Analysis on donor and isolation-related factors of successful isolation of human islet of Langerhans from human cadaveric donors

We analyzed the preexisting donor factors and isolation variables that affected isolation of human islets of Langerhans. Sixty-nine pancreata from cadaveric donors were analyzed for donor factors of age, gender, body mass index, cause of death as well as graft factors of cold ischemia time, pancreas status, distensibility during intraductal collagenase distension and time of collagenase expansion and digestion. Islet isolations that recovered >100,000 IEQ (n = 53) were compared to those generating less than 100,000 IEQ (n = 16) to analyze the factors affecting islet yield during donor harvest and isolation procedures. The mean islet recovery was 216.0 x 10(3) (IEQ) or 2840 (IEQ) per gram of pancreas. Mean purity was 54%. The success rate of islet isolation was 76%. Mean age was 31 years, and mean cold ischemia time was 6.9 hours. In univariate analysis, the status of the pancreas was the only significant factor for successful isolation, and gender, time of collagenase expansion and digestion were marginal factors. In stepwise multivariate logistic regression analysis of donor and isolation-related factors, donor gender, pancreas status and digestion time were significant factors. During the same period we performed three cases of clinical islet allotransplantation and one autotransplantation. This study confirmed that the same donor factors and variables in the isolation process can affect the ability to obtain successful human islet isolation. Enough experience and pertinent review of donor and isolation factors can make islet isolation consistent, supporting clinical islet transplantation without unnecessary cost.     

Toward maximizing the success rates of human islet isolation: influence of donor and isolation factors

In order to make islet transplantation a therapeutic option for patients with diabetes there is an urgent need for more efficient islet cell processing to maximize islet recovery. Improved donor management, organ recovery techniques, implementation of more stringent donor criteria, and improved islet cell processing techniques may contribute to enhance organ utilization for transplantation. We have analyzed the effects of donor and islet processing factors on the success rate of human islet cell processing for transplantation performed at a single islet cell processing center. Islet isolation outcomes improved when vasopressors, and in particular pitressin, and steroids were used for the management of multiorgan donors. Higher islet yields were obtained from adult male donors, BMI >25 kg/m2, adequate glycemic control during hospital stay, and when the pancreas was retrieved by a local surgical team. Successful isolations were obtained in 58% of the cases when > or = 4 donor criteria were met, and even higher success rates (69%) were observed when considering > or = 5 criteria. Our data suggest that a sequential, integrated approach is highly desirable to improve the success rate of islet cell processing.     

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 donor age on bovine pancreatic islet isolation

BACKGROUND: Pancreatic islets from pigs are largely used for experimental studies. However, pancreas harvesting requires modification of conventional slaughtering to reduce ischemia time. It has been shown that bovine pancreatic islets can be more easily obtained and they show satisfactory in vitro and in vivo function. To improve the isolation procedure we compared the effect of bovine donor age on islet isolation. METHODS: Islets were isolated by collagenase digestion and sequential sieving from calves (6 months of age) and from adult bovine (> 16 months of age). After isolation the number of islet equivalents was calculated and histological and immunohistochemical studies performed. The purity and viability of islet for each preparation was also estimated. In vitro function of islets was evaluated by static insulin secretion assay, and alginate encapsulated islets were transplanted in streptozotocin-induced diabetic rats for in vivo functional evaluation. RESULTS: A significantly higher number of islets were obtained from calf pancreas, compared with adult bovine pancreas. Hystological examination showed intact morphologic features of islets. The purity of islet preparations was higher from calf pancreas than from adult pancreas. Cell viability, and insulin production in presence of high glucose concentration, were not affected by donor age. All animals receiving microencapsulated islets from calves showed normoglycemia for prolonged periods (17-40 days). CONCLUSIONS: These results indicate that pancreatic islet isolation is more efficient from juvenile bovine than from adult. Calf pancreas is a good and convenient source of tissue for massive islet isolation for experimental studies.     

