ET-Kyoto Solution Research Articles

Effects of the preservation medium and storage duration of domestic cat ovaries on the maturational and developmental competence of oocytes <i>in vitro</i>

We examined the effectiveness of saline, Euro-Collins solution (EC), and ET-Kyoto solution (ET-K) as preservation media for the cold storage of feline ovaries. Ovaries were maintained in these media at 4°C for 24, 48, or 72 h until oocyte retrieval. The ET-K group exhibited a higher oocyte maturation rate than the saline group after 72 h of storage. Moreover, ET-K could sustain the competence of the feline oocytes to cleave after 48 h, and the morula formation rate of the ET-K group was higher than that of the other groups after 24 and 48 h. Furthermore, the ET-K group exhibited a higher blastocyst formation rate than the other groups after storage for 24 h, and only ET-K retained the developmental competence in blastocysts after 48 h of storage. In addition, regarding the cell numbers of the blastocysts, there was no significant difference among the tested groups. In conclusion, our results indicate that ET-K is a suitable preservation medium for feline ovaries.

Protective Effects of a Hydrogen-Rich Preservation Solution in a Canine Lung Transplantation Model

Molecular hydrogen (H2) has protective effects against ischemia-reperfusion injury in various organs. Because they are easier to transport and safer to use than inhaled H2, H2-rich solutions are suitable for organ preservation. In this study, we examined the protective effects of an H2-rich solution for lung preservation in a canine left lung transplantation (LTx) model. Ten beagles underwent orthotopic left LTx after 23 hours of cold ischemia followed by reperfusion for 4 hours. Forty-five minutes after reperfusion, the right main pulmonary artery was clamped to evaluate the function of the implanted graft. The beagles were divided into two groups: control group (n= 5), and H2 group (n= 5). In the control group, the donor lungs were flushed and immersed during cold preservation at 4°C using ET-Kyoto solution, and in the H2 group, these were flushed and immersed using H2-rich ET-Kyoto solution. Physiologic assessments were performed during reperfusion. After reperfusion, the wet-to-dry ratios were determined, and histology examinations were performed. Significantly higher partial pressure of arterial oxygen and significantly lower partial pressure of carbon dioxide were observed in the H2 group than in the control group (P= .045 and P < .001, respectively). The wet-to-dry ratio was significantly lower in the H2 group than in the control group (P= .032). Moreover, in histology examination, less lung injury and fewer apoptotic cells were observed in the H2 group (P < .001 and P < .001, respectively). Our results demonstrated that the H2-rich preservation solution attenuated ischemia-reperfusion injury in a canine left LTx model.

Hydrogen-Rich Preservation Solution Attenuates Lung Ischemia-Reperfusion Injury after Prolonged Cold Ischemia in a Canine Left Lung Transplant Model

Purpose Molecular hydrogen (H2) has been reported as having protective effects on ischemia-reperfusion injury (IRI) in various organs. However, most of the reports are on inhalation of H2. Our group has previously reported the protective effects of H2-rich solution on lung IRI in a rat hilar-clamp model (Takahashi M, et al. Eur J Cardiothorac Surg 2017; 51: 442-448.) and the protective effects of hydrogen-rich preservation solution on lung preservation in a rat left lung transplant (LTx) model (Saito M, et al. J Heart Lung Transplant 2018; 37: S226). In this study, we examined the protective effects of hydrogen-rich preservation solution on lung preservation in a canine left LTx model. Methods TOYO beagles underwent orthotopic left LTx after cold ischemia followed by reperfusion (RP) for 4 hours. Forty-five minutes after RP, the right main pulmonary artery was clamped for evaluating the implanted graft function. Beagles were divided into two groups: control group (CON, n=4) and hydrogen group (H2, n=4). In the CON groups, donor lungs were flushed and immersed for 23 hours cold preservation at 4 °C by the preservation solution (ET-Kyoto Solution). In the H2 groups, donor lungs were flushed and immersed by the hydrogen-rich ET-Kyoto Solution. Arterial blood gas analysis, lung mechanics, and pulmonary vascular resistance (PVR) were evaluated during RP. After RP, wet-to-dry ratio (W/D) was evaluated. Results Compared with the CON group, significantly higher partial pressure of arterial oxygen (Figure A) and significantly lower partial pressure of carbon dioxide in the H2 group was observed 4 hours after RP. Although not statistically significant, dynamic lung compliance was better and PVR was lower in the H2 group than in the CON group (Figure B, C). Furthermore, W/D in the H2 group was significantly lower than that in the CON group (Figure D). Conclusion Our results indicated that hydrogen-rich preservation solution attenuated IRI in a canine left LTx model.

