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

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

Microbial surveillance during human pancreatic islet isolation

OBJECTIVES: The study aim was to investigate the microbiological safety of islet isolation and transplantation. MATERIALS AND METHODS: Between 1996 and 2002, prospective microbiological screening was performed on all pancreata procured for islet transplantation. Pancreas transport media and postpurification preparations were screened for microbiological contamination. Prior to isolation, pancreata were washed with either Hanks solution (group I, n = 170) or decontaminated with antiseptic and antimicrobial drugs (group II, n = 45). RESULTS: Microbiological contamination of the pancreas preservation media was shown in 62%. Analysis of the contaminants showed 74% gram-positive, 21% gram-negative organisms, and 5% fungi. The donor condition or procurement center did not influence the contamination rate. Longer pancreas transport duration was significantly associated with bacterial contamination (P <.05). In group I, 16 (9.4%) of 170 islet preparations presented microbial contamination at the end of the isolation procedures. Gram-positive organisms were present in 10 (6%), gram-negative organisms in 4 (2.4%), and fungi in 2 (1.2%) preparations. Four islet preparations (2.4%) from pancreata with noninfected transport medium were positive on postpurification cultures, all with gram-positive organisms. In group II, only 2 of 45 islet preparations (4.4%) presented microbial contamination at the end of the isolation process. CONCLUSIONS: The rate of microbial contamination during pancreas procurement and transport is high. Significant contaminants present when beginning islet isolation become undetectable by the conclusion of isolation. Diminishing the bio-burden by pancreas decontamination reduces the risk of contamination of the final islet preparation.     

Microbial surveillance during human pancreatic islet isolation

The aim of the study was to investigate microbiological contamination rate during human pancreatic islet isolation. Between 1996 and 2002, pancreas preservation media and post-purification islet preparations were screened for microbiological contamination. After arrival in the laboratory, pancreata were washed prior to enzyme perfusion with either Hank's balanced salt solution (Group I, n = 170, 1996 to 2001) or decontaminated with polyvidonum-iodine, cefazoline, and amphotericine B (Group II, n = 45, 2001 to 2002). Microbiological contamination of preservation media was observed in 56% and 84% for Groups I and II, respectively. Analysis of contaminants revealed 74% Gram-positive, 21% Gram-negative bacteria and 5% fungi. Duration of transport had an influence on the rate of contamination (P < 0.05). After islet isolation, Group I presented microbial contamination of 16 islet preparations (9.4%) [i.e. Gram-positive bacteria (n = 10), Gram-negative bacteria (n = 4), and fungi (n = 2)]. In Group II, only 2 islet preparations (4.4%) presented microbial contamination. Microbial contamination during pancreas procurement occurs frequently. Most microorganisms are eliminated during islet isolation, and de novo contaminations during islet isolation are rare. Pancreas decontamination reduces the risk of infection of the final islet preparation.     

Detection of microbial contamination during human islet isolation

Current good manufacturing practice (cGMP) islet processing facilities provide an ultraclean environment for the safe production of clinical grade islets for transplantation into immunosuppressed diabetic recipients. The objective of this study was to monitor the rate of microbial contamination in islet products after implementation of good manufacturing practice conditions. Fluid samples for microbial contamination were collected at the following steps: from the pancreas transport solution upon arrival of the organ (n=157), after surface decontamination of the pancreas with antiseptic agents (n=89), from islet supernatant at the end of the isolation (n=104), and from islet supernatant as a final transplantable product after culture (n=53). Bacterial, fungal, and mycoplasma cultures were conducted for 2, 2, and 3 weeks, respectively. Microbial contamination was detected in 31% of transport solution. The contamination was not associated with the presence of the duodenum during the preservation, cold ischemia time, or procurement team (local vs. distant). Surface decontamination of the pancreas resulted in clearance of 92% of the microbial contamination. Six preparations at the end of the isolation revealed microbial growth. All were de novo contamination during the processing. Fifty-three preparations that met our release criteria in terms of product sterility were transplanted into type 1 diabetic patients. In two instances, positive culture of the islet preparation was reported after transplantation had occurred. No patient showed any clinical findings suggestive of infection or any radiological abnormalities suggestive of abscess; a single dose of antibiotic coverage was given routinely to recipients prior to islet infusion. Although transport solution carries a high risk of microbial contamination, most contaminants become undetectable during islet processing. Microbial contamination in final products is rare, but de novo contamination still occurs during processing even under cGMP conditions.     

Peri-insular presence of collagenase during islet isolation procedures.

