Establishment of a prolonged pancreas preservation model for islet isolation research in mice

Establishing a prolonged pancreas preservation model in a small animal is important for islet isolation research. Use of a rat pancreas model has been reported, but no published reports have used a mouse pancreas for prolonged cold preservation prior to islet isolation. For the model, a mouse is preferred over a rat because of its small size, well-known immune characterization, and variety of gene-modulated models. In the present study, we established a prolonged pancreas preservation model in a mouse for islet isolation research. The collagenase solution was injected successfully after 24 and 48 h cold preservations in University of Wisconsin solution, and islets could be isolated from both groups of preserved pancreata. The islet yields from the control, 24 h preserved, and 48 h preserved pancreata were 183.9 ± 13.9, 128.5 ± 15.5, and 24.6 ± 12.9 per pancreas, respectively. The propidium iodide-positive area assay was significantly increased in both preserved groups, and insulin secretion levels in response to 20.0 mM glucose and stimulation indices were significantly decreased in the 48 h preserved group. Inflammation-related genes mRNA levels were significantly upregulated in the 24 h preserved group, as previously shown in the human model. Thus, this model might be useful for prehuman islet isolation screening research, reserving research using human pancreata for the most promising approaches.     

Clinical islet isolation outcomes with a highly purified neutral protease for pancreas dissociation

Background: Pancreas dissociation is a critical initial component of the islet isolation procedure and introduces high variability based on factors including the enzyme type, specificity and potency. Product refinement and alterations to the application strategies have improved isolation outcomes over time; however, islet utilization from donor organs remains low. In this study we evaluate a low endotoxin-high activity grade neutral protease in clinical islet isolation.

Materials and Methods: The use of a non-collagenolytic enzyme, either thermolysin or high active neutral protease, was randomized in clinical islet isolations to evaluate efficacy. Additionally a retrospective comparison to neutral protease NB was conducted.

Results:The thermolysin group had lower trapped islet population and increased purity and post-culture islet mass in comparison to high active grade neutral protease. Comparison of neutral protease NB GMP grade to high active neutral protease displayed no measurable difference in islet mass or viability and transplantation outcomes at 1 mo post-transplant were favorable for both groups.

Conclusions: High activity neutral protease can generate clinical grade islets and may prove beneficial to islet function and viability based on a reduced endotoxin load but dosing of neutral protease requires ongoing optimization.     

Improvement of collagenase distribution with the ductal preservation for human islet isolation

A delivery of collagenase at the islet-exocrine interface is crucial for successful human islet isolation. In this study, we investigated how the ductal preservation method at the procurement site affected collagenase distribution. At first, we analyzed human islet isolation data among groups using Serva collagenase with or without ductal injection (DI) or using new Liberase MTF with DI. Then, to assess the distribution of collagenase, human pancreata were classified into two groups: without DI (no DI, n = 5) and with DI at the procurement site (DI, n = 5). Collagenase with 1% marking dye was perfused in the same manner as in our clinical isolation. The distension of the pancreas and the microscopic distribution of the dyed collagenase in pancreas sections were examined. For microscopic analysis, islets were counted and classified into three criteria: unreached, dye didn't reach the islet surface; surface, dye resided on the surface of the islet but not inside; and inside, dye was found inside the islet. As a result, DI groups substantially improved islet yields. In addition, Liberase MTF with DI significantly improved efficacy of pancreas digestion. All pancreata were well distended macroscopically. However, microscopically, the majority of islets in the no DI group were untouched by the dyed collagenase. Ductal preservation substantially improved dyed collagenase delivery on the surface of islets. In conclusion, delivery of collagenase on the surface of islets was unexpectedly insufficient without DI, which was substantially improved by DI. Thus, ductal preservation is a potent method to improve collagenase delivery and islet yields.     

Improvement of collagenase distribution with the ductal preservation for human islet isolation

A delivery of collagenase at the islet-exocrine interface is crucial for successful human islet isolation. In this study, we investigated how the ductal preservation method at the procurement site affected collagenase distribution. At first, we analyzed human islet isolation data among groups using Serva collagenase with or without ductal injection (DI) or using new Liberase MTF with DI. Then, to assess the distribution of collagenase, human pancreata were classified into two groups: without DI (no DI, n = 5) and with DI at the procurement site (DI, n = 5). Collagenase with 1% marking dye was perfused in the same manner as in our clinical isolation. The distension of the pancreas and the microscopic distribution of the dyed collagenase in pancreas sections were examined. For microscopic analysis, islets were counted and classified into three criteria: unreached, dye didn’t reach the islet surface; surface, dye resided on the surface of the islet but not inside; and inside, dye was found inside the islet. As a result, DI groups substantially improved islet yields. In addition, Liberase MTF with DI significantly improved efficacy of pancreas digestion. All pancreata were well distended macroscopically. However, microscopically, the majority of islets in the no DI group were untouched by the dyed collagenase. Ductal preservation substantially improved dyed collagenase delivery on the surface of islets. In conclusion, delivery of collagenase on the surface of islets was unexpectedly insufficient without DI, which was substantially improved by DI. Thus, ductal preservation is a potent method to improve collagenase delivery and islet yields.     