扩大标准的供者胰腺在胰岛移植中应用的动物实验

目的 通过系列的动物实验,从供者胰腺(简称:供胰)的热缺血时间、脂肪浸润程度和灌注损伤程度三个方面初步探讨扩大标准的供胰在胰岛移植中的应用. 方法 实验动物采用雄性Wistar大鼠.根据不同的干预冈素,将供胰分为3组,热缺血组、灌注水肿组和脂肪浸润组;另选正常胰腺作为正常对照组.胰岛提取采用原位灌注、胶原酶消化和Ficoll梯度密度离心法.获取的胰岛用双硫腙染色来计算分离的胰岛数目并判断所获取的胰岛纯度;丫啶橙/溴乙啶荧光染色法检测胰岛细胞的活率;体外葡萄糖刺激下胰岛素释放试验检测胰岛功能. 结果 胰腺热缺血15 min内对胰岛的提取量和活率无明显影响,纯度略有下降;热缺血30 min内胰岛的提取量、活率及纯度均明显下降,但胰岛体外功能良好;热缺血45 min时,胰岛体外功能不良.胰腺轻度和中度脂肪浸润时,胰岛提取量及胰岛素释放指数(SI)明显高于正常对照组,而重度脂肪浸润时,胰岛提取量较正常对照组明显降低,且体外功能不良,SI显著下降.胰腺轻度和中度水肿对胰岛提取量、活率、纯度及功能均无明显影响;胰腺重度水肿(含水量＞80%)时,胰岛的提取量、活率及纯度下降,体外功能不良,SI显著低于正常对照组. 结论 供胰质量是影响胰岛提取及功能的重要因素,重度脂肪浸润、严重灌注水肿、热缺血时间超过30 min均影响胰岛提取的数量、纯度及活率,对胰岛功能也有较大影响.     

Estimation of donor usability for islet isolation with the modified Ricordi method

BACKGROUND: The quality of donor pancreata is important for successful islet isolation. However, in some countries like Japan, the number of donor pancreata is low. Therefore, marginal donor pancreata have been used with less restrictive donor criteria. In order to use marginal donor pancreata, we established the modified Ricordi method. According to the United Network for Organ Sharing (UNOS) in 2005, more than 6000 pancreata were not clinically usable in the United States. In this study, we reevaluated donor usability based on the Japanese islet donor criteria. MATERIALS AND METHODS: We reviewed donor charts with well-documented cases in Texas from 2005 to 2006. We counted the number of pancreata for pancreas transplantation or islet transplantation. If not used clinically, the reason was also reviewed. Donors were reevaluated based on the Japanese islet donor criteria. RESULTS: We reviewed 236 donor charts, including 29 pancreata used for whole pancreas transplantations and 13 for islet isolation; therefore, 194 pancreata were not used. Among the 194 cases, we were able to identify the reasons that the pancreata were not used in 186 cases. When we applied the Japanese acceptance criteria, an additional 82 of 186 cases (44%) seemed suitable for islet isolations. CONCLUSIONS: With the modified Ricordi method, more than 2500 donor pancreata might be used for islet isolation in the United States when the Japanese criteria are applied.     

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.     

Successful islet transplantation from nonheartbeating donor pancreata using modified Ricordi islet isolation method

BACKGROUND: Current success of islet transplantation has led to donor shortage and the need for marginal donor utilization to alleviate this shortage. The goal of this study was to improve the efficacy of islet transplantation using nonheartbeating donors (NHBDs). METHODS: First, we used porcine pancreata for the implementation of several strategies and applied to human pancreata. These strategies included ductal injection with trypsin inhibitor for protection of pancreatic ducts, ET-Kyoto solution for pancreas preservation, and Iodixanol for islet purification. RESULTS: These strategies significantly improved both porcine and human islet isolation efficacy. Average 399,469+/-36,411 IE human islets were obtained from NHBDs (n=13). All islet preparations met transplantation criteria and 11 out of 13 cases (85%) were transplanted into six type 1 diabetic patients for the first time in Japan. All islets started to secrete insulin and all patients showed better blood glucose control without hypoglycemic loss of consciousness. The average HbA1c levels of the six recipients significantly improved from 7.5+/-0.4% at transplant to 5.1+/-0.2% currently (P<0.0003). The average insulin amounts of the six recipients significantly reduced from 49.2+/-3.3 units at transplant to 11+/-4.4 units (P<0.0005) and five out of six patients reduced to less than half dose. The first patient is now insulin free, the first such case in Japan. CONCLUSION: This demonstrates that our current protocol makes it feasible to use NHBDs for islet transplant into type 1 diabetic patients efficiently.     

