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.     

Testing combinations of protease inhibitor and preservation solution to improve islet quality and yield

Pancreas preservation using an oxygenated two-layer method (TLM) has been reported to improve islet yields, as has supplementation of Liberase with Pefabloc. We hypothesized that using both TLM and Pefabloc could enhance islet yield as compared with preservation in University of Wisconsin (UW) or Histidine-Tryptophan Ketoglutarate (HTK) solution. METHODS: Ninety-eight pancreata with no significant differences of age, body mass index, or cold ischemia time preserved randomly with UW (n = 40), TLM (n = 48), or HTK (n = 10) were processed with (n = 36) or without (n = 66) Pefabloc. RESULTS: The total islet equivalent (IEQ) from TLM-preserved pancreata processed with Pefabloc (n = 12) showed lower yields versus those processed without Pefabloc (n = 36): 216,120 +/- 27,906 vs. 301,427 +/- 21,447 IEQ (P < .05). Islets from 1 of 12 (8.33%) pancreata processed with Pefabloc in TLM were transplanted, in contrast with 15/36 TLM (41.67%) pancreata processed without it. Islet yields were not significantly different among pancreata preserved in UW and processed with Pefabloc (n = 17) versus without Pefabloc (n = 23): 342,693 +/- 45,588 versus 266,609 +/- 29,006 IEQ (P = .149). The number of transplants from UW-preserved pancreata was 3/17 (17.65%) when processed with Pefabloc and 4/23 (17.39%) without. Among the HTK group, there was no significant difference in islet yields between pancreata processed with (n = 7) versus without Pefabloc (n = 3): 248,227 +/- 65,294 versus 483,555 +/- 144,070 IEQ (P = .118). CONCLUSIONS: Pefabloc showed no benefit to improve islet yields. Pancreata preserved in TLM provided better transplant quality islets when processed in the absence of Pefabloc.     

Superiority of the two-layer method before islet isolation confirmed by in vivo viability assessment

BACKGROUND: Although the outcome of islet transplantation has improved, there remains a major obstacle in isolating viable islets from prolonged preserved pancreas. We previously reported that the two-layer cold storage method (TLM) improved the yield and in vitro function. In this study, we performed in vivo accurate functional analyses of islets from TLM-preserved pancreas and investigated pancreatic duct cell viability, which may critically affect islet isolation. METHODS: Rat islets isolated from fresh pancreas (group 1), after preservation in the University of Wisconsin (UW) solution (group 2) or by the TLM (group 3), were examined by assessing islet yields, stimulation indices, cure rates after transplantation to diabetic nude mice, and trypan blue uptake of pancreatic duct cells. RESULTS: TLM significantly improved the islet yield compared with UW cold storage. The cure rates after transplantation were 100%, 0%, and 80% for groups 1, 2, and 3, respectively. This indicates that islet viability was well maintained even after 24 hr of TLM preservation. The percentages of nonviable duct cells were 4.1%+/-1.9%, 48.3%+/-8.0%, and 26.1%+/-21.4% in groups 1, 2, and 3, respectively, showing that the TLM was superior to UW as seen by this duct cell viability assessment. CONCLUSIONS: The TLM used for pancreas preservation before islet isolation results in excellent islet function in addition to improved islet yield comparable to freshly isolated islets. The underlying mechanism may be duct cell viability maintained during TLM preservation. Therefore the TLM is an excellent preservation technique for isolating sufficient numbers of highly viable islets.     

