A new preservation solution increases islet yield and reduces graft immunogenicity in pancreatic islet transplantation

The aim of the study was to test a new preservation solution containing polyethylene glycol (S.C.O.T. solution) as pancreatic islet isolation medium both to increase the islet yield and to prolong the allograft survival. In a model of islet transplantation in diabetic mouse, islets were isolated with S.C.O.T. in experimental groups and with Hank's balanced salt solution (HBSS) in control groups. The use of S.C.O.T. solution improved the islet yield (596+/-27 IEQ/pancreas) as compared to HBSS (456+/-11 IEQ/pancreas) (P<0.001). Allograft survival was prolonged in experimental group (17.3+/-4.3 days) versus controls (7.3+/-3.6 days) in a full mismatch combination (P<0.001) and in absence of recipient immunosuppression. The same prolongation (10 days) was also found in a strongly alloreactive transgenic combination. It is hypothesized that a transitory phenomenon of immunocamouflage of the graft surface antigens occurs, as shown by immunofluorescence studies. The use of this new solution could improve the results of islet transplantation in humans.     

Influence of preservation solution on human islet isolation outcome

BACKGROUND: The influence of the preservation solution used for in situ perfusion of the donor and pancreas storage on islet isolation has received little attention. METHODS: In this prospective controlled trial, we compared the outcome of human islet isolation from pancreata perfused with University of Wisconsin (UW) solution or Celsior, an alternative colloid-free extracellular solution. RESULTS: At the 1-year interim analysis, the viability and insulin secretion of islets isolated from donors perfused with UW (n=19) or Celsior (n=5) were identical. However, total islet recovery (IEQ) and isolation yield (IEQ/g) were 1.8-fold and 2.1-fold inferior in the Celsior group (P<0.05 vs. UW). Overall, 13 (68%) of islet preparations were effectively transplanted from the UW group vs. none from the Celsior group (P=0.01). The clinical study was discontinued and the causes of these differences were further explored in the pig (n=14). In contrast to UW, Celsior induced cell swelling and pancreas edema after only four hours of cold storage. These abnormalities were delayed when the donor was perfused with Solution de Conservation d'Organes et de Tissus (SCOT), an extracellular solution containing polyethylene glycol. CONCLUSIONS: Our results suggest that colloid-free preservation solutions might be suboptimal for pancreas perfusion and cold storage prior to islet isolation and transplantation. Because pancreata are now frequently recovered for islet transplantation, preliminary experimental and clinical data about islet isolation should be obtained prior to the routine implementation of new preservation solutions for abdominal perfusion during multiorgan recovery.     

Comparison of modified Celsior solution and M-kyoto solution for pancreas preservation in human islet isolation

Since the successful demonstration of the Edmonton protocol, islet transplantation has advanced significantly on several fronts, including improved pancreas preservation systems. In this study, we evaluated two different types of organ preservation solutions for human islet isolation. Modified Celsior (Celsior solution with hydroxyethyl starch and nafamostat mesilate; HNC) solution and modified Kyoto (MK) solution were compared for pancreas preservation prior to islet isolation. Islet yield after purification was significantly higher in the MK group than in the HNC group (MK = 6186 ± 985 IE/g; HNC = 3091 ± 344 IE/g). The HNC group had a longer phase I period (digestion time), a higher volume of undigested tissue, and a higher percentage of embedded islets, suggesting that the solution may inhibit collagenase. However, there was no significant difference in ATP content in the pancreata or in the attainability of posttransplant normoglycemia in diabetic nude mice between the two groups, suggesting that the quality of islets was similar among the two groups. In conclusion, MK solution is better for pancreas preservation before islet isolation than HNC solution due to the higher percentage of islets that can be isolated from the donor pancreas. MK solution should be the solution of choice among the commercially available solutions for pancreatic islet isolation leading to transplantation.     

Improved islet yield and function with ductal injection of University of Wisconsin solution before pancreas preservation

