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.     

Key factors for human islet isolation and clinical transplantation

BACKGROUND: To further improve the outcome of clinical islet transplantation analysis of the impact of donor- and process-related factors could be of great importance. MATERIALS AND METHODS: Thirty-eight consecutive clinical islet transplantations were performed with consecutive islet isolations. Univariate analysis for donor- and isolation-related variables were correlated with recipient C-peptide levels at 2 and 4 weeks after transplantation. "Warm ischemia time" was defined as the time from start of University of Wisconsin solution perfusion in the donor until the pancreas was removed to the back table. RESULTS: Short "warm ischemia time" (WIT), low expression of tissue factor (TF) in pancreatic tissue, and high creatinine levels in the donor were variables related to high C-peptide values after islet transplantation. Furthermore, hospitalization length longer than 4 days was associated with low C-peptide levels. The number of islet equivalents (IEQ) did not correlate with the clinical outcome, possibly due to the fact that IEQ number was included in the release criteria for clinical islet transplantation CONCLUSIONS: Successful clinical islet transplantation is strongly correlated with donor and pancreas procurement factors rather than isolation process-related variables. "WIT" may induce TF expression in the pancreatic tissues. TF has been identified as the main trigger of the instant blood-mediated-inflammatory reaction in clinical islet transplantation. Therefore, assay of TF expression in pancreatic tissues could be applied as useful screening tool to identify "good" pancreata for clinical transplantation.     

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.     

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.     

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.     

Assessment of a novel two-component enzyme preparation for human islet isolation and transplantation

BACKGROUND: Biologic characteristics of enzyme products used for islet isolation are critical for the success of islet transplantation. In particular, lot-to-lot variability significantly affects the yields of the isolation procedure. In this study, we have evaluated a new enzyme preparation in which highly purified collagenase can be blended with separately supplied neutral protease in a predetermined ratio. METHODS: We compared the results of human-islet isolations performed either with Collagenase NB1 supplemented with Neutral Protease NB (group I, n=9) or with Liberase (group II, n=9). RESULTS: Endotoxin contents of the enzyme preparations were lower in group I. Total islet yields were similar in both groups, but islet equivalents per gram of pancreas was higher in group I (4,020+/-1,240 vs. 2,360+/-1,350; P<0.05). Islet morphology was improved in group I with significantly higher proportion of free and intact islets (71+/-9% vs. 52+/-14%; P<0.01). In vitro function was improved and apoptosis rate was lower in group I. CONCLUSIONS: This new enzyme blend was as efficient as Liberase in terms of islet yields and showed improvements in islet morphology, viability, and in vitro function. The possibility to control lot-to-lot variability and the low endotoxin contents make Collagenase NB1 a promising product for human-islet isolation.     

Donor and isolation variables predicting human islet isolation success

BACKGROUND: Recent advances in the fields of islet transplantation and in vitro islet cell expansion place a renewed emphasis on the optimization of islet isolation from cadaveric human donor organs. We retrospectively analyzed 171 islet isolations to identify variables that predict islet yield and isolation success. METHODS: Cadaveric human donor pancreata were procured and processed according to established protocols. Donor-, procurement-, and isolation-related variables were analyzed for correlation with islet yield and isolation success (> or =250,000 islet equivalents). RESULTS: Univariate analysis suggested correlations between islet yield and donor age (P<0.005), body surface area (P<0.005), duration of enzymatic digestion (P<0.001), and pancreatic beta-cell volume (P<0.05). Donor sex (P<0.01), procurement team (P<0.05), and peridigestion serine protease inhibition (P<0.05) affected islet yield, whereas enzyme lot (P<0.01) and pancreatic fatty infiltration (P<0.05) influenced isolation success. By logistic regression, donor sex and age, and duration of enzymatic digestion could predict a successful isolation with 72% accuracy. The use of Liberase CI improved islet yield (P<0.05) in young donors (< or =25 years). CONCLUSIONS: While donor-related variables are useful in predicting islet yield, these are likely surrogates for pancreatic beta-cell volume. Enzyme lot, and the associated duration of enzymatic digestion (P<0.05), appears to be key determinants of isolation success.     

