Magnetic resonance imaging: a tool to monitor and optimize enzyme distribution during porcine pancreas distention for islet isolation

Porcine islet xenotransplantation is emerging as a potential alternative for allogeneic clinical islet transplantation. Optimization of porcine islet isolation in terms of yield and quality is critical for the success and cost‐effectiveness of this approach. Incomplete pancreas distention and inhomogeneous enzyme distribution have been identified as key factors for limiting viable islet yield per porcine pancreas. The aim of this study was to explore the utility of magnetic resonance imaging (MRI) as a tool to investigate the homogeneity of enzyme delivery in porcine pancreata. Traditional and novel methods for enzyme delivery aimed at optimizing enzyme distribution were examined. Pancreata were procured from Landrace pigs via en bloc viscerectomy. The main pancreatic duct was then cannulated with an 18‐g winged catheter and MRI performed at 1.5‐T. Images were collected before and after ductal infusion of chilled MRI contrast agent (gadolinium) in physiological saline. Regions of the distal aspect of the splenic lobe and portions of the connecting lobe and bridge exhibited reduced delivery of solution when traditional methods of distention were utilized. Use of alternative methods of delivery (such as selective re‐cannulation and distention of identified problem regions) resolved these issues, and MRI was successfully utilized as a guide and assessment tool for improved delivery. Current methods of porcine pancreas distention do not consistently deliver enzyme uniformly or adequately to all regions of the pancreas. Novel methods of enzyme delivery should be investigated and implemented for improved enzyme distribution. MRI serves as a valuable tool to visualize and evaluate the efficacy of current and prospective methods of pancreas distention and enzyme delivery.     

Morphological changes of porcine islets of Langerhans after collagenase and HBSS infusion of the pancreas

Hilling DE, Rijkelijkhuizen JK, Marang‐van de Mheen PJ, Töns A, Terpstra OT, Bouwman E. Morphological changes of porcine islets of Langerhans after collagenase and HBSS infusion of the pancreas. Xenotransplantation 2010; 17: 413–417. © 2010 John Wiley & Sons A/S. Abstract: Background: A remarkable change in porcine islet morphology was observed after infusion of the pancreas with collagenase. The aim of the present study was to quantify these morphological changes and to assess whether these changes were due to the volume expansion caused by the collagenase entering the islet or the result of its digestive effects. Methods: This study was performed in pancreata of 28 crossbred pigs. First, eight pancreata were intraductally injected with collagenase by a continuous controlled pressure of 180 mmHg. Pancreas samples before collagenase infusion were used as controls. All tissue samples, both before and after infusion, were stained with anti‐insulin. To quantify the morphological change of the islets, the mean beta cell/endocrine content ratio of the infused and not‐infused tissue samples was compared. In a second experiment, 20 pancreata were similarly assessed after intraductal injection with Hank’s balanced salt solution (HBSS). Results: In both the collagenase‐ and HBSS‐infused groups, mean beta cell/endocrine content ratio was lower than in the control samples. The observed decline in the beta cell/endocrine content ratio was not significantly different between collagenase‐ and HBSS‐infused pancreata. This suggests that the lower beta cell/endocrine content ratio and thus the morphological change in the infused tissue samples is caused by volume expansion of the fluid entering the islet and that the digestive effect of collagenase plays no or only a minor role. Conclusion: Morphological changes of islets are observed after infusion of pancreata with collagenase and HBSS, most likely caused by volume expansion due to fluid entering the islets.     

Design of a simple, in vitro method for evaluating the efficiency of crude Clostridium histolyticum collagenase and its components for porcine islet isolation

Abstract: The wide variability of different batches of crude collagenase is one of the major factors influencing the isolation of porcine islets of Langerhans. In order to enable the production of reproducible collagenase batches, however, it is first necessary to determine which of the many components of crude collagenase are required for porcine islet isolation. For this to be done, experiments must control for the inter-pancreatic variation seen with porcine pancreata, and this requires methodology that enables many different crude collagenases or collagenase components to be tested on any one pancreas. The aim of this study was to assess four different in vitro methods for evaluating different batches of crude collagenase. Each of the methods involved placing blocks of either distended or undistended pancreas in centrifuge tubes, adding crude collagenase in various concentrations dissolved in Hanks' and incubating the tubes in a waterbath at 35°C. The remainder of the pancreas was simultaneously processed using the semi-automated method. Every 10 min, samples were taken from both the semi-automated circuit and the relevant centrifuge tubes and assessed for number of cleaved islets, cleavage index, degree of fragmentation and quality of exocrine digestion. A correlation was made between each in vitro method and the "gold standard" semi-automated method. We conclude that it is possible to use an in vitro method to evaluate crude collagenase that correlates well with the digestion occurring in the Ricordi chamber and that enables the testing of many batches or components of crude collagenase on any one porcine pancreas.     