Impact of Ex Vivo Administration of Mesenchymal Stem Cells on the Function of Kidney Grafts From Cardiac Death Donors in Rat

BackgroundMesenchymal stem cells (MSCs) have been applied to the treatment of various diseases, and MSC administration in marginal donor grafts may help avoid the ischemia–reperfusion injury associated with solid organ transplants. Given the reports of side effects after intravenous MSC administration, local MSC administration to the target organ might be a better approach. We administered adipose tissue–derived MSCs (AT-MSCs) ex vivo to donor rat kidneys obtained after cardiac death (CD). MethodsUsing male Lewis rats (8–10 weeks), and a marginal transplant model of 1hr CD plus 1hr sub-normothermic ET-Kyoto solution preservation were conducted. AT-MSCs obtained from double-reporter (luciferase–LacZ) transgenic Lewis rats were injected either systemically (1.0 × 106 cells/0.5 mL) to bilaterally nephrectomized recipient rats that had received a marginal kidney graft (n = 6), or locally via the renal artery (500 μL ET-Kyoto solution containing the same number of AT-MSCs) to marginal kidney grafts, which were then preserved (1 hour; 22°C) before being transplanted into bilaterally nephrectomized recipient rats (n = 8). Serum was collected to assess the therapeutic effects of AT-MSC administration, and the recipients of rats surviving to Day 14 were separately evaluated histopathologically. Follow-up was by in vivo imaging and histological LacZ staining, and tumor formation was evaluated in MSC-injected rats at 3 months. ResultsSystemic injection of MSC did not improve recipient survival. In vivo imaging showed MSCs trapped in the lung that later became undetectable. Ex vivo injection of MSCs did show a benefit without adverse effects. At Day 14 after RTx, 75% of the rats in the AT-MSC–injected group (MSC[+]) had survived, whereas 50% of the rats in the AT-MSC–non-injected group (MSC[−]) had died. Renal function in the MSC(+) group was improved compared with that in the MSC(−) group at Day 4. LacZ staining revealed AT-MSCs attached to the renal tubules at 24 hours after RTx that later became undetectable. Histopathologic examination showed little difference in fibrosis between the groups at Day 14. No teratomas or other abnormalities were seen at 3 months.

Clinical application of ET-Kyoto solution for lung transplantation.

Because of the severe donor shortage in Japan, even after the revision of the Organ Transplant Law in 2010, the frequency of recovery of extended criteria lungs has increased in Japan. We developed a new lung preservation solution, "ET-Kyoto solution," to enhance lung preservation, to minimize primary graft dysfunction (PGD) and to improve the post-transplant outcomes. In this study, we retrospectively analyzed our results of lung transplantation using the ET-Kyoto solution. From 2002 to 2012, 26 patients underwent transplantation of lungs preserved with ET-Kyoto solution from brain-dead donors. We retrospectively reviewed the post-transplant pulmonary function and long-term survival. The graft performance was assessed by the PGD grading system. The mean graft ischemic time was 483.8 ± 19.0 min. The oxygenation capacity after reperfusion and recovery of respiratory function were both acceptable despite the long ischemic time. The survival rate at 5 years after transplantation was 85.1 %. Lungs preserved by ET-Kyoto solution had satisfactory postoperative lung function, despite the long preservation time, with excellent long-term survival. The results were acceptable for the use of grafts with a long ischemic time.