Effect of JNK inhibitor during islet isolation and transplantation

Although islet transplantation has been remarkably improved by the Edmonton protocol, the insulin independence rate after islet transplantation from one donor pancreas has remained low. The c-Jun NH2-terminal kinases (JNKs) are classic stress-activated protein kinases; many cellular stresses have been shown to stimulate JNK activation. JNK in the pancreas is activated during brain death, pancreas procurement, and organ preservation, and its activity is progressively increased during the isolation procedure. Moreover, JNK activity in the transplanted liver after islet transplantation increases markedly within 24 hours. In this study, we show the effect of a JNK inhibitor during islet isolation and transplantation. Use of the JNK inhibitor in pancreas preservation, islet culture, and/or islet transplantation prevents islet cell apoptosis and improves islet graft function. These findings suggest that inhibition of JNK could prevent the impairment of islet cells and improve outcomes after pancreatic islet transplantation.     

Evaluating the effect of serine proteases on collagenase activity during human islet isolation

Inconsistencies in human islet yields after collagenase digestion have been attributed to the activation of endogenous enzymes of the donor pancreas. It has been suggested that pancreatic serine proteases contribute to the proteolysis of collagenase. This study defined the effects of endogenous enzymes within the pancreas on pancreas dissociation during collagenase digestion. Levels of collagenase activity from samples taken throughout several steps in islet isolation procedures, both with and without the addition of the serine protease inhibitor Pefabloc, were determined by a spectrophotometric assay using N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala as the substrate. Results clearly demonstrated that the level of collagenase activity remains stable throughout the isolation procedure despite differences in the donor factors from several cadaveric donor pancreases. This was further demonstrated by observing no difference in activity levels after incubating commercial collagenase preparations with serine proteases and analyzing by means of collagenase activity and SDS-PAGE. These data show that the presence of serine proteases does not affect the level of collagenase activity; however, they likely damage the islet cells upon prolonged digestion of the pancreatic tissue. Further efforts at examining exogenous and endogenous enzyme levels may result in the development of an enzyme cocktail that is both stable and effective for digesting the human pancreas while preserving islet function and viability.     

Analysis of donor factors affecting human islet isolation with current isolation protocol

BACKGROUND: Appropriate donor selection is one of the keys for successful human islet isolation. Previous studies identified several critical donor factors; however, significant improvements in current human islet isolation protocols make reevaluation of donor factors necessary. STUDY DESIGN: Review was performed on 31 human islet isolations. Islet isolations were conducted using the standard automated islet isolation method with three protocol revisions that included the two-layer method (TLM) of pancreas preservation prior to islet isolation, usage of purified collagenase mixture Liberase, and continuous density gradient for islet purification. Factors leading to successful isolations (islet yield > 100,000 IE and static incubation stimulation index greater than 2.0) were analyzed. The impacts of various risk factors were also examined. RESULTS: Donors in the successful islet isolation group had a significantly lower incidence of elevated peak transaminases and creatinine levels, lower usage of norepinephrine or cardiac arrest, less prolonged hospitalization (> 96 hours), and less prolonged preservation time of donor pancreata (>25 hours). The TLM extended acceptable preservation time of donor pancreata from 8 to 25 hours. When donors had no risk factor, the success rate was 14/16 (87.5%). In sharp contrast, when donors had two or more risk factors, the success rate was 0/7 (0%; P <.001). CONCLUSION: Risk factors for human islet isolation with the current islet isolation protocol were identified. The decision to process pancreata based on review of donor factors should improve the consistency of human islet isolations and transplantation for curing type 1 diabetes.     

Pancreatic donor operation for islet cell isolation and transplantation

Preservation of B-cells, following organ collection and cold ischaemia, was defined on 19 donor organs by means of histological tests and testing for insulin content in pancreatic tissue. The degree of preservation declined by 50 per cent after two hours of cold ischaemia. The Velcro technique and uncrushed organ digestion were used for isolation. Islet yield was calculated by determination of insulin levels in isolated tissue (nmol/ml) and in the islet (pmol/islet). The average islet yield was 2 076 islets to one gram pancreas, that is ten per cent of the original amount. Studies conducted into in-vitro insulin secretion from isolated islets have revealed damage to organs due to handling, especially to organs which had undergone extended cold ischaemia.     

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)     

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.     

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.