Successful pancreas preservation by a perfluorocarbon-based one-layer method for subsequent pig islet isolation

BACKGROUND: The oxygenation of human pancreas by the two-layer method (TLM) during cold storage was recently established for clinical islet transplantation. Simplification of TLM would facilitate the application of perfluorocarbon (PFC) as a regularly used preservation solution for subsequent islet transplantation. The present study examined whether PFC can be used in a one-layer method (OLM) for long-term pancreas preservation before isolation of adult pig islets. METHODS: Resected pancreases were intraductally flushed with cold University of Wisconsin solution and immediately processed (n=6) or subjected to 7-hour storage by OLM (n=8) or TLM (n=10). Subsequently, pancreases were intraductally distended with collagenase NB-8 supplemented with neutral protease. Isolation and purification were performed as previously described. RESULTS: Compared with unstored pancreases (3,670+/-740 islet equivalents [IEQ]) purified islet yield in TLM-stored organs (2,080+/-290 IEQ, P<0.05) was significantly decreased in contrast with OLM-preserved pancreases (3,110+/-520 IEQ, NS). No differences were observed between groups regarding purity (>90%), trypan-blue exclusion (>95%), adenosine triphosphate content, and mitochondrial viability of islets. Stimulation index during static glucose incubation (20 vs. 2.8 mm) was decreased after storage by TLM (1.81+/-0.20, P<0.05) but not by OLM (2.27+/-0.57) if compared with unstored pancreases (2.47+/-0.36). However, transplantation into diabetic nude mice resulted in sustained normoglycemia of recipients of either group until nephrectomy of graft-bearing kidneys was performed. CONCLUSIONS: This study demonstrates that PFC alone can be used in a one-layer procedure for successful pig-pancreas preservation. This simplification can facilitate the broad application of PFC as pancreas preservation solution without reducing its benefits demonstrated by TLM.     

Adaption of neutral protease activity for islet isolation from the long-term two-layer method-stored pig pancreas

BACKGROUND: Islet release from the pancreas is mediated by both collagenase and neutral protease (NP), a critical effector of islet integrity. To prove the hypothesis that adjustment of NP reduces islet damage after prolonged ischemia, adult pig pancreata were digested after 7-hour preservation by the two-layer method (TLM) using a 2-component enzyme blend consisting of collagenase NB-8 and NP. METHODS: After intraductal University of Wisconsin (UW) flush resected pancreata were distended with 4.4 PZ-U/g of UW-dissolved Serva collagenase either before (TLM-preloaded, n = 7) or after (TLM-postloaded, n = 10) cold storage, or for immediate processing (n = 6). NP was adjusted after preliminary experiments to respectively 1.1, 0.2, or 0.8 DMC-U/g for unstored, TLM-preloaded, or postloaded organs. RESULTS: Purified islet yield decreased from 3670 +/- 730 islet equivalents (IEQ)/g in unstored pancreata to 1800 +/- 180 and 2080 +/- 290 IEQ/g in TLM-preloaded or postloaded organs, respectively (P < .05). Although purity was always >90%, IEQ recovery was significantly decreased in TLM-preloaded pancreata. Quality control revealed consistently high viability as determined using trypan-blue exclusion (>95%) or formazan production. Compared with unstored organs (2.47 +/- 0.36; P < .05), glucose stimulation index was reduced in TLM-preloaded (1.48 +/- 0.15) and TLM-postloaded pancreata (1.81 +/- 0.20). Normoglycemia in diabetic nude mice transplanted with islets from TLM-preloaded pancreata was transient in contrast to sustained function in the other groups. CONCLUSIONS: Significant amounts of viable pig islets can be isolated after prolonged TLM preservation by reducing NP activity. Nevertheless, early enzyme administration prior to long-term storage deteriorates islet graft function.     

Improved Islet Yields From Macaca Nemestrina and Marginal Human Pancreata After Two-Layer Method Preservation and Endogenous Trypsin Inhibition