Luminescence-Based Assay to Screen Preservation Solutions for Optimal Ability to Maintain Viability of Rat Intestinal Grafts

BackgroundSegmental intestinal transplantations from living, genetically related donors provide advantages compared with those from cadaveric subjects. However, successful preservation during ischemic cold storage is critical for living donor grafts. Thus, the development of preservation solutions that maintain graft viability is essential for success. Herein we have reported application of a cell-based viability assay in multiwell plates to assess the effectiveness of various solutions to preserve intestinal grafts. MethodsFreshly isolated intestinal chips from luciferase transgenic rats were placed in 96-well tissue culture plates for incubation at 4°C for 24 hours in various preservation solutions: ET-Kyoto (ET-K), University of Wisconsin (UW) solution, Euro-Collins (EC) solution, histidine-tryptophan-ketoglutarate (HTK) solution, lactated Ringer’s (LR) solution, or saline. ResultsAs indicated by a higher level of luminescence, intestinal chips preserved in UW, HTK, or ET-K solution contained more viable cells, than those preserved in EC, LR, or saline solution. After exposure to the preservation solutions for 1 hour, the mucosal layer chips showed lower cell viability than the muscle layer chips. ConclusionOur data demonstrated that ET-K and UW solutions used together with intestinal chips of Luciferase transgenic rat and in vivo imaging provided optimal viability during ischemic cold storage prior to transplantation. Further development of preservation conditions to minimize the loss of viability of intestinal grafts before clinical transplantation is essential to improve outcomes.

Improvement of Hepatocyte Recovery from Liver Tissue Suffered from Warm Ischemia by Citrate Phosphate Dextrose (CPD) Added Euro-Collins Perfusion Solution

Because hepatocyte cell lines lack sufficient native functions, they are not suitable for hepatocyte transplantation, pharmaceutical research and biological study. Thus, primary human hepatocyte is attracting researchers interest these days. However, when we use surgically resected liver tissues or livers from non-heart beating donors as cell sources, unavoidable warm ischemia time interferes the efficacy of the collagenase digestion, and reduces the live cell recovery. We speculated that the impairment of collagenase digestion is due to microembolism that is formed by warm ischemia. To improve recovery rate of hepatocytes, the different perfusion solution was evaluated in rat. Nine-week old male SD rats were anesthetized with enflurane inhalation and received midline abdominal incision. Then, portal vein and hepatic artery were separated gently from surrounding connective tissue and 22G cannula was inserted into portal vein. The warm ischemia was started by ligating both portal vein and hepatic artery with 3-0 suture at the lower part of the cannulated point. Two perfusion solutions, Euro-Collins and ET-Kyoto solution, and two anti-coagulative agents, heparin and citrate phosphate dextrose solution (CPD), were examined comparing with the Seglen's original Ca2+-Mg2+-free Hank's solution (Seglen, Methods Cell Biol 13:29 1976). In result, Seglen's solution showed 2.27 ± 0.53 × 108 live cell yield (n=11) in non-warm ischemia control, but only 0.38 ± 0.17 × 108 (n=3) cells in 10min warm ischemia group (P=0.00001). Addition of heparin in Seglen's solution did not improve cell recovery very much (0.71 ± 0.40 × 108 (n=4)). In 10min warm ischemia groups, CPD added Euro-Collins solution brought about the best recovery, 1.41 ± 0.50 × 108 (n=7) cells that is 3.7 times to Seglen's solution (P=0.041). In 15min warm ischemia groups, CPD added Euro-Collins showed 1.37 ± 0.28 × 108 (n=3) cells recovery that is 1.69 time greater than CPD added ET-Kyoto solution although there was no statistical significance. Addition of heparin to Euro-Collins or ET-Kyoto solution showed only 0.19 ± 0.07 × 108 (n=3) cells and 0.16 ± 0.03 × 108 (n=3) cells respectively. Therefore, heparin is not likely to be suitable for hepatocyte isolation from warm ischemia liver. Actually, macroscopic observation also demonstrated good blood removal by CPD added solutions, suggesting restoration of better hepatic microcirculation. The present results demonstrate that CPD would be more effective on ensuring the microcirculation in the liver tissue suffered from warm ischemia to enable collagenase digestion. Our present protocol will be benefitial to isolate hepatocytes not only from surgically resected liver tissue but also livers from non-heart beating donors. In conclusion, CPD, not heparin, added Euro-Collins solution would be suitable for the hepatocyte isolation from liver tissue suffered from warm ischemia.