Reportedly, higher islet yields are obtained by ductal collagenase administration and subsequent digestion of the pancreas than by the chopped tissue collagenase digestion technique. However, the exact mechanism of islet isolation is not known. This study aims to understand the underlying mechanism of a favorable effect of ductal collagenase administration. To this end, we investigated if the higher yields can be explained by a different distribution of the collagenase enzymes in the pancreatic tissue after ductal application as compared to during chopped tissue digestion. India ink was used to mimic and visualize the distribution of collagenase in histological sections of pancreases of several species. Ink particles were seen around and even within the islets both after ductal application and during chopped tissue collagenase digestion. Thus, collagenase enzymes are not restricted to the exocrine tissue compartment with either technique. In view of this observation, we compared the efficacy of both techniques in islet isolation procedures in paired experiments in rats. Both techniques gave similar islet yields to those reportedly obtained with the ductal collagenase method. However, with either technique, the islet yield was only approximately 50% of the endocrine volume of the pancreas, indicating that a substantial loss of islet tissue had occurred. We conclude that, irrespective of the route of collagenase administration, collagenase enzymes are present in the peri-insular space during islet isolation procedures. This is pertinent in view of the finding that both methods have similar islet yields in rats and that collagenase digestion, as such, is associated with loss of islet tissue.     

Endogenous pancreatic enzyme activity levels show no significant effect on human islet isolation yield

Despite advances in human islet isolation, islet yield remains inconsistent and unreliable. In recent studies, it has been suggested that serine proteases, in particular trypsin, have been shown to have a damaging effect on islet yield. This study evaluated enzyme activity levels throughout 42 human islet isolation procedures. Trypsin, chymotrypsin, and elastase activity was determined spectrophotometrically using suitable chromophoric substrates. The results of the islet isolations were rated as successful (n = 19) or unsuccessful (n = 23) based on the islet yield and functionality. The enzyme activity profiles of the isolations were compared. No significant differences in donor-related variables were found in this study. However, in the successful isolations, a significantly greater amount (85.6 +/- 1.9%; p = 0.0017) of the pancreas was digested in a significantly shorter digestion time (19.7 +/- 0.6 min; p = 0.0054) compared with 74.8 +/- 2.5% of digested tissue in 22.6 +/- 0.7 min in the poor isolations. This study showed no significant effect of serine protease levels on the outcome of islet isolations, regardless of enzyme inhibitor supplementation. These data suggest that serine protease activity does not sufficiently affect islet yield. However, the data show that the most successful human islet isolations are achieved when the maximum amount of tissue is digested in the shortest amount of time. This suggests that further understanding of the isolation process should focus on the role of the collagenase digestion solution in the dissociation of the endocrine-exocrine tissue connection.     

Islet of Langerhans isolation from pediatric and juvenile donor pancreases

Islet grafts isolated from young donors allow superior functional outcomes but are often associated with poor islet isolation yields. The objective of this study was to comparatively analyze the outcomes of islet isolation between young and older donors. We retrospectively analyzed 564 pancreas isolations performed at our institution. Isolation outcomes were compared between donors aged ≤20 years (n = 42, YD) and >20 years (n = 522, OD). Isolation procedure was identical in both groups. Prepurification percentage of embedded islets was higher in YD (44.3 ± 22.7% vs. 24.9 ± 20.9%, P < 0.001). This led to a lower recovery rate in YD (48% vs. 76%, P = 0.002) and hence lower postpurification IEQ/g pancreas in YD (2 412 ± 1 789 IEQ/g vs. 3 194 ± 1 892 IEQ/g, P = 0.01). Final yield was 180 982 ± 128 073 IEQ in YD and 244 167 ± 134 137 IEQ in OD, (P = 0.006). In vitro function was markedly, albeit nonsignificantly, higher in YD (SI: 4.5 ± 5.1 vs. 3.0 ± 5.7, P = 0.350). Proportion of transplanted preparations was similar in both groups, 38% (16/42) in YD vs. 43% (224/522) in OD, P = 0.628. In spite of isolation and purification difficulties, pancreases from young donors allowed similar islet transplantation rates as older donors. Efforts should be directed at improving islet extraction in these donors to realize their full potential for islet transplantation.     

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.     

Factors that affect human islet isolation

More than 10,000 IEQ/kg recipient weight of islets is often necessary to achieve insulin independence in patients with type 1 diabetes mellitus. Several studies have identified high donor body mass index (BMI) and pancreas size as important factors for the success of human islet isolation. However, the donor shortage underscores the need to improve isolation outcomes from lower BMI pancreas donors and/or small pancreata. The aim of this study was to identify the critical factors that affect isolation outcome. We analyzed the data from 207 isolations performed from 2002 to 2006 with respect to donor characteristics, pancreas condition, and processing variables. More than 3000 IEQ/g pancreas weight was considered to be an acceptable isolation outcome. This goal was obtained from donors with a BMI >30 kg/m2 (P = .002). The pancreatic surface integrity was also a significant factor (P = .02). Moreover, longer digestion times (P = .04) and a greater proportion of trapped islets negatively affected success rates (P = .004). As previously reported, pancreata from high BMI donors were suitable for islet isolation and transplantation, as they yielded higher total islet particle numbers and higher IEQ/g. Although BMI and pancreas size are not controllable due to the organ donor shortage, factors such as pancreatic surface integrity, shorter digestion time, and lower proportions of trapped islets were found to be significant to obtain higher success rates. The development of better protocols and systematic training of processing/procurement teams will be of assistance to increase the number of successful human islet isolations.     