We tested whether two-layer method (TLM) pancreas preservation and trypsin inhibition (Pefabloc) during processing allows longer preservation while retaining or improving viable islet recovery. Non-marginal primate (Macaca nemestrina) and marginal human (ischemic or preservation-injured) pancreata were processed with a research-oriented pan technique (Seattle method). Organs were processed upon arrival (+/- Pefabloc), or after TLM or University of Wisconsin solution (UW) preservation (+ Pefabloc). Islet yield, viability, and function were assessed. Pefabloc increased M. nemestrina islet yields from 9696 +/- 1749 IE/g to 15 822 +/- 1332 IE/g (p < 0.01). Two-layer method preservation (< 6 h) further increased yields, to 23 769 +/- 2773 IE/g (vs. + Pefabloc; p < 0.01). Similarly, Pefabloc increased marginal human islet yields from 2473 +/- 472 IE/g to 4723 +/- 1006 IE/g (p < 0.04). This increase was maintained after lengthy TLM preservation (> 30 h; 4801 +/- 1066 IE/g). We also tested the applicability of TLM preservation (23.5 +/- 3.2 h) to the processing of marginal human pancreata by the Edmonton/Immune Tolerance Network clinical protocol. Islet yield and function approached published results of pancreata processed 4.8 +/- 0.8 h after organ recovery (p = 0.06). Pefabloc, and TLM vs. UW preservation, prolonged the tolerable interval between organ recovery and islet isolation. Islet yield, viability, and functionality improved from both marginal and nonmarginal pancreata.     

Improved pancreatic islet isolation outcome in autologous transplantation for chronic pancreatitis

Total or partial pancreatectomy followed by autologous islet transplantation is a therapeutic option for the treatment of refractory chronic pancreatitis (CP). Maximization of islet yields from fibrotic and inflamed organs is crucial for prevention of posttransplant diabetes. We adapted technical advancements developed for islet allotransplantation toward islet autotransplantation. Eight patients (two men, six women; ages 24-58 years) underwent total (n = 7) or partial (n = 1) pancreatectomy for the treatment of CP refractory to maximal medical management. Pancreata were preserved in UW solution (UW group) in initial three cases and the last five pancreata were preserved with pancreatic ductal injection followed by ET-Kyoto/oxygenated PFC solutions (DI+TLM group). Islets were isolated by modified Ricordi method and were purified only in one case. All islet infusions were performed under general anesthesia via direct vein injection into the portal venous system with pressure monitoring. Total islet yields (129,314 ± 51,627 vs. 572,841 ± 116,934 IEQ, p < 0.04), islet yield/pancreas weight (1,233 ± 359 vs. 6,848 ± 847 IEQ/g, p < 0.003), and islet yield/patient body weight (1,951 ± 762 vs. 7,305 ± 1,531 IEQ/kg, p < 0.05) were significantly higher in the DI+TLM group when compared to the UW group. Pellet size was also higher (5.3 ± 0.3 vs. 13.5 ± 3.4 ml) in the DI+TLM group, suggesting that this method of preservation effectively protected pancreatic tissue against autolysis. First month posttransplant basal C-peptide and the secretory unit of islet transplant objects (SUITO) index were also higher in the DI+TLM group when compared to the UW group (2.0 ± 0.3 vs. 1.4 ± 0.4 ng/ml and 42.6 ± 12.7 vs. 14.6 ± 5.6, respectively). There were no technical complications related to the infusion. Our results suggest that higher islet yields can be achieved even from chronically inflamed and fibrotic organs using DI+TLM. The techniques applied for islet isolations from normal pancreata are showing promise for fibrotic pancreata from CP patients.     

Effect of the two-layer (University of Wisconsin solution-perfluorochemical plus O2) method of pancreas preservation on human islet isolation,as assessed by the Edmonton Isolation Protocol