Effects Of Static Subnormothermic Preservation Following Warm Ischemia on Ischemia-Reperfusion Injury and Long-Term Outcome of the Renal Allograft in CLAWN Miniature Swine

Objective: Normothermic organ machine perfusion has been suggested in place of hypothermic preservation for kidneys from donors after cardiac death and other marginal donors. Here we attempted to evaluate the efficacy of static subnormothermic preservation on renal grafts exposed to periods of warm ischemia time (WIT) in a preclinical large animal model using MHC inbred miniature swine. Methods: Nine MHC-inbred CLAWN miniature swine received MHC-matched renal allografts with 12 days of FK506 (days 0-11). In Group 1, two animals received kidneys without WIT. In Groups 2 and 3, the grafts were subjected to 60-min of WIT, and then preserved at 4°C (Group 2; n=3) or 22°C (Group 3; n=4) for 60-min in ET-Kyoto solution. In 2 additional groups (4 and 5), WIT was extended to 2 hours. Renal function was monitored by serum creatinine (sCr) and biopsies. Serum levels of TNF-α and IL-6 were measured to characterize inflammatory responses. Results: In Group 1, both animals survived over 60 days with stable renal function and minimal rise in sCr (2.4 ± 0.8mg/dl around day 2). In Group 2, all animals showed moderate elevations of sCr (4.1 ± 0.8mg/dl), peaking at a median of 2 days; renal function recovered by day 7 in all three animals and remained stable for over 60 days in 2 animals. sCr in the remaining animal gradually increased after 30 days and the animal was sacrificed at day 60 (sCr=20.5 mg/dl). In Group 3, sCr increased to 3.6±1.0mg/dl, peaking at day 2 and recovered by day 6. One animal died from infection at day 24, but the other 3 recipients survived 60 days. Systemic early pro-inflammatory cytokine levels (TNF-α, IL-6) increased in both Group 2 and Group 3, peaking 6 hours after reperfusion (Group 2 vs. 3; TNF-α: 152 ± 9 vs. 122 ± 6 pg/ml; IL-6: 2357 ± 367 vs. 1823 ± 487 pg/ml). Although there was no statistical difference in peak levels of sCr, renal function as well as the peak cytokine concentration for animals receiving 22°C (vs 4°C) preservation was better across all three parameters. One animal underwent 2-hr of WIT plus 1-hr 4°C preservation (Group 4) and died of irreversible graft non-function on day 5. In contrast, 2 animals, underwent 2-hr of WIT plus 1-hr of 22°C preservation (Group 5) and maintained graft function >45 days (ongoing) although both had transient rise in sCr around day 3. Conclusion: We demonstrated that subnormothermic (22°C) organ preservation of kidney grafts exposed to 60-min of WIT was as effective as hypothermic (4°C) preservation, in miniature swine. These data also suggest a possible benefit of subnormothermic preservation for organs which experience of up to 2-hr of WIT. To our knowledge, this is the first demonstration of the applicability of static subnormothermic storage in preclinical large animals. Although further optimization is required, this preservation method may contribute to an expansion of the donor pool through improvement of extended criteria organs.

Development of Perfusate for Composite Tissue Allograft: New Maneuvers for Ischemic Golden Period

Objective: In the management of traumatic limb amputation, it is important to consider ischemic time and reperfusion injury by free radicals when blood supply is reestablished. These considerations must be weighed even more seriously in macroreplantation cases. To have the greatest chance for success, replantation operations require a minimizing unnecessary ischemic injury. This abstract reports a successful case of hand replantation after long ischemia time and the development of perfusate for Composite Tissue Allograft. Design and method: A 55-year-old man suffered complete amputation of his left hand 0.5 inches distal from the wrist. He arrived at our hospital 4 hours and 20 minutes after his accident. We performed three simple maneuvers before hand replantation to shorten the warm ischemia time and minimize muscle necrosis. 1. Blood was aspirated from the femoral artery and infused through the radial artery. 2. The dorsalis pedis artery and vein were then anastomosed to the radial artery and vein. 3. One hour prior to replantation, University of Wisconsin solution was used to flush the hand vasculature. The aim of these maneuvers was maintain blood pH and electrolyte stability. Microsurgical replantation started 10 hours after injury. Reperfusion was achieved approximately 12 hours after injury. Results: The amputated hand survived completely. There was no evidence of reperfusion injury. Conclusions: We achieved a successful microsurgical complete hand replantation despite 12 hours of ischemic time. This illustrates that it is possible to extend the ischemic golden period. After this experience, we develop “Dynamic Preservation Method” which focuses the oxygen supply. This is a special combination of ET-kyoto solution (existing organ storage solution), artificial hemoglobin and oxygen nanobubble. This persusate may overcome an ischemic time limit which is the one of the problems of the allotransplantation.