Efficacy of human islet isolation from the tail section of the pancreas for the possibility of living donor islet transplantation

BACKGROUND: Islet transplantation is on the rise for the treatment of type 1 diabetes. Apparent donor shortages could be alleviated through use of living donor pancreata. A critical issue for using a section of pancreas from living donors is whether islet yields would be sufficient for transplantation. METHODS: After obtaining human pancreata, islets were isolated from the head section (n=20, head group), tail section (n=23, tail group) or whole pancreas (n=24, whole group). Islets were isolated by enzymatic digestion followed by purification, then assessed for yields, purity, morphology, functionality, and insulin content. RESULTS: Fifteen of twenty cases (75%) in the head group, all cases (100%) in the tail group, and 23 of 24 cases (96%) in the whole group were successfully completed for islet isolation. Islet yield per gram pancreas was significantly higher in the tail group compared with both the head and whole groups (head, 1,472+/-326 IE/g; tail, 4,256+/-574 IE/g; whole, 2,424+/-506 IE/g). Total islet yield from the head group was significantly lower compared with both tail and whole groups (head, 75,016+/-18,933 IE; tail, 197,469+/-28,236 IE; whole, 208,207+/-43,414 IE), and the tail group showed similar islet yield to the whole group. The whole group showed significantly lower purities and the head group showed significantly lower morphologic scores. There were no significant differences in viability, function, and insulin content among the three groups. CONCLUSIONS: The tail section of the human pancreas is suitable for islet isolation. The living donor islet transplantation may be feasible using only this section of the pancreas for the first transplantation to reduce hypoglycemic unawareness for small recipients, which might be followed by the second islet transplantation from cadaveric donor.     

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.     

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.     

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.     

The effect of donor factors on human islet yield and their in vivo function

BACKGROUND: A major problem in the islet field is the high selectivity exercised in accepting cadaveric pancreas for islet isolation. This practice is based on experience that indicates that islet yield and posttransplant function are related to donor demographics and injury mechanisms. OBJECTIVE: To examine factors influencing islets recovery and in vivo function with emphasis on donor-related factors. METHODS: Islets were isolated from 99 human donor pancreata, and islet yield was reported as islet equivalent per gram pancreatic tissue. Donor, procurement, and isolation factors were collected for each isolation and correlation statistics were performed between these variables and islet yield. RESULTS: Results indicated a differential effect of enzyme mixes on yield with Collagenase P digestion most suitable for increased ischemic time (R2 = 0.1; P < .08), Liberase with small donor pancreas size and elevated preprocurement glucose (R2 = 0.15; P < .02), and Serva with female donors (R2 = 0.17; P < .06). Islets from 29 isolations were further tested by transplantation under the kidney capsule of immune-deficient NOD-SCID mice. Although all 29 preparations had acceptable in vitro perfusion parameters indicating viability, only 19 functioned in vivo with serum levels of insulin >5 U/mL and C peptide >1.5 ng/mL. No significant differences in donor, procurement, and isolation factors were evident between the islet preparations that functioned in vivo and those that were nonfunctional. CONCLUSIONS: These data demonstrate that although yield is affected by a variety of donor factors and enzyme mixes, these factors do not affect islet in vivo function.     

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.     

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.     

Validation of the scoring system for standardization of the pancreatic donor for islet isolation as used in a new islet isolation center

BACKGROUND: The aim of this study was to evaluate the effectiveness of the Edmonton Donor Scoring System for use in our much less active islet center. Because the ability to recognize an appropriate donor may help to achieve consistent and predictable success of pancreatic islet isolation, it should lead to increased effectiveness and lower cost. MATERIAL AND METHODS: Charts of 36 consecutive pancreas donors were reviewed to assess the donor points (DP). DP ranged from 0 to 100 based on donor age, body mass index, cause of death, social and medical history, hospital stay, vasopressor dosages, laboratory tests, cold ischemia time and procurement team, as well as pancreas size, consistency, fat content, damage, and quality of procurement and packing. RESULTS: Successful isolation was achieved in 39% of donors (14 of 36), a value similar to that achieved in Edmonton (40%). We used the optimal cutoff value (DP = 79) proposed by the Edmonton group. Sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 66%, 75%, 57%, 82% and 72%, respectively. Successful islet isolation from poor or marginal donors (DP < 49.5 and 50 to 59.5) was 0% and 28.6% respectively; it was 63% and 100% in optimal donors (DP = 80 to 89.5 and 90 to 100). We concluded that islet isolation success correlated with the previously proposed donor scoring system. CONCLUSIONS: The Donor Scoring System can be successfully implemented regardless of the level of activity of an experienced isolation center. This system permits identification of a suitable donor prior to organ processing. It may guide a center's donor selection strategy based on its goals and its budget.