BACKGROUND: Ensuring sufficient islet yield from preserved pancreases is a critical step in clinical islet transplantation. The aim of this study was to investigate whether pancreatic ductal injection, performed at procurement, using a small volume of preservation solution before cold storage (ductal preservation method) would improve islet yield and function from rat pancreases preserved for 6 and 24 hr. MATERIALS AND METHODS: Islets were isolated from Lewis rats. Pancreases were classified into five groups: fresh (group 1); preserved for 6 hr in University of Wisconsin solution without and with ductal preservation (groups 2 and 3); and preserved for 24 hr in University of Wisconsin solution without and with ductal preservation (groups 4 and 5). We assessed islet yield, function, and viability of pancreatic ductal cells. RESULTS: Islet yields per pancreas in groups 1 to 5 were 2010+/-774, 674+/-450, 1418+/-528, 527+/-263, and 1655+/-618 (islet equivalent) (+/-SD), respectively. Stimulation indices in groups 1 to 5 were 11.97+/-3.17, 6.48+/-4.04, 12.44+/-5.65, 2.56+/-2.03, and 5.55+/-2.71. Functional success rates in groups 1 to 5 were 100%, 0%, 100%, 0%, and 66.7%. Percentages of nonviable pancreatic duct cells in groups 1 to 5 were 3.8+/-2.7%, 59.7+/-4.4%, 19.5+/-7.3%, 64.7+/-4.5%, and 17.2+/-2.6%. In all experiments, the differences were significant between the groups without versus the groups with ductal preservation (P<0.05, group 2 vs. group 3 and group 4 vs. group 5). CONCLUSIONS: Ductal preservation improved islet yield and function after 6 and 24 hr of preservation. Well-preserved pancreatic ducts maintained good distribution of collagenase solution.     

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.     

Experimental pancreatic preservation prior to islet isolation and transplantation

Preservation of the Lewis rat pancreas prior to islet isolation was accomplished by initial intraductal distension with the University of Wisconsin (UW) hydroxyethyl starch-lactobionate solution to which collagenase had been added, followed by simple cold storage at 4 degrees C for 0, 3, 12, 24, and 48 hr (n = 16-21 at each interval). The pancreases were then processed by digestion and mechanical dispersion to produce free islets of Langerhans. The mean islet yields (+/- standard errors of the means) were controls = 819 +/- 58 (n = 21), 3 hr = 867 +/- 51 (n = 20), 12 hr = 770 +/- 71 (n = 16), 24 hr = 805 +/- 62 (n = 18), and 48 hr = 722 +/- 55 (n = 16). None of these means differed significantly. The islets from pairs of donor pancreases (mean dose of islets = 1586 +/- 72) were transplanted intraportally into single isogeneic recipients with streptozotocin-induced diabetes (plasma glucose greater than 400). The preservation interval directly influenced the outcome of these islet isografts in the following manner: (i) Rates of functional success (nonfasting glucose less than 200 mg/dl) were 100% after storage times of 0 hr (n = 10), 3 hr (n = 8), and 12 hr (n = 8); 86% with a storage time of 24 hr (n = 7); and 0% after 48 hr (n = 8). (ii) Return of euglycemia was increasingly delayed with increasing preservation intervals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preservation of pancreas in the University of Wisconsin solution supplemented with AP39 reduces reactive oxygen species production and improves islet graft function

Ischemia-reperfusion injury (IRI) results in increased rates of delayed graft function and early graft loss. It has recently been reported that hydrogen sulfide (H₂S) protects organ grafts against prolonged IRI. Here, we investigated whether the preservation of pancreas in University of Wisconsin (UW) solution supplemented with AP39, which is a mitochondrial-targeted H₂S donor, protected pancreatic islets against IRI and improved islet function. Porcine pancreata were preserved in the UW solution with AP39 (UW + AP39) or the vehicle (UW) for 18 h, followed by islet isolation. The islet yields before and after purification were significantly higher in the UW + AP39 group than in the UW group. The islets isolated from the pancreas preserved in UW + AP39 exhibited significantly decreased levels of reactive oxygen species (ROS) production and a significantly increased mitochondrial membrane potential as compared to the islets isolated from the pancreas preserved in the vehicle. We found that the pancreas preserved in UW + AP39 improved the outcome of islet transplantation in streptozotocin-induced diabetic mice. These results suggest that the preservation of pancreas in UW + AP39 protects the islet grafts against IRI and could thus serve as a novel clinical strategy for improving islet transplantation outcomes.     

Two-layer method in short-term pancreas preservation for successful islet isolation