Islet isolation from adult designated pathogen-free pigs: use of the newer bovine nervous tissue-free enzymes and a revised donor selection strategy would improve the islet graft function

Jin S‐M, Shin JS, Kim KS, Gong C‐H, Park SK, Kim J‐S, Yeom S‐C, Hwang ES, Lee CT, Kim S‐J, Park C‐G. Islet isolation from adult designated pathogen‐free pigs: use of the newer bovine nervous tissue–free enzymes and a revised donor selection strategy would improve the islet graft function. Xenotransplantation 2011; 18: 369–379. © 2011 John Wiley & Sons A/S. Abstract: Background: In clinical trials using adult porcine islet products, islets should be isolated from the designated pathogen‐free (DPF) pigs under the current good manufacturing practice (GMP) regulations. Our previous studies suggested that male DPF pigs are better donors than retired breeder pigs and histomorphometrical parameters of donor pancreas predict the porcine islet quality. We aimed to investigate whether the use of the newer bovine nervous tissue–free enzymes and a revised donor selection strategy could improve the islet graft function in the context of islet isolation with DPF pigs. Methods: Using 30 DPF pigs within a closed herd, we compared the islet yield of porcine islets isolated with Liberase PI (n = 11, as a historical control group), Liberase MTF C/T, which is a GMP‐grade enzyme (n = 12), and CIzyme collagenase MA/BP protease (n = 7). We analyzed the relationship between the diabetes reversal rate of recipient NOD/SCID mice (n = 75) and histomorphometric parameters of each donor pancreas as well as donor characteristics. Results: Proportion of islets larger than 200 μm from the biopsied donor pancreas (P = 0.006) better predicted islet yield than age (P = 0.760) or body weight (P = 0.371) of donor. The proportion of islets larger than 200 μm from the biopsied donor pancreas was not related to the sex of the donor miniature pig (P = 0.358). The islet yield obtained with the three enzymes did not differ, even after stratification of the donor with the histomorphometric parameters of the biopsied donor pancreas and the sex of donor. The use of the newer bovine nervous tissue–free enzymes (P < 0.001), a higher proportion of large islets in donor pancreas (P = 0.006), and a male sex of the donor (P = 0.025) were independent predictors of earlier diabetes reversal. Conclusions: Use of the newer bovine nervous tissue–free enzymes including a GMP‐grade enzyme resulted in better islet quality than that of islet isolated using Liberase PI. To obtain high‐quality islet from DPF pigs, the donor should be male pig and histomorphometrical parameters from donor pancreas should be considered.     

Impact of adverse pancreatic injury at surgical procurement upon islet isolation outcome

The consequence of a pancreas injury during the procurement for islet isolation purpose is unknown. The goal of this work was to assess the injuries of the pancreata procured for islet isolation, and to determine their effect on the islet yield. Between January 2007 and October 2013, we prospectively documented every injury of the pancreata processed in our centre for islet isolation. Injuries involving the main duct were classified as major, the others as minor. Donors’ characteristics and islet yields were compared between the groups of injuries. A pancreas injury was identified in 42 of 452 pancreata received for islet isolation (9.3%). In 15 cases, the injury was major (3.3% of all pancreata). Although a minor injury did not affect the islet yield, a major injury was significantly associated with unfavourable outcomes (postpurification mean islet equivalent of 364 ± 181, 405 ± 190 and 230 ± 115 × 10³ for absence of injury, minor injury and major injury, respectively). A major injury was significantly more prevalent in lean and short donors. We recommend assessing the quality of the pancreas in the islet isolation centre before starting the isolation procedure. Each centre should determine its own policy based on its financial resources and on the wait list.     

Current practices of donor pancreas allocation in the UK: future implications for pancreas and islet transplantation*

Recent refinements in technique mean islet cell transplantation offers the chance of a cure to an increasing patient cohort with diabetes. Such developments put pressure upon the scarce resource of donor organs, with potential competition between the modalities of cellular and solid organ transplantation. This questionnaire based study examines current patterns of donor pancreas procurement and use. Reasons for non procurement are studied together with the attitudes of transplant professionals to pancreas allocation. The minority of potentially useful pancreata are currently made available to either whole pancreas or islet transplant programs. Whilst professionals appreciate the role of each modality, there is a need to define criteria for pancreas allocation to avoid under use of donor organs.     

