Toshiaki Tsujimura

Human islet transplantation from pancreases with prolonged cold ischemia using additional preservation by the two-layer (UW solution/perfluorochemical) cold-storage method

Background. A two-layer (University of Wisconsin solution/perfluorochemical [UW/PFC]) cold-storage method delivers sufficient oxygen to the pancreas during preservation and restores the ischemically damaged pancreas. In this study, we determined whether the additional preservation by the two-layer method could improve islet recovery from human pancreases with prolonged cold storage in UW. Methods. Human pancreases were procured from cadaveric organ donors and preserved by the two-layer method (UW/PFC) for 2.9±0.7 hours (mean±SEM) at 4°C after 11.8±1.5 hours of cold storage in UW (UW/PFC group, n=7), or by cold UW alone for 11.3±0.3 hours (UW group, n=14). The selected pancreases met the criteria of having at least 10 hours of cold storage in UW. All were processed by using a standard protocol of Liberase perfusion with Pefabloc by way of the duct, gentle mechanical dissociation, and Ficoll gradient purification. Transplanted islets were selected with the criteria of the Edmonton protocol (>5,000 islet equivalents [IE]/kg recipient body weight). Results. The islet recovery was significantly increased in the UW/PFC group compared with the UW group (349.2±44.1×103 and 214.0±31.0×103 IE, respectively;P <0.05). This resulted in islet yields of 4.6±1.0×103 IE/g of pancreas in the UW/PFC group compared with 2.0±0.3×103 IE/g of pancreas in the UW group (P <0.05). Five of 7 cases (71%) in the UW/PFC group and 5 of 14 cases (36%) in the UW group were transplanted. The islet grafts in the UW/PFC group improved the ability of glycemic control and decreased exogenous insulin administration in all recipients. Conclusions. Improvements in methods to preserve and recover ischemically damaged human pancreases before islet isolation and transplant could be extremely beneficial to the field of clinical islet transplantation. This preliminary study shows that additional short preservation by the two-layer (UW/PFC) cold-storage method can significantly improve islet recovery and increase opportunities of islet transplantation from human pancreases after prolonged cold ischemia.

Influence of pancreas preservation on human islet isolation outcomes: impact of the two-layer method.

Background. Human pancreas preservation for islet transplantation holds additional challenges and considerations compared with whole pancreas transplantation. The purpose of this study was to clarify the limitations of the University of Wisconsin (UW) solution and the potentials of the two-layer method (TLM) for pancreas preservation before human islet isolation. Methods. We retrospectively evaluated human islet isolation records between January 2001 and February 2003. One hundred forty-two human pancreata were procured from cadaveric donors and preserved by means of the UW solution (n=112) or TLM (n=30). Human islet isolations were performed using a standard protocol and assessed by islet recovery and in vitro function of islets. Results. Eight to ten hours of cold ischemia in the UW solution is a critical point for successful islet isolations. It is difficult to recover a sufficient number of viable islets for transplantation from human pancreata with more than 10 hours of cold storage in the UW solution. The overall islet recovery in the TLM group was significantly higher than in the UW group. With 10 to 16 hours of cold storage, the success rates of islet isolations remained at 62% in the TLM group but decreased to 22% in the UW group. Transplanted islets in the TLM group worked well in the recipients. Conclusions. There are time limitations for using the UW solution for pancreas preservation before human islet isolation. The TLM is a potential method to prolong the optimal cold storage time for successful islet isolations.

Short-term storage of the ischemically damaged human pancreas by the two-layer method prior to islet isolation.