BACKGROUND: We sought to determine whether the two-layer method (TLM) offers advantages over UW storage solution for locally procured pancreata with cold ischemia time of <8 hours for successful islet isolation. METHODS: From October 2003 through February 2005, 22 human pancreata were procured locally from cadaveric donors and preserved using UW solution (n = 11) or TLM (n = 11). RESULTS: Donor characteristics were similar in the two groups, with no statistical difference. Cold ischemia time was 4.5 +/- 0.6 (2.5 to 8) hours in the UW and 5.1 +/- 0.5 (3 to 8) hours in TLM group (P > .05). Organs preserved with TLM were exposed to PFC for 4 +/- 0.5 (2 to 7.5) hours. After TLM preservation, 8 of 11 (72%) pancreata yielded >300,000 IEQ pancreatic islets, which met all criteria for clinical transplantation; after UW cold storage, only 3 of 11 isolations were equally successful (27%) (P < .05). Mean IEQ was higher in the TLM than in the UW group: 349,000 +/- 37,000 vs 277,800 +/- 34,000; IEQ/g was also higher at 5100 +/- 760 vs 3000 +/- 570, respectively (P < .05). Islet quality, characterized by purity, viability, and insulin SI, did not differ statistically in the two groups: 67 +/- 4 vs 74 +/- 4%, 87 +/- 2 vs 83 +/- 4%, and 4 +/- 0.7 vs 4.8 +/- 1, respectively (P > .05). CONCLUSIONS: The Two Layer Method for locally procured human pancreata with cold ischemia time lower than 8 hours offers significant advantage over UW cold storage increasing the pancreatic islet isolation yield and the isolation success rate.     

Influence of strain and age differences on the yields of porcine islet isolation: extremely high islet yields from SPF CMS miniature pigs

BACKGROUND: Porcine pancreas is a potential source of material for islet xenotransplantation. However, the difficulty in isolating islets, because of their fragility and the variability of isolation outcome in donor age and breed, represents a major obstacle to porcine islet xenotransplantation. In this study, we compared the islet isolation yield of specific pathogen-free (SPF) Chicago Medical School (CMS) miniature pigs with that of another miniature pig breed and market pigs from a local slaughterhouse. METHODS: Nine adult CMS miniature (ACM) pigs (>12 months), six young CMS miniature (YCM) pigs (6-7 months), four adult Prestige World Genetics (PWG) miniature (APM) pigs (>12 months), and 13 adult market (AM) pigs from a local slaughterhouse were used for islet isolation. RESULTS: The islet yield per gram of pancreas from ACM pigs (9589 +/- 2823 IEQ/g) was significantly higher than that from APM pigs (1752 +/- 874 IEQ/g, P < 0.05), AM pigs (1931 +/- 947 IEQ/g, P < 0.05), or YCM pigs (3460 +/- 1985 IEQ/g, P < 0.05). Isolated islets from ACM pigs were significantly larger than those from AM pigs or YCM pigs. The in vitro and in vivo function of isolated islets showed no difference among experimental groups. The pancreases of ACM pigs contained higher mean islet volume density percentages and larger size of islets than those of AM or APM pigs. CONCLUSIONS: We isolated extremely high yields of well-functioning islets from ACM pigs bred under SPF conditions. SPF CMS miniature pigs should be one of the best porcine islet donors for clinical porcine islet xenotransplantation.     

新型胰岛分离液对小鼠胰岛分离效果的研究

目的  探讨新型胰岛分离液（IPS液）在小鼠胰岛分离中的分离效率及胰岛保护作用。方法  将消化后的小鼠胰腺按体积平均分为两组（IPS组和UW组）,分别应用IPS-Optiprep液及UW-Optiprep液进行连续梯度密度离心分离胰岛,比较两组分离液的分离纯化效率和分离后的胰岛活性。将诱导成功的糖尿病小鼠随机分为3组：实验组（n=10）,接受采用IPS-Optiprep液分离纯化的胰岛移植;对照组（n=10）,接受采用UW-Optiprep液分离纯化的胰岛移植;假性移植组（n=5）,仅给予手术但并不进行胰岛移植。分析3组小鼠术后血糖水平以及测实验组和对照组小鼠术后21 d的腹腔葡萄糖耐量试验的血糖水平。比较两种分离液的配制成本。结果  与UW组相比,IPS组的IPS-Optiprep液可提供更高的胰岛当量（IEQ）、胰岛纯度、回收率及胰岛完整度。胰岛形态观察可见,IPS组胰岛被膜完整,直径明显大于UW组。UW组纯化后的胰岛活性率高于IPS组[（88±5）%比（84±3）%,P < 0.01]。与UW-Optiprep液相比,IPS-Optiprep液可获得相当的在体胰岛功能。IPS-Optiprep液可显著降低胰岛分离纯化成本。结论  新型胰岛分离液IPS-Optiprep液在胰岛分离提纯中显示出较好的分离效率,增加了胰岛的纯度、完整度与回收率,并显著降低了纯化成本,但对胰岛细胞活性的保护作用稍逊,可能与胰岛的高完整度及IPS-Optiprep液中的内毒素有关。     

Cold-storage preservation of the canine and rat pancreas prior to islet isolation