从人胚胎胰腺中分离获得巢蛋白表达阳性的细胞

目的 探讨从人胚胎胰腺组织分离巢蛋白（Nestin)阳性细胞并进行体外传代培养的方法。方法 取引产的16、18、20周人胚胎胰腺组织，分离胰岛样细胞簇（Ialet-like cell clusters,ICCs），进行体外培养。用免疫组织化学和逆转录-聚合酶链反应（RT-PCR）方法检测巢蛋白、胰十二指肠同源盒基因-1（Pancreatic and duodenal bomeobox gene-1,PDX-1/IPF-1)以及胰岛内分泌激素胰岛素和胰升糖素（Glucagon)的表达。结果 ICCs经体外培养可获得巢蛋白表达阳性细胞，这种细胞可进一步形成球状细胞簇，除有巢蛋白阳性细胞外，还有PDX-1，胰岛素和胰升糖素表达阳性的细胞。并且细胞可连续传代，结论 从人胚胎胰腺组织中可分离获得巢蛋白表达阳性细胞，这种细胞可在体外增殖并有向胰岛内分泌细胞分化的能力。     

Prophylactic trypsin inhibition during the isolation procedure guarantees reproducible, high porcine islet yields

Abstract: For isolating islets from the porcine pancreas, we established a semiautomated digestion method. Although the isolation technique was standardized and collagenase of controlled quality was used, until now the reproducibility of high islet yields was unsatisfactory. Our hypothesis was that pancreatic trypsin was responsible for this failure. The aim of this study was to investigate the effect of endogenous trypsin on islet yield. Our results demonstrate that a high trypsin level correlates with poor islet yield, whereas low trypsin activity always correlates with high islet yield. Specific inhibition of trypsin results in low trypsin activity and reproducible, high islet yields.     

Effective islet isolation method with extremely high islet yields from adult pigs

Achieving good islet isolation is one of the most important factors for successful islet transplantation. Porcine pancreas is suitable for islet isolation research due to its anatomical and physiological similarities to human pancreas. In this study, we evaluated a new porcine islet isolation method designed to maximize islet yield and compared it with our previous open pan method and the standard method using a Ricordi chamber (Ricordi method). We performed 15 porcine islet isolations, five each with the new method, the open pan method, and the Ricordi method. The new method features several important improvements. Pancreata remain uncut and are kept intact during collagenase intraductal injection, a large filtration chamber to handle whole pancreata, low concentration of collagenase (Liberase HI) for digestion, and large plastic containers for large-scale islet purification. All isolated islets were assessed for yield, purity, viability and in vitro function. Islets isolated with this new method were transplanted under the kidney capsules of SCID mice with chemically induced diabetes for in vivo functional assessment (n = 8). With the new method, we obtained on average more than 1,000,000 islet equivalents (IE) (1,236,266 +/- 213,486 IE) (mean +/- SE) before purification and 800,000 IE (879,815 +/- 222,729 IE) after purification from one adult pig. Islet yield per pancreas was significantly higher compared with our previous open pan method (30,666 +/- 11,532 IE, p < 0.01) and the Ricordi method (317,073 +/- 86,093 IE, p < 0.05). All mice, transplanted with 1000 islets from the new method, returned to normoglycemia within 4 days after transplantation. Our new method makes it possible to obtain extremely high porcine islet yield with good function. It should produce useful information for human islet isolation and transplantation, and might be applied to single donor clinical xenogeneic transplantation.     

Successful human islet isolation utilizing recombinant collagenase

The enzymatic dissociation of acinar tissue by collagenase is a substantial step in the isolation of pancreatic islets. Although essential collagenase components have been purified, the variability in the activity of different batches limits long-term reproducibility of isolation success. The utilization of purified recombinant proteases would solve this problem. In the present study, pancreases from multiorgan donors were dissociated by means of digestion-filtration using either Liberase HI (n = 51) or a recombinant collagenase blend (n = 25). No significant differences were found regarding islet yield before and after purification, the percent of exocrine-attached islets, and final purity. However, the ratio between islet equivalents and islet numbers indicated a lesser fragmentation in islets isolated with recombinant collagenase (P < 0.01). In contrast, viability was slightly higher in islets isolated with Liberase (92.3 +/- 0.8 vs. 85.6 +/- 2.9%; P < 0.05). Insulin release during static glucose incubation was not different between experimental groups. Islet transplantation into diabetic nude mice resulted in sustained normoglycemia in either group until the graft was removed. These results demonstrated that viable human islets can be isolated using recombinant collagenase. Final optimization of this enzyme blend would offer continuous reproducibility of isolation success.     