BACKGROUND: Current techniques for isolating islets require that pancreata stored with University of Wisconsin solution (UW) are processed within 12 hours of cold storage. In this study, we hypothesized that the two-layer method (TLM) could extend the acceptable preservation period of pancreata before islet isolation and increase islet yields. METHODS: In the first experimental set, eight pancreata were maintained for an average of 8.3+/-1.2 hours in UW and transferred into the TLM for an additional 14.3+/-1.1 hours for a total cold ischemic period of 22.6+/-1.6 hours (prolonged TLM). Four pancreata were maintained as a control group in UW alone for a total of 21.3+/-2.0 hours. In the second experimental set, six pancreata were maintained for an average of 6.4+/-1.8 hours in UW followed by 4.8+/-0.8 hours with the TLM for a total preservation time of 11.3+/-2.5 hours (short TLM). The control organs for the short TLM group were stored for an average of 9.5+/-1.3 hours in UW alone. Islets were isolated and evaluated according to the Edmonton protocol. RESULTS: Between each group of the two experimental sets, there was no significant difference in donor-related factors (i.e. gender, age, body mass index [BMI], etc.). The TLM as compared with UW preservation resulted in a significant increase in islet yields postpurification for both short (3,353+/-394 islet equivalents [IE] vs. 2,027+/-415 IE; mean+/-SEM) and prolonged (2,404+/-503 IE vs. 514+/-180 IE) periods of storage. Furthermore, islet yields after prolonged storage with the TLM were not significantly different from organs maintained for only a short period with UW (P=0.17). The quality of islets as assessed by size, postculture viability, survival rates, insulin content, and insulin secretion were similar for each of the four groups. CONCLUSION: In comparison with UW organ preservation, exposure of pancreata to the TLM result in greater islet yields and extended preservation times.     

Efficacy of the oxygen-charged static two-layer method for short-term pancreas preservation and islet isolation from nonhuman primate and human pancreata

Previous reports indicate that the two-layer method (TLM) of human pancreas preservation is superior to University of Wisconsin solution (UW) when pancreata are preserved for extended periods (i.e., >24 h) prior to islet isolation. In this study, the efficacy of using the TLM for preserving pancreata for short periods (i.e., <13 h) was evaluated using both nonhuman primate and human pancreata preserved with a TLM kit precharged with oxygen. An oxygen precharged TLM (static TLM) was established and compared with the original TLM with continuous oxygen supply. For the static TLM, the perfluorochemical was fully oxygenated and the oxygen supply removed prior to pancreas preservation. In the primate model, pancreata were preserved by the static TLM, the original TLM, and UW for 5 h prior to islet isolation. In the human model, pancreata were preserved with the static TLM or the original TLM or UW for 4-13 h. Both primate and human pancreata were processed by intraductal collagenase injection and digestion followed by continuous density gradient purification to isolate islets. Islets were assessed for islet yield, purity, viability, and in vitro functionality. In the primate model, islet yield, viability, and in vitro functionality were significantly improved by both the static TLM and the original TLM with similar results. Postculture islet yields were 23,877 +/- 3619 IE/g in the static TLM, 21,895 +/- 3742 IE/g in the original TLM, and 6773 +/- 735 IE/g in UW. In the human model, both the static TLM and the original TLM significantly increased islet yield compared with UW with postculture islet yields of 2659 +/- 549 IE/g in the static TLM, 2244 +/- 557 IE/g in the original TLM, and 1293 +/- 451 IE/g in UW. Nonhuman primate and human pancreata stored in the static TLM, immediately upon procurement, yield isolated islets of a substantially higher quantity than when pancreata are stored in UW. Thus, the use of the static TLM should replace the use of UW for storage of pancreata during transport prior to islet isolation.     

The effect of two-layer (University of Wisconsin solution/perfluorochemical) preservation method on clinical grade pancreata prior to islet isolation and transplantation

BACKGROUND: Research-grade pancreata preserved by the two-layer method (TLM) compared to organs stored with University of Wisconsin (UW) solution prior to islet isolation result in significantly better islet yields. However, it is unknown whether the TLM improves islet yields from pancreata that meet the criteria for the selection of clinical-grade organs. METHODS: Six clinical-grade pancreata were preserved for 4.8 +/- 0.5 hour in UW and three clinical-grade pancreata were preserved by the TLM for 11.7 +/- 2.0 hour. The local team procured all pancreata. All donors were hemodynamically stable without norepinephrine usage and length of hospitalization was less than 96 hour. Causes of death were either head trauma or cerebrovascular accident. Islets were isolated and evaluated according to the Edmonton protocol. RESULTS: The TLM as compared to UW resulted in a significant increase in islet yields (3415 +/- 227 vs 2006 +/- 337 IE/g pancreas, P <.03). The quality of islets as assessed by visual score was significantly better in the TLM group (8.7 +/- 0.2 vs 7.3 +/- 0.3, P <.02) but other parameters (viability, survival rate after culture, insulin content, stimulation index) were similar between the two groups. We transplanted all three islet preparations in the TLM group but only two of six preparations from the UW group. CONCLUSION: Compared to UW, exposure of pancreata to the TLM resulted in greater islet yields and extended preservation times with clinical grade pancreas. Pancreata should be preserved by the TLM prior to islet isolation even for donors that meet clinical grade organ selection criteria. The Human Islet Transplantation in Seattle (HITS) Consortium is supported in part by a grant from the Juvenile Diabetes Research Foundation International. The HITS consortium is an islet transplant program involving the University of Washington, Pacific Northwest Research Institute, the Puget Sound Blood Center, Fred Hutchinson Cancer Research Center, Swedish Hospital, and the Virginia Mason Research Center.     