A two-layer cold storage method (TLM) allows sufficient oxygen delivery to pancreata during preservation and resuscitates the viability of ischemically damaged pancreata in the canine pancreas transplant model. In this study, we applied a short-term preservation of the TLM to human pancreata after prolonged cold ischemia prior to islet isolation, and investigated the mechanisms of resuscitation of the ischemically damaged human pancreas by the TLM. Human pancreata were procured from cadaveric donors and preserved by the TLM for 3.2 ± 0.5 h after 11.1 ± 0.9 h of cold storage in UW (TLM group), or by cold UW alone for 11.0 ± 0.3 h (UW group). Islet isolations of all pancreata were performed using the Edmonton protocol. Islet recovery and in vitro functional viability of isolated islets were significantly increased in the TLM group compared with the UW group. According to the criteria of the Edmonton protocol, 10/14 cases (71%) in the TLM group were transplanted to patients with type I diabetes mellitus compared with only 5/21 cases (24%) in the UW group. In the metabolic assessment of human pancreata, levels of energetic parameters (ATP, total adenylates, and energy charge) were significantly increased, and malondialdehyde (MDA) levels were significantly decreased after the TLM preservation. There was no observable change in the incidence or degree of mitochondrial injury after the TLM preservation. Additional short-term storage by the TLM resuscitates the ischemically damaged human pancreas by regenerating the energetic status and prevents further damage by oxidative stress, ultimately leading to improvements of islet recovery and in vitro function. Use of the TLM following prolonged storage in UW provides an excellent adjunctive protocol for treating human pancreata for the rigors of the islet isolation process.

Human Pancreas Preservation Prior to Islet Isolation

Islet transplantation has become a feasible treatment option for patients with type 1 diabetes mellitus after improvements regarding less diabetogenic immunosuppression and preparation of sufficient quantities of highly viable islets for transplantation. While the current techniques of multiorgan harvesting and transportation of organs to distant hospitals often result in 12 h or more of cold storage time before transplantation, University of Wisconsin (UW) solution has proven to be effective in pancreas preservation, leading to successful clinical whole pancreas transplantation, and human pancreatic grafts can be preserved by UW solution for periods exceeding 24 h. In the case of clinical islet transplantation, however, prolonged cold ischemia in UW solution before islet isolation significantly reduced recovery of viable islets, and it is still difficult to recover sufficient numbers of islets from a single cadaveric donor pancreas to achieve insulin independence after transplantation. Therefore, preserv...

Syk protein-tyrosine kinase is involved in neuron-like differentiation of embryonal carcinoma P19 cells

Syk has been implicated in activated immunoreceptors to downstream signaling events in hematopoietic cells. Here we report that Syk is expressed in neuron‐like cells and involved in neuron‐like differentiation of embryonal carcinoma P19 cells. Immunoblot, RT‐PCR, and Northern analysis indicated that Syk is expressed in mouse brain, PC12 and P19 cells. In addition, Syk was found to be tyrosine phosphorylated during neuron‐like differentiation of P19 cells. Furthermore, adenovirus‐mediated overexpression of Syk induced supernumerary neurite formation and extracellular signal‐regulated kinase (ERK) activation in P19 cells. These results suggest that Syk plays an important role in signaling steps leading to ERK activation in P19 cells.

Ameliorating Injury During Preservation and Isolation of Human Islets Using the Two-Layer Method With Perfluorocarbon and UW Solution

This study assessed the effects of a two-layer method (TLM), using perfluorocarbon and UW solution, on the quality of human pancreata following storage and islet yield/function after isolation. In part A, TLM was applied immediately after procurement and the energetic profile was compared to a group treated with UW solution only (control) throughout 24-h storage. In part B, cadaveric human pancreata were procured and subjected to a TLM after cold storage in UW solution (TLM group) or UW solution (control group). Energetics, lipid peroxidation, and islet recovery/function were assessed after preservation at 4°C. In part A, after 9-h storage, the energetic profile (ATP, ATP/ADP, energy charge) for the TLM group was superior to controls. In part B, TLM treatment resulted in consistently greater ATP, ATP/ADP, and energy charge values than with storage in UW solution alone (p < 0.05). UW treatment resulted in 40% greater peroxidative damage than in the TLM group (p < 0.05). Islet recovery and functional viability were 30–40% higher following TLM treatment (p < 0.05). These data support the hypothesis that islet viability and yields can be significantly improved using a brief period of TLM treatment following conventional UW storage; reduced energetic and oxidative stress are implicated as potential mechanisms.