Technical improvement of human pancreatic islet isolation

INTRODUCTION: A key factor for successful islet isolation is to place the optimal amount of enzyme into the pancreatic ducts prior to starting digestion of pancreatic glands. To improve this procedure, we introduced novel techniques to identify and repair tissue damage resulting in leakage of collagenase solution. MATERIALS AND METHODS: One hundred twelve standardized consecutive islet isolations were for the effects of dye and glue on islet yield, islet function using a perifusion assay, and the possibility of clinical transplantation. One group of pancreata (n = 26) obtained en bloc together with duodenum were carefully detached with ligation of accessory ducts in an isolation unit (WPD group), whereas the pancreata were dissected from the duodenum in the operating room in the other 86 isolations. In 28 of 86 isolations, whole glands were used (WP group), while only the body and tail area were applied in the remaining 58 isolations (PP group). RESULTS: Both dye and glue effectively prevented leakage of collagenase from the gland. Both islet yield and success rate were higher with these tools without adverse effects on islet function or collagenase activity. The success rate of isolations and islet yield were significantly higher in the WPD group (P = .02 and .001, respectively). CONCLUSIONS: Dye and glue may be useful tools to improve human islet isolation procedures. In addition, the use of the whole pancreas further improves the outcome.     

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.     

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.     

Structural and functional changes resulting from islet isolation lead to islet cell death.

BACKGROUND: Islet isolation exposes the islet to a variety of cellular stresses and disrupts the cell-matrix relationship--events known to be associated with apoptosis. The purpose of this study was to determine whether islet isolation leads invariably to islet cell death and to specify the mechanisms involved. METHODS: Canine islets were isolated using Liberase CI and purified using a centrifuge. Islets were sampled for up to 5 days in culture and analyzed by routine histology, electron microscopy, immunocytochemistry, and reticulin staining for basement membrane. Apoptosis was assessed by cell death enzyme-linked immunosorbent assay and terminal deoxynucleotidyl transferase-mediated decoxyuridine triphosphate nick and labeling (TUNEL) assay. Activation of the prosurvival ERK1/2 and proapoptotic p38 and JNK were determined by immunoblotting. RESULTS: Immediately after isolation, the peri-insular basement membrane was absent, and integrin-alpha 5 expression diminished. DNA fragmentation rose from 2.5 +/- 1.8 (arbitrary units) on the day of isolation to 42.4 +/- 6.7 48 hours later (P < .05), coinciding with the appearance of pyknotic nuclei and apoptotic bodies. The apoptotic index determined by TUNEL assay increased from 5% +/- 1% on the day of isolation to 60% +/- 2% on day 5 (P < .01), and most of the affected cells were beta-cells. Finally, the p38 and JNK activity were elevated relative to ERK1/2. CONCLUSIONS: During isolation, islet cells undergo profound changes in structure and function, resulting in beta-cell apoptosis. These findings suggest that strategies directed to the manipulation of the cell-matrix relationship and the modulation of mitogen-activated protein kinase signal transduction may offer a valuable new approach to improving islet transplant outcome.     

Improved human islet isolation by a tube method for collagenase infusion.

BACKGROUND: Collagenase infusion into the pancreatic duct is an essential step in human islet isolation. We developed a new method for ductal canulation and collagenese infusion. METHODS: A total of 53 pancreata were divided into two groups: group 1 (n=23), the new tube method, and group 2 (n=30), the standard angiocatheter method. In group 1, a polyethylene tube was inserted into the duct and pushed to the tail. The tail was first expanded, followed by expansion of the body and then the head, by pulling out the tube. RESULTS: Total islet number and number/g pancreas (mean+/-SE) were significantly higher in group 1 (481,123+/-43,218 and 8,010+/-722) (mean+/-SE) than in group 2 (300,974+/-35,122 and 5,090+/-515, P<0.01). Total islet equivalent number and islet equivalent number per gram pancreas were also significantly higher in group 1 (319,176+/-39,354 and 5,455+/-652) than in group 2 (202,022+/-23,331 and 3,722+/-468, P<0.04). Islet purity and fragmentation showed no differences between the groups. CONCLUSIONS: The tube method improved islet yields. We recommended this method for human islet isolation.