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: shinichi@kuhp.kyoto-u.ac.jp 75

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[2,3]. The TLM consists of two kinds of solutions; one is PFC and the other is a standard organ preservation solution (typically University of Wisconsin solution (UW)). Pancreata preserved in the TLM are oxygenated through the PFC and substrates are supplied by the UW [2]. ANIMAL RESEARCH As shown in the canine model, the TLM has the capability to store pancreata for 96 hours and UW for 72 hours [4]. During TLM preservation, pancreas grafts have been shown to continuously generate ATP [5,6]. ATP levels within the canine pancreas, after TLM preservation, correlate directly with the success of pancreas transplantation [7,8]. ATP is generated during TLM preservation by the utilization of adenosine [9] from the UW solution and is used to drive sodium=potassium pump [10]. This in turn generates proteins, including heat shock protein 32 (hemeoxygenase-1) and heat shock protein 70 [11]. The TLM also improves the blood circulation in the pancreas after transplantation [12,13]. After 90 minutes of warm ischemic injury, none of the canine pancreas grafts were viable; however, when we preserved the damaged grafts with the TLM for 24 to 48 hours, all grafts became viable [14,15]. This is an attractive effect because we could potentially use pancreata from nonheart beating donors. The TLM could resuscitate warm ischemic injured pancreata for 5 hours at 20C, for 24 hours at 4C but could not resuscitate at 37C [16]. One factor that may have a role in resuscitating warm ischemic damage is thromboxane A2 synthesis inhibitor OKY046, which has been demonstrated to improve blood circulation when combined with TLM [13,17]. The effect of resuscitation is correlated with pancreatic tissue ATP level after TLM [8]. The mechanism of resuscitation and prolongation of preservation period seems to be different [18]. The TLM improved canine islet function compared to that of UW alone after 3-hour preservation and improved both yield and function after 24-hour preservation [19]. The TLM might improve islet yield where there is warm ischemic injury to canine [20], non-human primate and human pancreata [21]. Non-human primate data suggests that an additional 5-hour preservation using the TLM substantially increased islet yields without affecting functionality [22]. CLINICAL APPLICATION The TLM was used clinically for whole pancreas transplantation with favorable results [23]. In this first clinical trial of 10 cases, there were no adverse events related to the TLM. Furthermore, the morphologic

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quality of the human pancreas grafts after reperfusion was better than pancreas stored in UW alone. In addition, there were no acute rejection episodes of pancreata preserved by the TLM. However, recently it was revealed that there was no significant difference in expression of MHC class I and II on isolated islets from fresh pancreata and pancreata preserved with the TLM in the rodent model [24]. As of yet, there is no evidence of immune modulatory effect by the TLM. Nonetheless, this first clinical trial made it possible to use the TLM for pancreas preservation before clinical islet isolation.

THE TLM AS AN IMPORTANT FACTOR FOR SUCCESSFUL ISLET TRANSPLANTATION In 2000, the University of Alberta demonstrated that allogeneic islet transplantation could cure type 1 diabetes with islets isolated from 2 human donor pancreata [25]. Their results stimulated the clinical trial of islet transplantation in the US and Europe. At the same time, researchers immediately noticed the importance of improvement of the efficacy of the islet isolation process to achieve the successful single donor islet transplantation. Despite the clear advantage of pancreas preservation with the TLM before islet isolation in the canine model, several groups have been skeptical of this potential benefit for human pancreata due to the difference of thickness between the canine and human pancreas [26]. In 2000, we presented the advantage of utilizing TLM preservation prior to human islet isolation using pancreata from braindead donors for the first time [27]. In this experiment human pancreata were preserved for a long period (>24 hours) in order to show the difference between the TLM and UW. This experiment clearly demonstrated the benefit of the TLM of human pancreas before islet isolation. During this time period, there were still some questions about the benefits of the TLM before islet isolation. These were: 1) Is the TLM beneficial for short-term preservation? 2) Is the TLM able to resuscitate cold ischemic injury? 3) Is the TLM able to improve the isolation results from a marginal donor? 4) Is the continuous oxygenation essential for the TLM? 5) Finally, is the TLM capable to contribute to the cure of type 1 diabetic patient using only one pancreas? Knowing the success of the University of Alberta group, we conducted the randomized comparison between the TLM and UW for shortterm and long-term preservation before islet isolation with Edmonton islet isolation protocol [28]. This experiment demonstrated that the TLM improved the islet isolation even for short-term preservation using the most successful Edmonton islet isolation protocol.