Cold-storage preservation of the canine pancreas prior to islet isolation has previously been noted to reduce the intrasplenic islet autograft success rate; but the mechanism of this deleterious effect has not been determined. We undertook a study in both outbred dogs and Lewis (RT1-1) rats to determine the influence of cold-storage preservation interval, preservation solution, and flushing technique on islet yield and islet viability. The preservation solutions used were those that had proved most efficacious in preserving segmental canine pancreases--namely, the modifications of silica gel fractionated plasma (SGF-III and SGF-IV) and an hydroxyethylstarch/lactobionate solution (UW-1). In the first set of experiments, the traditional vascular flush was used; this was followed by storage at 4 degrees C. After brief periods of preservation (3 hr in the rat, 12 hr in the dog) there was a significant (P less than 0.006) reduction in islet yield. The reduced yields were similar with each solution tested, were made worse with increasing intervals of storage, and resulted in a significant reduction in autograft success rate. The second set of experiments examined the effect of using an intraductal flush prior to preservation, along with the effect of adding collagenase to the preservation fluid. Islet yields were maintained at control values in both animal models using preservation intervals of up to 24 hr. These islet yields produced auto- or isograft success rates similar to those obtained by transplanting freshly obtained tissue; verifying adequate islet viability. We recommend that a pre-storage ductal flush technique be used for cold-storage preservation of the pancreas prior to islet isolation and transplantation.     

Novel cell‐permeable p38‐MAPK inhibitor efficiently prevents porcine islet apoptosis and improves islet graft function

During islet transplantation, mitogen‐activated protein kinase (MAPK) p38 is preferentially activated in response to the isolation of islets and the associated inflammation. Although therapeutic effects of p38 inhibitors are expected, the clinical application of small‐molecule inhibitors of p38 is not recommended because of their serious adverse effects on the liver and central nervous system. Here we designed peptides to inhibit p38, which were derived from the sites on p38 that mediate binding to proteins such as MAPK kinases. Peptide 11R‐p38I₁₁₀ significantly inhibited the activation of p38. To evaluate the effects of 11R‐p38I₁₁₀, porcine islets were incubated with 10 µmol/L 11R‐p38I₁₁₀ or a mutant form designated 11R‐mp38I₁₁₀. After islet transplantation, blood glucose levels reached the normoglycemic range in 58.3% and 0% of diabetic mice treated with 11R‐p38I₁₁₀ or 11R‐mp38I₁₁₀, respectively. These data suggest that 11R‐p38I₁₁₀ inhibited islet apoptosis and improved islet function. Peptide p38I₁₁₀ is a noncompetitive inhibitor of ATP and targets a unique docking site. Therefore, 11R‐p38I₁₁₀ specifically inhibits p38 activation, which may avoid the adverse effects that have discouraged the clinical use of small‐molecule inhibitors of p38. Moreover, our methodology to design “peptide inhibitors” could be used to design other inhibitors derived from the binding sites of proteins.     

胰导管保护法提高大鼠胰岛获取量和功能的研究

目的 探讨胰导管保护法(即在胰腺获取时向胰导管内注射少量含胰酶抑制剂的UW液,TIUW液)能否提高冷保存后的大鼠胰腺的胰岛获取量和功能.方法 大鼠胰腺分为5组.组1:新鲜胰腺(n=10);组2:未应用胰导管保护法仅用TIUW液保存6 h的胰腺(n=10);组3:应用胰导管保护法且用TIUW液保存6 h的胰腺(n=10);组4:未应用胰导管保护法仅用TIUW液保存24 h的胰腺(n=10);组5:应用胰导管保护法且用TIUW液保存24 h的胰腺(n=10);双硫腙染色鉴定胰岛形态并计数,锥虫蓝染色评估胰岛死亡率,葡萄糖刺激试验评价胰岛离体功能,糖尿病裸鼠胰岛移植评价胰岛在体功能.结果 分离纯化后胰岛获取量分别为590±127、272±50、454±65、253±56、447±66(胰岛当量±标准差);胰岛死亡率分别为(5.7±4.2)%、(18.3±6.5)%、(11.7±4.2)%、(26.3±5.6)%、(15.0±5.3)%;刺激指数分别为7.32±2.32、4.81±1.17、7.56±2.44、2.88±1.00、5.71±1.90;糖尿病治愈率分别为100%、0%、100%、0%、70%.所有结果均显示差异有统计学意义(P＜0.05,组2与组3和组4与组5).结论 胰导管保护法提高了冷保存后大鼠胰腺的胰岛获取量和功能.