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.     

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.     

Resuscitation 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. This study determined the effect of additional preservation of ischemically damaged human pancreata by the TLM before islet isolation. Human pancreata were procured from cadaveric organ donors and preserved by the TLM for 3.2 +/- 0.5 hours (mean +/- SEM) at 4 degrees C after 11.1 +/- 0.9 hours of cold storage in University of Wisconsin solution (UW) (TLM group), or by cold UW alone for 11.0 +/- 0.3 hours (UW group). Islet isolations of all pancreata were performed using the Edmonton protocol. Islet recovery and in vitro function of isolated islets were significantly increased in the TLM group compared with the UW group. In the metabolic assessment of human pancreata, ATP levels were significantly increased after the TLM preservation. This study showed that additional short-term preservation by the TLM resuscitates the viability of ischemically damaged human pancreata before islet isolation, leading to improvements in islet recovery and in vitro function of isolated islets.     

Influence of collagenase loading on long-term preservation of pig pancreas by the two-layer method for subsequent islet isolation

BACKGROUND: The introduction of the two-layer method (TLM) for long-term human pancreas preservation revealed the enormous potential of TLM to improve graft function of isolated islets. It is still unclear whether pig islets can be successfully isolated from pancreases after prolonged cold ischemia. To clarify this question, pig pancreases were subjected to 7-hour preservation by University of Wisconsin solution (UWS) storage or TLM. Another aim was to verify whether TLM can be synergistically combined with intraductal collagenase injection before cold storage. METHODS: After intraductal flush with UWS, organs were distended with 4.4 PZ-U/g of UWS-dissolved collagenase NB-8 and neutral protease adjusted to respectively 1.1, 0.2, 0.5, or 0.8 DMC-U/g for pancreases freshly procured (n=6) or distended with enzymes before (TLM preloaded, n=7) or after cold storage (UWS storage, n=4; TLM postloaded, n=10). RESULTS: Purified islet yield decreased from 429,200+/-86,700 islet equivalents (IEQ) in unstored pancreases to respectively 37,670+/-19620, 210,400+/-22900 and 238,000+/-26600 IEQ in UWS-stored (P<0.01), TLM-preloaded, or postloaded organs (P<0.05). Purity (>90%), viability (>95%), and insulin content were not different between groups. Islets from UWS-stored pancreases fragmented extensively, preventing further assessment of in vivo function. Compared with other experimental groups, islets from TLM-preloaded organs were characterized by enhanced basal and stimulated insulin release. Sustained normoglycemia was observed in diabetic nude mice transplanted with islets from TLM-postloaded or unstored pancreases in contrast with transient function in TLM-preloaded islets. CONCLUSIONS: This study demonstrates that significant amounts of intact pig islets can be isolated after prolonged pancreas preservation by TLM. Enzyme administration before TLM preservation decreases islet graft function.     