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The University of Alberta group addressed the question about the resuscitation of the cold ischemic injury by the TLM. They isolated islets from the human pancreata as soon as possible or preserved the human pancreas for about an additional 3 hours with the TLM before isolation. They clearly demonstrated that the additional TLM preservation improved the islet yields (349,000 IE vs. 214,000 IE; TLM vs. UW) and the success rate of transplantation (71% vs. 36%; TLM vs. UW) [29]. The University of Miami group addressed the question about the benefit of the TLM for the marginal donor. They isolated islets from older donors (>50years) pancreata in 15 cases with the TLM and 18 cases with UW. They clearly demonstrated that the TLM improved the islet yields (306,000 IE vs. 149,000 IE; TLM vs. UW) and the success rate of transplantation (53% vs. 11%; TLM vs. UW) with marginal donors [26]. The favorable results from the University of Alberta and University of Miami stimulated the desire to use the TLM before islet isolation at other islet isolation centers. However, the necessity of continuous oxygenation hindered the prevalence of the TLM. We postulated that since PFC is able to contain extremely high levels of oxygen, continuous oxygenation is not necessary once the PFC is fully oxygenated. Therefore we developed oxygen static charge TLM (static TLM) and examined the effect of this system [30]. We were able to show the static TLM was as efficient as the regular TLM and significantly better than UW [30]. Currently, many islet isolation centers use the static TLM before islet isolation. Eventually, the University of Minnesota group demonstrated the successful single donor islet transplantation with the TLM. They were able to cure 12 out of 14 type 1 diabetic patients with the islets from single donor pancreas preserved by the TLM [31]. Since they didnt compare with the UW, the benefit of the TLM was not clear; however, they were able to show the best islet transplantation results with the TLM. The results from University of Alberta, University of Miami, University of Minnesota and ours pushed the TLM to the standard pancreas preservation method before islet isolation. The TLM has become a standard in the medical use of artificial blood.

FUTURE RESEARCH FOCUS The TLM clearly improved human islet yields; however, the mechanisms are not clear. Recently, Iwanaga et al. demonstrated that the TLM could maintain the zymogen granules and in sharp contrast UW destroyed them [32]. Destroyed zymogen granules would lead to the activation of trypsin and cause the over digestion of isolated islets. Or the activated trypsin would break the pancreatic duct and cause a disturbance with collagenase delivery during perfusion through the pancreatic duct. Both the

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inhibition of trypsin activity (22) and conservation of the pancreatic duct contributed to an increase in islet yields (33). The TLM may have a major effect on both mechanisms by contributing to the preservation of zymogen granules and pancreatic duct. Even 28 hour TLM before islet isolation showed to be beneficial, the optimal preservation period is still unknown. Recently, it was demonstrated that 12 times of UW storage might be an optimal length of time for TLM preservation before islet isolation (34). We have demonstrated that more than 24-hour preservation by the TLM is not beneficial before islet isolation. Since pancreatic tissue ATP levels correlate the results of islet isolation (35), real time measurement of pancreatic ATP levels might help to identify the optimal preservation period. Iwanaga et al. demonstrated that TLM using a new preservation solution, Unisol, improved the viability of isolated islets compared to UW based TLM in canine model (36). Since UW solution inhibits the activity of collagenase, this new preservation solution might further improve the human islet isolation. Thus clarification of the mechanisms and optimization of the TLM before human islet isolation would further contribute to the islet transplantation.

CONCLUSION Nowadays, Perfluorocarbon is routinely used for pancreas preservation prior to islet isolation with significant impact on curing type 1 diabetes.

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