Islet alloautotransplantation: Allogeneic pancreas transplantation followed by transplant pancreatectomy and islet transplantation

Simultaneous pancreas–kidney (SPK) transplantation is an important treatment option for patients with type 1 diabetes (T1D) and end‐stage renal disease (ESRD). Due to complications, in up to 10% of patients, allograft pancreatectomy is necessary shortly after transplantation. Usually the donor pancreas is discarded. Here, we report on a novel procedure to rescue endocrine tissue after allograft pancreatectomy. A 39‐year‐old woman with T1D and ESRD who had undergone SPK transplantation required emergency allograft pancreatectomy due to bleeding at the vascular anastomosis. Islets were isolated from the removed pancreas allograft, and almost 480 000 islet equivalents were infused into the portal vein. The patient recovered fully. After 3 months, near‐normal mixed meal test (fasting glucose 7.0 mmol/L, 2‐hour glucose 7.5 mmol/L, maximal stimulated C‐peptide 3.25 nmol/L, without insulin use in the preceding 36 hours) was achieved. Glycated hemoglobin while taking a low dose of long‐acting insulin was 32.7 mmol/mol hemoglobin (5.3%). When a donor pancreas is lost after transplantation, rescue β cell therapy by islet alloautotransplantation enables optimal use of scarce donor pancreata to optimize glycemic control without additional HLA alloantigen exposure.     

Preservation of the donor pancreas for whole pancreas and islet transplantation

Ridgway D, Manas D, Shaw J, White S. Preservation of the donor pancreas for whole pancreas and islet transplantation.
Clin Transplant 2010: 24: 1–19. © 2009 John Wiley & Sons A/S.
Abstract:  Whole pancreas and islet cell transplantation are both reliant upon the procurement and preservation of a high quality donor pancreas for a successful outcome. In the climate of a reducing donor pool it is imperative that donor optimization, meticulous surgical retrieval and evidence based methods of preservation are practiced to ensure optimal graft quality. Moreover expanded criteria donors and novel methods of pancreas preservation have the potential to expand the number of usable grafts and increase the availability of these transplant modalities to suitable patients with diabetes. This article provides a review of the current literature surrounding donor management, surgical technique and the various technologies of organ preservation applicable to the donor pancreas.     

Minimization and withdrawal of steroids in pancreas and islet transplantation

For reducing the corticosteroid (CS)-related side-effects, especially cardiovascular events, CS-sparing protocols have become increasingly common in pancreas transplantation (PT). Lympho-depleting induction antibodies, such as rabbit anti-thymocyte globulin (rATG) or alemtuzumab, have been widely used in successful trials. The results of various CS-sparing protocols combining calcineurin inhibitors (CNI) and mycophenolate or sirolimus, have been mixed for rejection and survival rates. Most of the studies were uncontrolled trials of low-risk patients, therefore the grade of evidence is limited. Large-scale prospective studies with long-term follow up are necessary to assess risks and benefits of CS-sparing regimens in PT before recommending such strategies as standard practice. Islet allo-transplantation for patients with brittle type 1 diabetes mellitus, less invasive and safer procedure than PT, has been attempted since late 1980s, but diabetogenic immunosuppressants at maintenance, mainly CS and high-dose CNI, prevented satisfactory results (10% insulin-independence at 1-year post-transplant). Since 2000, CS-free and CNI-reducing protocols, including more potent induction [daclizumab, OKT3gamma1(ala-ala) anti-CD3 antibody, rATG] and maintenance (sirolimus, mycophenolate) agents, have significantly improved short-term outcomes whereas long-term are still inadequate (from 80% to 20% insulin-independence from 1- to 5-year post-transplant). Main limitations are allo- and autoimmunity, immunosuppression-related islet and systemic toxicity and transplant site unsuitability, which tolerogenic protocols and biotechnological solutions may solve.     