Application of the two-layer method on pancreas digestion results in improved islet yield and maintained viability of isolated islets

Background. Oxygenation of the pancreas during preservation by the two-layer method (TLM) has shown beneficial effects in islet transplantation. Here, we apply this concept (oxygenation) to the isolation process. Methods. Rat pancreases were digested using four different methods. Pancreases were digested with preoxygenated perfluorocarbon (PFC) in group 2 and without it in group 1. Additionally, adenosine was included in the collagenase solution in subgroups B but not in subgroups A. Islet yields and viability were compared between groups. Results. Tissue oxygen tension in group 1 was essentially zero during digestion, but rapidly reached around 300 mm Hg and was maintained in group 2. The tissue adenosine triphosphate (ATP) level in rat pancreas just after laparotomy (control) was 4.2±0.7 &mgr;mol/g dry weight; after digestion, it was 0.12±0.03 &mgr;mol/g, 0.70±0.10 &mgr;mol/g, 0.30±0.18 &mgr;mol/g, and 2.90±0.80 &mgr;mol/g in groups 1A, 1B, 2A, and 2B, respectively. No significant differences were observed between group 2B and control (P=0.19). Islet yields (IEQ/pancreas) were 1600±400, 1400±400, 1300±400, and 2400±100 in groups 1A, 1B, 2A, and 2B, respectively. The islet yield of group 2B was significantly higher than other groups (P<0.05). The cure rate after transplanting 200 islets into athymic nude mice did not differ (80% in all groups). The stimulation indices in the four groups were also the same. Conclusions. Tissue ATP levels after digestion were well maintained using TLM with adenosine digestion method. Consequently, greater numbers of islets could be retrieved. The new method was at least equivalent to islet function isolated by conventional method. Clinical study is therefore warranted.

Improvement of pancreatic islet isolation outcomes using glutamine perfusion during isolation procedure.

During procurement, isolation, and transplantation, islets are exposed to high levels of oxidative stress triggering a variety of signaling pathways that can ultimately lead to cell death. Glutamine is an important cellular fuel and an essential precursor for the antioxidant glutathione. The aim of this study was to examine the role of intraductal glutamine administration in facilitating recovery of isolated rat islets from pancreases subjected to a clinically relevant period of warm ischemia. Islets were isolated in Sprague-Dawley (SD) rats (n= 18 per group). Pancreata in groups 1 and 2 were procured immediately while groups 3 and 4 were subjected to 30-min warm ischemia. Groups 2 and 4 were treated intraductally with 5 mM glutamine prior to pancreatectomy. Exposure to 30-min warm ischemia significantly reduced islet yield [groups 1 & 2 (nonischemia): 503 ± 29 islets/rat vs. groups 3 & 4 (ischemia): 247 ± 26 islets/rat; p < 0.05]. Intraductal glutamine treatment significantly improved islet yield when pancreata were subjected to 30-min warm ischemia [144 ± 16 islets/rat without glutamine (group 3) vs. 343 ± 36 islets/rat with glutamine (group 4), p < 0.05]. Glutamine also significantly improved islet viability (values were 50 ± 4% in group 4 vs. 27 ± 3% in group 3, p < 0.05). Similarly, glutathione (reduced) levels were significantly elevated in both glutamine-treated groups; however, this increase was greatest in tissues exposed to ischemia (2.76 ± 0.04 nmol/mg protein in group 4 vs. 1.66 ± 0.04 nmol/mg protein in group 3, p < 0.05). Intraductal glutamine administration considerably improves the islet yield, viability, and augments endogenous glutathione levels in pancreata procured after a clinically relevant period of ischemia. Intraductal administration of glutamine at the time of digestive enzyme delivery into the harvested pancreas may represent a simple yet effective tool to improve islet yields in clinical isolations.

Ameliorating Small Bowel Injury Using a Cavitary Two-Layer Preservation Method with Perfluorocarbon and a Nutrient-Rich Solution

The aim of this study was to improve small bowel (SB) quality during cold storage by combining two proven preservation strategies involving perfluorocarbon (PFC) and a novel luminal amino acid‐rich solution.

Successful 24‐h Preservation of Canine Small Bowel Using the Cavitary Two‐layer (University of Wisconsin Solution/Perfluorochemical) Cold Storage Method

We previously developed the two‐layer cold storage method (TLM), which allows sufficient oxygen delivery to the canine pancreas during preservation, and successfully achieved 96‐h preservation. In this study, we applied a modified TLM (cavitary TLM) to small bowel preservation in a canine heterotopic transplant model.