Use of Modified ET-Kyoto Solution (ET-Kyoto/Neutrophil Elastase Inhibitor) Significantly Improves Islet Yield and Survival

Introduction: Islet transplantation is a minimal invasive approach for β-cell replacement. However, a sufficient number of islets derived from two or more donor pancreas are usually required to achieve insulin independence. Thus, there is a need for novel strategies that increase islet yield, maintain high islet quality, and protect transplanted islet grafts. Indeed, the islet isolation procedure itself can lead to tissue destruction and activation of cellular and non-cellular components of the pancreas, including resident neutrophils, macrophages and T cells, which probably play an important role in impairment of islet survival. In the present study, we focused on the role of neutrophils, in particular neutrophil elastase (NE), against both islets during islet isolation and survival, because NE, released from activated neutrophils can directly cause tissue injury, and may also amplify inflammatory responses in acute organ injury. Objectives: The objectives of in this study were to determine whether the addition of NE inhibitor [sivelestat (ONO-5046)] to the islet isolation solution improves islet yield and viability. We also investigated the cytoprotective effects of sivelestat in islet recipients. Materials and methods:Islet isolation: Fresh islets were isolated from male C57BL/6 mice using either UW, UW containing 20 μM of sivelestat (ie: S-UW), ET-Kyoto or ET-Kyoto containing 20 μM of sivelestat (ie: S-Kyoto), respectively. Islet viability assay: To determine the amelioration of islet viability, TMRE assay was performed. Glucose-Stimulated Insulin Release: To determine the changes in the endocrinological potency of isolated islets, static glucose challenge was performed. Measurement of neutrophil elastase activity: To determine whether neutrophils are activated and release neutrophil elastase during pancreas digestion, neutrophil elastase activity was measured by the incubation with N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide as the substrate. Islet transplant experiments: The recipient 10-12-week-old male Balbc/A mice were rendered diabetic by a injection of streptozotocin. 500 of freshly isolated islets were transplanted under the kidney capsule. After transplantation, non-fasting blood glucose level was monitored. Moreover, to assess the beneficial effects of monotherapy with intraperitoneal sivelestat, sivelestat was administered at 100 mg/kg/day/for one day before transplantation and every day until 14 days after transplantation. To determine the effects of sivelestat administration on glucose tolerance, IPGTT was conducted at one week post-transplantation. Results: Data are summarized in the Table.[Tanemura et al. result table]Conclusion: We succeeded in isolating large numbers of islets using a new preservation solution, S-Kyoto solution, and in significant prolongation of islet graft survival in recipient mice treated with sivelestat. Therefore, treatment with NE inhibitors is potentially suitable for better harvest of transplantable islets and long-term islet allograft survival, allowing successful management of diabetes with islets from a single donor.

Mo1503 Indications for Total Pancreatectomy and Islet Auto-Infusion Beyond Chronic Pancreatitis With Intractable Pain

Background/Aims Total pancreatectomy with islet auto-infusion (TP + IAI) is effective in selected patients with chronic pancreatitis (CP) who have intractable pain unresponsive to medical and interventional therapies. IAI can maintain adequate glycemic control, possibly insulin independence, and has been used in selected cases following total pancreatectomy for IPMN, pancreas trauma or pancreas necrosis with persistent leak. We report our experience with TP + IAI for indications beyond chronic pancreatitis. Methods TP + IAI has been performed since 2006 at BUMC. Pancreata are preserved using chilled ET-Kyoto solution and using the oxygen-charged static two layer method. Digestion is by the modified Ricordi method, and purified when over 10 ml of tissue is obtained and then injected into the portal vein. A SUITO index of > 10 and islet yield of 500,000 correlates with increased insulin independence. Patients who had the procedure for a diagnosis other than chronic pancreatitis and intractable pain were selected from the IRB approved database. Results Thirty seven patients had a TP + IAI since 2006; 34 patients had CP confirmed by CT/MRI and/or EUS/ERCP, endoscopic secretin stimulated pancreas function testing (ePFT) and histology. Three patients had the procedure for other indications and are reported. Patient 1: 32 y/o F with idiopathic recurrent acute pancreatitis (IRAP)resulting in multiorgan failure (MOF), ARDS and ventilator dependency with each attack. EUS/ERCP were not diagnostic of CP, ePFT was normal. No evidence of endocrine/exocrine failure. No genetic mutations found. Decision to perform TP + IAI after last admission with 2 month hospitalization with MOF. Patient 2: 31 y/o M with hereditary chronic pancreatitis (HP) with PRSS1 (R122H) mutation, mother with CP and PRSS1, 2 family members with CP, 2 family members with pancreas cancer (<55 y/o). Intermittent pain exacerbations treated mostly at home. Decision to perform procedure due to known mutation and family history of cancer. Patient 3: 62 y/ o F with ampullary adenoma, recurrent high grade dysplasia despite repeated ampullectomies complicated by pancreas necrosis, and distal pancreatectomy with persistent leak. Decision to perform procedure as a completion pancreatectomy was expected. The results are summarized in the table. Conclusions The pt with IRAP had a higher c-peptide, SUITO index and islet yield compared to the patient with HP, but post-procedure c-peptide and glycemic control were similar. Despite purity of the pancreas extract ductal cells could have been injected into the portal vein, which was explained to the patient with HP and who consented. As TP + IAI becomes more routine studies are needed to understand its application beyond treatment of intractable pain and glycemic control in CP. TP + IAI for indications other than intractable pain in chronic pancreatitis