国产胶原酶在小鼠胰岛分离中的效果研究#br#

目的 研究一种国产胶原酶在小鼠胰岛分离中的分离效果,探索该胶原酶在胰岛分离中应用的 可行性。方法 将国产胶原酶分别配成不同浓度的胶原酶溶液和含中性蛋白酶的胶原酶的溶液,经胰管逆行灌注胶原酶的方法进行小鼠胰岛分离,采用Ficoll液对胰岛进行纯化,计数所得的胰岛细胞团,培养 6 h后检测活性,对最佳分离结果的胰岛做同系糖尿病小鼠的肾被膜下移植,监测术后血糖和进行组织学 检查,以进口Sigma胶原酶V为对照。结果 国产胶原酶在小鼠胰腺消化时间、所得胰岛数量和活性效果 上跟进口胶原酶V有一定差距（P＜0.01）,但含中性蛋白酶的国产胶原酶组在小鼠胰岛分离数量和当量上 与进口胶原酶组无差异（P＞0.05）,分离的胰岛在体内移植后降血糖效果与进口胶原酶组无差异（P＞0.05）。 结论 国产胶原酶结合中性蛋白酶可用于小鼠胰岛的分离。     

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

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

Purity of islet preparations and 5‐year metabolic outcome of allogenic islet transplantation

In allogenic islet transplantation (IT), high purity of islet preparations and low contamination by nonislet cells are generally favored. The aim of the present study was to analyze the relation between the purity of transplanted preparations and graft function during 5 years post‐IT. Twenty‐four patients with type 1 diabetes, followed for 5 years after IT, were enrolled. Metabolic parameters and daily insulin requirements were compared between patients who received islet preparations with a mean purity <50% (LOW purity) or ≥50% (HIGH purity). We also analyzed blood levels of carbohydrate antigen 19‐9 (CA 19‐9)—a biomarker of pancreatic ductal cells—and glucagon, before and after IT. At 5 years, mean hemoglobin A1c (HbA1c levels) (P = .01) and daily insulin requirements (P = .03) were lower in the LOW purity group. Insulin independence was more frequent in the LOW purity group (P < .05). CA19‐9 and glucagon levels increased post‐IT (P < .0001) and were inversely correlated with the degree of purity. Overall, our results suggest that nonislet cells have a beneficial effect on long‐term islet graft function, possibly through ductal‐to‐endocrine cell differentiation. ClinicalTrial.gov NCT00446264 and NCT01123187.     

Islet damage during isolation as assessed by miRNAs and the correlation of miRNA levels with posttransplantation outcome in islet autotransplantation

High‐quality pancreatic islets are essential for better posttransplantation endocrine function in total pancreatectomy with islet autotransplantation (TPIAT), yet stress during the isolation process affects quality and yield. We analyzed islet‐enriched microRNAs (miRNAs) ‐375 and ‐200c released during isolation to assess damage and correlated the data with posttransplantation endocrine function. The absolute concentration of miR‐375, miR‐200c, and C‐peptide was measured in various islet isolation steps, including digestion, dilution, recombination, purification, and bagging, in 12 cases of TPIAT. Posttransplantation glycemic control was monitored through C‐peptide, hemoglobin A_1c, insulin requirement, and SUITO index. The amount of miR‐375 released was significantly higher during enzymatic digestion followed by the islet bagging (P < .001). Mir‐200c mirrored these changes, albeit at lower concentrations. In contrast, the C‐peptide amount was significantly higher in the purification and bagging steps (P < .001). Lower amounts of miR‐375 were associated with a lower 6‐month insulin requirement (P = .01) and lower hemoglobin A_1c (P = .04). Measurement of the absolute quantity of miRNA‐375 and ‐200c released during islet isolation is a useful tool to assess islet damage. The quantity of released miRNA is indicative of posttransplantation endocrine function in TPIAT patients.     

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.     

Pancreas preservation fluid microbial contamination is associated with poor islet isolation outcomes – a multi‐centre cohort study

The microbiological safety of islet preparations is paramount. Preservation medium contamination is frequent, and its impact on islet yield and function remains unclear. Microbiological samples collected during islet isolations from 2006 to 2016 were analyzed and correlated to isolation and allo‐ and autotransplantation outcomes. Microbial contamination of preservation medium was found in 64.4% of processed donor pancreases (291/452). We identified 464 microorganisms including Staphylococcus (253/464, 54.5%), Streptococcus (31/464, 6.7%), and Candida species (25/464, 5.4%). Microbial contamination was associated with longer warm and cold ischemia times and lower numbers of postpurification islet equivalents, purity, transplant rate, and stimulation index (all P < 0.05). Six percent of the preparations accepted for transplantation showed microbial contamination after isolation (12/200); 9 of 12 were Candida species. Six patients were transplanted with a sample with late microbial growth discovered after the infusion. Insulin independence rate was not affected. This risk of transplanting a contaminated islets preparation was reduced by half following the implementation of an additional sampling after 24 h of islet culture. Pancreas preservation fluid microbial contamination is associated with lower transplant rate and poorer in vitro function, but not with changes in graft survival. Culture medium testing 1 day after isolation reduces the risk of incidental transplantation with contaminated islets.     

