Preservation of the human pancreas before islet isolation using a two-layer (UW solution???perfluorochemical) cold storage method1

Recent progress in clinical islet transplantation resulting from improvements in less diabetogenic immunosuppression and preparation of sufficient quantities of highly viable islets for transplantation now provides an attractive treatment option for selected patients with type 1 diabetes mellitus (1–3). However, it is still difficult to recover sufficient numbers of islets from a single cadaveric donor pancreas to achieve insulin independence after transplantation. Therefore, preservation of the human pancreas has been an important factor in the subsequent success of clinical islet transplantation. University of Wisconsin (UW) solution has proven to be effective in pancreas preservation, leading to successful whole-pancreas transplantation in experimental animal models and clinically, and human pancreatic grafts can be preserved by UW solution for periods exceeding 24 hr (4). In the case of clinical islet transplantation, islets are isolated from the cadaveric donor pancreas using intraductal enzyme loading, enzymatic and mechanical dissociation, and purification of the islets from the exocrine tissues. The entire process may be critically affected by pancreas storage conditions before isolation—by either alteration in endogenous pancreatic enzyme activity or alteration in differential density between the exocrine and endocrine components of the pancreas. This creates additional challenges compared with pancreas transplantation. The authors have shown that 16 hr was the upper limit for cold storage of human pancreases before islet isolation (5), and more than 8 hr of cold storage before human islet isolation significantly reduced islet yield and functional viability (5–7). Current techniques of multiorgan recovery and the need to transport organs from distant hospitals to centralized islet processing facilities often result in more than 12 hr of cold storage time before islet isolation. Efforts to improve the transportation of the pancreas within an optimal 8-hr time course have led to considerable additional expense for dedicated jet transportation and challenging logistical difficulties. Improvements in human pancreas preservation before islet isolation remain one of the obstacles to the expansion of islet transplantation. In the late 1980s, Kuroda et al. developed a two-layer cold storage method using perfluorocarbon (PFC) and UW solution for whole-pancreas preservation (8–10). PFC is a hyperoxygen carrier designed to release oxygen into the surrounding tissue more effectively. PFC differs from hemoglobin preparations in that it is a totally synthetic compound formed on a liquid hydrocarbon base. In contrast to hemoglobin, oxygen is not chemically bound to the PFC carrier. PFC takes up and releases oxygen following Henry’s linear law, on the basis of the partial pressure of the gas, rather than Barcroft’s sigmoid curve described for hemoglobin (11). Unlike hemoglobin, acidosis, alkalosis, and temperature seem to have no or little effect on the oxygen delivery of PFC, allowing this compound to be used effectively during cold storage of organs. The two-layer (UW solution–PFC) cold storage method continuously supplies sufficient oxygen to a pancreas during preservation and reduces cold ischemic injury (8–10). Furthermore, they have shown that canine pancreases subjected to 90 min of warm ischemia were resuscitated during preservation by the two-layer method at 4°C for 24 to 48 hr (12,13). One of the mechanisms of this method is to augment adenosine triphosphate (ATP), which maintains cellular integrity and controls ischemic cell swelling (14). Endogenous substrate for ATP synthesis is eliminated during ischemia. However, during the preservation by the two-layer method, ATP is synthesized within the ischemically damaged pancreas by means of the direct phosphorylation of adenosine contained in the UW solution (14). Because ATP is an essential source of energy to repair damaged cells (15), it is likely that ATP regeneration plays a key role in restoration of the ischemically damaged pancreas during preservation. In the canine autotransplant model, the two-layer method was applied to preserve pancreases before islet isolation (16,17). Although the canine pancreases preserved by simple cold storage in UW solution for 3 hr resulted in significantly decreased functional success compared with those without preservation, those preserved by the two-layer method for 3 hr resulted in almost the same islet recovery and functional success as those without preservation. When the canine pancreases were preserved by the two-layer method for 24 hr, islet yields slightly decreased compared with that without preservation but were three times as high as with simple cold storage in UW solution for 24 hr, and the functional success rate greatly improved compared with simple cold storage in UW solution for 24 hr (56% vs. 0%) (16). Furthermore, with preservation by the two-layer method for 24 hr, the canine pancreases subjected to 60 min of warm ischemia resulted in almost the same islet yield as those without warm ischemia (17). This showed the effect of the two-layer method in restoring the ischemically damaged pancreas. The authors recently applied the two-layer (UW solution–PFC) method additionally to preserve the human pancreas with prolonged cold ischemia (>10 hr) before islet isolation (Table 1) (18). In spite of the prolonged cold storage (14.7±1.4 hr [mean±SEM] of total cold ischemic time), the authors obtained 349.2±44.1×103 islet equivalents with the additional preservation (2.9±0.7 hr) by the two-layer method, and five of seven isolated islets (71%) were transplanted into patients with type 1 diabetes with the criteria of the Edmonton protocol. In pancreas grafts with more than 16 hr of cold ischemia, three of five preparations were able to be transplanted. In cases with cold storage in UW solution, the authors obtained 214.0±31.0×103 IE with 11.3±0.3 hr of cold storage in UW solution and only 5 of 14 cases (36%) underwent transplantation. Furthermore, in the viability assessments, the additional preservation by the two-layer method had a positive effect on the insulin secretory activity of the purified human islets compared with simple cold storage in UW solution alone. In addition, all grafts preserved by the two-layer method improved the ability of glycemic control and decreased exogenous insulin administration in all patients; indeed, in one case, insulin independence was achieved after single-donor islet transplantation. The authors’ preliminary study shows that additional short preservation by the two-layer method can significantly improve human islet recovery and increases the chances to obtain sufficient mass and quality of purified human islets from the pancreas after prolonged cold ischemia. This may therefore increase the donor pool by resuscitation of the ischemically damaged human pancreas and prolong the preservation time. In future investigations, the authors will apply the two-layer method to preserve human pancreases immediately after the procurement for more than 12 hr before islet isolation and determine the mechanism of protective effect of the two-layer method on human pancreas preservation before islet isolation. Table 1: Table 1.Human islet isolation after preservation by the two-layer method or by simple cold storage in UW solutionaAcknowledgments. The authors thank the Human Organ Procurement and Exchange (HOPE) program for their assistance in the identification and recovery of human cadaveric pancreases, and the technical staff members of the human islet isolation laboratory, University of Alberta.

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