Comparison of Ulinastatin, Gabexate Mesilate, and Nafamostat Mesilate in Preservation Solution for Islet Isolation

For islet transplantation, maintaining organ viability after pancreas procurement is critically important for optimal graft function and survival. We recently reported that islet yield was significantly higher in the modified ET-Kyoto (MK) solution, which includes a trypsin inhibitor (ulinastatin), compared with the UW solution, and that the advantages of MK solution are trypsin inhibition and less collagenase inhibition. In this study, we compared ulinastatin with other trypsin inhibitors, gabexate mesilate, and nafamostat mesilate, in preservation solution for islet isolation. Ulinastatin was easily dissolved in ET-Kyoto solution, while ET-Kyoto with gabexate mesilate and nafamostat mesilate became cloudy immediately after addition. Although there were no significant differences in islet yield among the three groups, viability was significantly higher for the MK group than for the GK group or the NK group. The stimulation index was significantly higher for the MK group than for the GK group. In summary, there are no other trypsin inhibitors that are more effective than ulinastatin. Based on these data, we now use ET-Kyoto solution with ulinastatin for clinical islet transplantation.

Improving Efficacy of Clinical Islet Transplantation with Iodixanol-Based Islet Purification, Thymoglobulin Induction, and Blockage of IL-1β and TNF-α

Poor efficacy is one of the issues for clinical islet transplantation. Recently, we demonstrated that pancreatic ductal preservation significantly improved the success rate of islet isolation; however, two transplants were necessary to achieve insulin independence. In this study, we introduced iodixanol-based purification, thymoglobulin induction, and double blockage of IL-1β and TNF-α as well as sirolimus-free immunosuppression to improve the efficacy of clinical islet transplantation. Nine clinical-grade human pancreata were procured. Pancreatic ductal preservation was performed using ET-Kyoto solution in all cases. When the isolated islets met the clinical criteria, they were transplanted. We utilized two methods of immunosuppression and anti-inflammation. The first protocol prescribed daclizumab for induction, then sirolimus and tacrolimus to maintain immunosuppression. The second protocol used thymoglobulin for induction and tacrolimus and mycophenolate mofetil to maintain immunosuppression. Eternacept and anakinra were administered as anti-inflammatory drugs. The total amount of insulin required, HbA1c, and the SUITO index were determined to analyze and compare the results of transplantation. All isolated islet preparations (9/9) met the criteria for clinical transplantation, and they were transplanted into six type 1 diabetic patients. All patients achieved insulin independence with normal HbA1c levels; however, the first protocol required two islet infusions (N = 3) and the second protocol only required a single infusion (N = 3). The average SUITO index, at 1 month after a single-donor islet transplantation, was significantly higher in the second protocol (49.6 ± 8.3 vs. 19.3 ± 6.3, p < 0.05). Pancreatic ductal preservation, iodixanol-based purification combined with thymoglobulin induction, and blockage of IL-1β and TNF-α as well as sirolimus-free immunosuppression dramatically improved the efficacy of clinical islet transplantations. This protocol enabled us to perform successful single-donor islet transplantations. Further large-scale studies are necessary to confirm these results and clarify the mechanism of each component.