A comparison of cold storage solutions for pancreas preservation prior to islet isolation

BACKGROUND: Several solutions are used to preserve the pancreas prior to islet isolation. This study sought to assess whether the type of solution had an impact on the isolation outcome. METHODS: We reviewed data from 125 islet isolation procedures performed from January 2002 to January 2005. Pancreata were preserved in University of Wisconsin (UW) (n = 101), Celsior (CS) (n = 19), or IGL-1 (n = 5) solutions. Islet isolation results and transplantation rates were compared between groups. RESULTS: UW, CS, and IGL-1 groups were similar according to donor's age, weight, and body mass index. Weight of undigested pancreas was 20 +/- 13.1, 21.4 +/- 15.7, and 17.4 +/- 8.7 g for UW, CS, and IGL-1, respectively (P > .2). Final total number of IEQ was 267,000 +/- 132,000, 277,000 +/- 155,000, and 311,000 +/- 163,000, respectively (P > .4). Success rate (defined as >250,000 IEQ) was 55.5%, 52.9%, and 60% for UW, Celsior, and IGL-1 (P > .9); the transplantation rate was 42.2% for UW, 36.8% for Celsior, and 80% for IGL-1 preservation (P > .2). CONCLUSIONS: In this preliminary study, UW, Celsior, and IGL-1 solutions demonstrated similar islet isolation results. The new IGL-1 solution appears promising.     

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.     

A meta-analysis for comparison of the two-layer and university of Wisconsin pancreas preservation methods in islet transplantation

Conflicting results have been reported on the effectiveness of the two-layer method (TLM) compared with the University of Wisconsin (UW) method for preserving pancreata. The objective of this study was to compile the evidence for or against any difference in human islet yield and viability between these two. PubMed (January 2000 to May 2008) and Cochran Library searches were performed and 17 studies were included for the meta-analysis. Data on donor characteristics, preservation time, and outcomes were abstracted. Studies were subgrouped based on how TLM was used (UW + TLM or TLM alone), on mean cold ischemic time (CIT) (>20 h or <20 h), and on whether special chemical was used (yes or no). Meta-analysis of all studies and subgroups was performed and the pooled standardized mean differences (SMD) with 95% confidence intervals (CI) were reported. Overall, the use of TLM significantly increased islet yield [SMD, 0.74 (0.44-1.04)] and viability [SMD, 0.63 (0.14-1.12)]. The beneficial effects of TLM on islet yield were more evident when TLM was used following UW storage or when prolonged CIT was used. TLM used alone, shorter CIT, and no chemical use all resulted in similar islet viability between TLM and UW groups. Beneficial effects of TLM on islet viability were demonstrated only when TLM was used following UW storage, or with prolonged CIT, or with chemical use. In conclusion, the TLM was beneficial for prolonged pancreas preservation before human islet isolation; however, benefit of the TLM for short-term preservation was not clear.     

Pancreas storage in oxygenated perfluorodecalin does not restore post-transplant function of isolated pig islets pre-damaged by warm ischemia

BACKGROUND: Cold storage in oxygenated perfluorodecalin (PFD) restores transplant function of ischemically damaged dog pancreata and reduces the impact of cold ischemia on recovery of isolated human islets. Whether PFD storage can improve islet isolation from pancreata exposed to significant warm ischemia (WI) is unclear yet. The present study aimed to clarify this question in adult pigs. METHODS: After exsanguination, the intestine was removed immediately or left in the cavity for 30 min of WI. Resected pancreata were intraductally flushed with cold University of Wisconsin solution. Subsequently, pancreata were processed immediately by digestion-filtration (group I: 0 min WI, n=6; II: 30 min WI, n=6) or first stored for 3 h in oxygenated PFD (III: 0 min WI+PFD, n=5; IV: 30 min WI+PFD, n=6). RESULTS: Pancreata subjected to 30 min of WI yielded significantly less islets compared with the corresponding non-ischemic organs (I vs. II, P<0.01; III vs. IV, P<0.05). Oxygenation did not ameliorate the loss in islet yield (II vs. IV, NS). Ischemic islets were characterized by depleted ATP stores (388+/-73 (I) vs. 133+/-22 ng/1000 IEQ (II), P<0.01) and diminished insulin response to glucose calculated as stimulation index (SI; 2.47+/-0.36 (I) vs. 0.25+/-0.17 (II), P<0.05). PFD storage of ischemic organs partially restored ATP content (217+/-23 ng/1000 IEQ, II vs. IV, P<0.05) and glucose SI (1.60+/-0.09, II vs. IV, P<0.05) to a significant extent that reached the level of corresponding PFD-stored, non-ischemic pancreata (III vs. IV, NS). Sustained normoglycemia was exclusively observed in diabetic nude mice transplanted with islets isolated from non-ischemic organs. The significantly reduced graft function of ischemic islets (I vs. II, III vs. IV, P<0.001) was not increased by pancreatic oxygenation (II vs. IV, NS). CONCLUSIONS: The present study suggests that pancreas short-term storage in oxygenated PFD improves in vitro but not the in vivo function of ischemically damaged pig islets.     