Hypothermic oxygenated machine perfusion of the human pancreas for clinical islet isolation: a prospective feasibility study

Due to an increasing scarcity of pancreases with optimal donor characteristics, islet isolation centers utilize pancreases from extended criteria donors, such as from donation after circulatory death (DCD) donors, which are particularly susceptible to prolonged cold ischemia time (CIT). We hypothesized that hypothermic machine perfusion (HMP) can safely increase CIT. Five human DCD pancreases were subjected to 6 h of oxygenated HMP. Perfusion parameters, apoptosis, and edema were measured prior to islet isolation. Five human DBD pancreases were evaluated after static cold storage (SCS). Islet viability, and in vitro and in vivo functionality in diabetic mice were analyzed. Islets were isolated from HMP pancreases after 13.4 h [12.9–14.5] CIT and after 9.2 h [6.5–12.5] CIT from SCS pancreases. Histological analysis of the pancreatic tissue showed that HMP did not induce edema nor apoptosis. Islets maintained >90% viable during culture, and an appropriate in vitro and in vivo function in mice was demonstrated after HMP. The current study design does not permit to demonstrate that oxygenated HMP allows for cold ischemia extension; however, the successful isolation of functional islets from discarded human DCD pancreases after performing 6 h of oxygenated HMP indicates that oxygenated HMP may be a useful technology for better preservation of pancreases.     

Histomorphological characteristics of the porcine pancreas as a basis for the isolation of islets of Langerhans

Abstract: Isolation of porcine islets of Langerhans for future clinical xenotransplantation in type I diabetic patients is limited by the difficulty to isolate sufficiently functioning islets and by their particular fragility. Seven domestic pig races and the wild boar were investigated histologically to obtain detailed information on islet profiles, numbers, sizes, and volume density of the endocrine tissue. Theoretically calculated islet numbers were correlated with those obtained after islet isolation. Results: 1) Porcine islets are round, oval, dumbbell-like and triangular, and, as expected, occur in all intermediate profiles. Round and oval islets predominate. These profiles are also detected in crude islet preparations, which indicates that they can withstand the enzymatic digestion process. 2) The total number of islets varies greatly in all eight pig races, with, e.g., the wild boar showing twice as many islets (approx. 500/cm² tissue section) as the minipig (approx. 250/ cm²). 3) The tail of the pancreas, which is usually used for islet isolation, accounts for about 50% of the pancreas mass. 4) The majority of islets, 64.3% (range 59.8–68.9%), of the seven domestic races are 50–100 μm in diameter. With its 86% the wild boar is a remarkable exception. German landrace pigs show the greatest number (2%) of large islets (250 to >300 μm. 5) With 3.4%, German landrace pigs reveal the greatest islet volume density of all pig races (range 2.6–4.2%). 6) Only 25% of all islets are surrounded by a collagen “capsule” that covers >75% of the islet surface. Roughly 33% of all islets are “capsulated” by collagen type I, III, and IV fibers to an extent of <25%. 7) In young pure bred pigs (7–12 month old) only 3.0–10.6% of all theoretically available islets can be isolated during the enzymatic digestion. However, much better islet yields (30.1%) can be obtained from adult hybrid pigs (3 years old) much better islet yields (30.1%) can be obtained than from young hybrid pigs (7.6%). It may be concluded from our results that histological parameters-besides those already known (pH of the isolation medium, trypsin inhibition by Pefabloc-may have a greater influence on porcine pancreatic islet isolation than was assumed so far.     

Impact of ischemia time on islet isolation success and posttransplantation outcomes: A retrospective study of 452 pancreas isolations

Many variables impact islet isolation, including pancreas ischemia time. The ischemia time upper limit that should be respected to avoid a negative impact on the isolation outcome is not well defined. We have performed a retrospective analysis of all islet isolations in our center between 2008 and 2018. Total ischemia time, cold ischemia time, and organ removal time were analyzed. Isolation success was defined as an islet yield ≥200 000 IEQ. Of the 452 pancreases included, 288 (64%) were successfully isolated. Probability of isolation success showed a significant decrease after 8 hours of total ischemia time, 7 hours of cold ischemia time, and 80 minutes of organ removal time. Although we observed an impact of ischemia time on islet yield, a probability of isolation success of 50% was still present even when total ischemia time exceeds 12 hours. Posttransplantation clinical outcomes were assessed in 32 recipients and no significant difference was found regardless of ischemia time. These data indicate that although shorter ischemia times are associated with better islet isolation outcomes, total ischemia time >12 hours can provide excellent results in appropriately selected donors.