Body Mass Index Reflects Islet Isolation Outcome in Islet Autotransplantation for Patients with Chronic Pancreatitis

Total pancreatectomy with autologous islet cell transplantation (TP with AIT) is an effective treatment for chronic pancreatitis patients with severe abdominal pain. Body mass index (BMI) of the pancreatic donor is proven to be a useful predictor for islet isolation and transplantation outcomes in allogenic islet transplantation. However, the association between BMI and islet isolation outcome and/or metabolism after AIT was previously unclear. Twelve patients who received TP with AIT at our hospital were included in this study. All pancreata were preserved with both pancreatic ductal injection and oxygen-charged static two-layer method using ET-Kyoto solution. The cohort was divided into two groups: low BMI group (BMI <23 kg/m(2), n=5) and high BMI group (BMI ≥23, n=7). The high BMI group had a significantly higher islet yield per gram than the low BMI group both in pancreas postdigestion and in final product (postdigestion: 7330 ± 539 vs. 3509 ± 563 IE/g; p<0.001; final product: 6555 ± 585 vs. 3476 ± 546 IE/g; p=0.004). For islet yield in final product per patient body weight, the high BMI group also had significantly higher islet yield than the low BMI group (7997 ± 779 vs. 4175 ± 750 IE/kg, p=0.007). Insulin independence rate in the high BMI group (71%) was also higher than that low BMI group (40%), but it did not reach statistical significance. Pancreata from patients with higher BMI could obtain higher islet yield in the setting of autologous islet cell transplantation for chronic pancreatitis.

Reconstruction of Pancreatic Duct for Previous Puestow Operation for an Islet Autotransplantation

Reconstruction of Pancreatic Duct for Previous Puestow Operation for an Islet Autotransplantation

Super-High-Dose Islet Transplantation Is Associated With High SUITO Index and Prolonged Insulin Independence: A Case Report

Introduction One of the current issues of clinical islet transplantation is the difficulty to achieve a prolonged insulin-free status. Functional islet mass gradually decreased after transplantation. We developed the SUITO index, which reflects engrafted islet mass. The SUITO (Secretory Unit of Islet Transplant Objects) index more than 26.0 is associated with an insulin-free status. In this study, we have experienced that super-high-dose islet transplantation maintained insulin-free status and a high SUITO index for a prolonged period. Materials and methods Two islet isolations were performed in February 2007 and January 2008. Ductal injections were performed at the procurement site using the ET-Kyoto solution and pancreata preserved by a two-layer method. Islets were isolated using the modified Ricordi method. Both isolated islets were transplanted into a type 1 diabetic patient. Efficacy of islet transplantation was assessed by the amount of insulin requirements and SUITO index. Results Islet yields were 514,467 islet equivalents (IE) and 872,174 IE, with purities of 49% and 85% for the first and second islet transplantations, respectively. The patient received a total of 24,327 IE/kg body weight. The immunosuppression was based on the Edmonton protocol. After the second islet transplantation, the average SUITO index for the following 1 month was 48.5, and the patient became insulin-free. At postoperative day 1006, the SUITO index was 44.6 and the patient maintained an insulin-free status with excellent glycemic control. Conclusion Super-high-dose islet transplantation was associated with an high SUITO index and prolonged insulin independence.

Seven Consecutive Successful Clinical Islet Isolations with Pancreatic Ductal Injection

Inconsistent islet isolation is one of the issues of clinical islet transplantation. In the current study, we applied ductal injection to improve the consistency of islet isolation. Seven islet isolations were performed with the ductal injection of ET-Kyoto solution (DI group) and eight islet isolations were performed without the ductal injection (standard group) using brain-dead donor pancreata. Isolated islets were evaluated based on the Edmonton protocol for transplantation. The DI group had significantly higher islet yields (588,566 +/- 64,319 vs. 354,836 +/- 89,649 IE, p < 0.01) and viability (97.3 +/- 1.2% vs. 92.6 +/- 1.2%, p < 0.02) compared with the standard group. All seven isolated islet preparations in the DI group (100%), versus only three out of eight isolated islet preparations (38%) in the standard group met transplantation criteria. The islets from the DI group were transplanted into three type 1 diabetic patients and all three patients became insulin independent. Ductal injection significantly improved quantity and quality of isolated islets and resulted in high success rate of clinical islet transplantation. This simple modification will reduce the risk of failure of clinical islet isolation.