Pancreas preservation by the 2-layer cold storage method before islet isolation protects isolated islets against apoptosis through the mitochondrial pathway

BACKGROUND: Apoptosis in isolated islets has been implicated in primary nonfunction or early graft failure after islet transplantation. Recently, pancreas preservation by the 2-layer method (TLM) before islet isolation has been proved to improve the islet yield, quality, and transplant results not only in experimental models, but also in clinical settings. We examined the influence of TLM on apoptosis of isolated islets. METHOD: Rat islets freshly isolated and after pancreas preservation by TLM or conventional cold storage in University of Wisconsin solution (UW) were examined and compared. Islet apoptosis was assessed by TUNEL and annexin V assays. The apoptosis pathways involved were investigated by measurement of caspase 3, 8, and 9 activities and by immunoblotting for total and phosphorylated c-Jun NH2-terminal kinase (JNK) and p38. RESULTS: Islet apoptosis in the UW group was significantly increased compared with the fresh and TLM groups. Both caspase 3 and 9 activities in the UW group were higher than in the fresh and TLM groups with an approximate increase of 2- to 3-fold. On the other hand, there was no significant difference in caspase 8 activity among these 3 groups. JNKs were strongly activated both in the TLM and UW groups; although they were not activated in the fresh group, p38 was activated to almost the same levels in these 3 groups. CONCLUSIONS: Pancreas preservation by TLM before islet isolation protects isolated islets against apoptosis mainly through the mitochondrial pathway. Pancreas storage before islet isolation even with TLM triggers activation of JNKs in isolated islets.     

Long-term preservation of the pig pancreas by a one-layer method for successful islet isolation

Background

Pancreas preservation by two-layer method (TLM) was recently established for clinical islet transplantation. The extensive use of TLM would require enormous efforts to solve logistical and technical problems. Omitting University of Wisconsin solution (UW) as second layer would facilitate the regular application of oxygenated perfluorocarbon; (PFC). To clarify whether long-term pancreas preservation is feasible by this simplified procedure, pancreases from retired breeder pigs were subjected to 7-hour preservation utilizing PFC alone in a one-layer method (OLM, n = 8) or in combination with UW (TLM, n = 10).

Methods

Resected pancreata were intraductally flushed with cold UW. Subsequently, pancreata were promptly processed (n = 6) as previously described or stored by TLM or OLM.

Results

Compared to unstored (429200 ± 86700 IEQ) and OLM-stored pancreases (338600 ± 42100 IEQ), (P = ns vs unstored) postpurification islet yield decreased after TLM storage (238000 ± 26600 IEQ, P < .05). No significant differences were found regarding purity (>90%), adenosine triphosphate (ATP) content, and viability as determined by formazan production and trypan-blue exclusion (>95%). Glucose stimulation index of freshly isolated islets (2.5 ± 0.4) was significantly decreased after TLM storage (1.8 ± 0.2, P < .05) but not after OLM storage (2.3 ± 0.6). Islet transplantation in diabetic nude mice demonstrated sustained graft function in all experimental groups.

Conclusions

This study demonstrates that viable pig islets can be successfully isolated after prolonged ischemia utilizing PFC alone for oxygenation of cold-stored pig pancreases. The easy handling of OLM could facilitate the regular application of PFC as pancreas preservation solution.