Comparison of Trypsin Inhibitors in Preservation Solution for Islet Isolation

Islet transplantation has recently emerged as an effective therapy and potential cure for type 1 diabetes mellitus. Recent reports show that the two-layer method (TLM), which employs oxygenated perfluorochemical (PFC) and University of Wisconsin (UW) solution, is superior to simple cold storage in UW for pancreas preservation in islet transplantation. Moreover, we recently reported that islet yield was significantly higher in the ET-Kyoto solution with ulinastatin (MK)/PFC preservation solution compared with the UW/PFC preservation solution in the porcine model and that the advantages of MK solution are trypsin inhibition and less collagenase inhibition. In this study, we compared ulinastatin with another trypsin inhibitor, Pefabloc, in preservation solution for islet isolation. Islet yield before purification was higher in the MK/PFC group compared with the ET-Kyoto with Pefabloc (PK)/PFC group. The stimulation index was higher for the MK/PFC group than for the PK/PFC group. These data suggest that ET-Kyoto with ulinastatin was the better combination for pancreas preservation than ET-Kyoto with Pefabloc. Based on these data, we now use ET-Kyoto solution with ulinastatin for clinical islet transplantation.

Preservation Solutions Alter Mrp2-Dependent Bile Flow in Cold Ischemic Rat Livers

Previously, decreased organic anion transport through multidrug resistance protein 2 (Mrp2) was observed without any notable cell lysis, even when the livers were stored for 8 hours in University of Wisconsin solution (UW). The aim of this study was to examine the bile flow and its constituents, markers of graft dysfunction without necrosis in cold ischemic livers, using the following preservation media: UW, ET-Kyoto solution (ET-K) and histidine-tryptophan-ketoglutarate solution (HTK). Rat livers were stored at 4 degrees C for 8 hours in the preservation media, and reperfused to collect the bile and determine their constituents. Glycyrrhizin (GL) and/or glutathione (GSH) were added to the media as necessary. The transport efficiency of Mrp2 was assessed by the biliary excretion of 5-carboxyfluorescein (CF), a fluoroprobe excreted from Mrp2. The Intracellular distribution of Mrp2 was determined by immunostaining. Livers stored for 8 hours exhibited significantly decreased bile production and biliary glutathione (GSH) levels without notable cell lysis. CF excretion was significantly delayed in all solutions. However, these markers were remarkably improved by the redistribution of Mrp2 from the cytoplasm to the canalicular membrane, when the livers were exposed to UW in the presence of GL. Moreover, livers exposed to the Kyoto and HTK solutions increased their bile production and organic anion transport in the presence of GL and GSH. These results suggest that the addition of GL and GSH to preservation solutions improves bile production and biliary organic anion transport by increasing Mrp2 localization to the bile canaliculi in post-cold ischemic livers. (248 words).

Islet cell transplantation for the treatment of type 1 diabetes in the USA

Islet cell transplantation (ICTx) is one of the most effective treatments for type 1 diabetes and is less invasive compared to whole organ transplantation. The US has been the leader in the research and clinical applications of ICTx for the last 40 years. ICTx requires complex procedures, including pancreas procurement and preservation; pancreas digestion; islet purification; and transplantation. Even with the dramatic progresses in each of the procedures listed above, there are still challenges to make ICTx the standard therapy. These challenges are: (1) obtaining enough islets from a single donor and (2) preventing graft loss due to allogenic rejection and recurrence of autoimmune islet destruction. A new preservation strategy for pancreata and pancreatic ducts using ET-Kyoto solution as well as a new islet purification method using iodixanol has substantially improved islet yields. Continuous research to improve the efficacy of islet isolation will solve the issue of obtaining enough islets from a single donor. Immunological tolerance is an ideal solution for the issue of rejection and autoimmune recurrence and a regulatory T cell strategy seems promising. Moreover, the SUITO index is a simple and powerful tool to assess engrafted islet mass and is, therefore, useful for evaluating the efficacy of new immunosuppressant strategies. Once ICTx becomes a standard treatment, the donor shortage will become the next challenge. Marginal or living donor islet transplantations could help alleviate this issue; however, bio-artificial islet transplantation with animal islets could be the ultimate solution.