Current indications for pancreas or islet transplant

Pancreas or islet transplantation can provide good glycaemic control and insulin independence. Pancreas transplantation has been associated with improvement in diabetic retinopathy, nephropathy, neuropathy and vasculopathy, but has the associated morbidity of major surgery. Both forms of therapy require long-term immunosuppression and its attendant risks and both achieve insulin independence rates of about 80% at 1 year. Pancreas transplantation at the same time as a renal transplant is a worthwhile option to employ, especially if the diabetes has been difficult to control. Diabetes associated with frequent severe hypoglycaemia or extreme lability, despite optimization of diabetes management, may benefit from either pancreas or islet transplant alone with the latter being the lower-risk procedure. More quantitative measures of hypoglycaemia and lability are now available to facilitate the assessment of the severity of these problems with glucose control. Diabetic patients with renal involvement (macroproteinuria, but no major elevation of creatinine) and unstable diabetes may be helped with an islet or pancreas transplant, but this approach should still be considered experimental and such a transplant may hasten the need for renal replacement therapy. In the setting of well-controlled diabetes and intact renal function, it is difficult to justify pancreas or islet transplant alone given the risks of immunosuppression.     

An analysis of the role of collagenase and protease in the enzymatic dissociation of the rat pancreas for islet isolation

Summary Crude Clostridium histolyticum collagenase is widely used for the enzymatic degradation of pancreatic extracellular matrix in order to isolate the islets of Langerhans. The variable enzymatic composition of crude collagenases is a critical issue which contributes to the poor reproducibility of islet isolation procedures. In this study, the separate contributions of collagenase and protease to the islet isolation process were analysed by testing various combinations of purified collagenase and purified protease in rat pancreas dissociations under conditions which eliminated all other proteolytic activity. Under these conditions, complete tissue dissociation by purified collagenase required 99±10 min, whereas increasing amounts of protease progressively reduced this time to a minimum of 36±1 min. Histochemical analysis of the dissociation process showed that protease enhanced the degradation of all four major components of the extracellular matrix: collagen was degraded more completely, while proteoglycans, glycoproteins and elastin were degraded at a higher rate. Pancreas dissociation under the present, strictly controlled conditions resulted in a high yield of viable islets: 4.2–5.0 l islet tissue volume (3,300–3,800 islets) were isolated per g pancreas in the presence of a high or low protease concentration, respectively. Prolonged dissociation in the presence of protease resulted in a dramatic decrease in islet yield which correlated with the observation that the enzyme accelerated islet disintegration. It is concluded that the collagenase-induced dissociation of the extracellular matrix is facilitated by protease. Our study shows that high yields of viable islets can be obtained under controlled enzymatic conditions, provided that the exposure of islets to protease is limited.     

Impact of donor-related variables on islet isolation outcome in dogs

Summary Clinical human islet transplantation programmes are considerably hampered by the variability of islet isolation outcome. The effects of the islet content of the pancreas and other donor-related variables on isolation outcome have not been evaluated systematically so far — either in large animals, or in man. We studied the impact of interindividual differences in age, body weight and pancreatic islet content on the outcome of collagenase isolation of islets from the splenic pancreas of beagle dogs (n=31). The islet volume of the splenic pancreas amounted to a mean (± SEM) 15.7±0.9 l per gramme pancreas, and varied three-fold (from 8.4 to 27.3 l). Isolated islet yield was 7.6±0.7 l/g and varied nine-fold (1.8–16.3 l). Animals also varied in age eight-fold (867 months) and body weight two-fold (8.6–18.3 kg). Differences in body weight and age explained 60% of variance in the fractional islet volume of the pancreas and 50% of the variance in islet yield (p<0.001). Fractional islet volume of the splenic pancreas also explained 50% of the variance in islet yield (p<0.001). We conclude that the outcome of islet isolation may be predictable after controlling for the variable islet content of pancreases, and other donor-related variables, and suggest that similar studies should be done in man.     

Islet vs. pancreas transplantation in Brazil: Defining criteria for pancreas allocation decision

Background: Many studies have evaluated whether there are characteristics related to pancreas donors and the islet isolation process that can influence in pancreatic islet yield. However, this analysis has not yet been performed in Brazil, one of the world leaders in whole pancreas organ transplantation (WOPT), where pancreas allocation for pancreatic islet transplantation (PIT) has no officially defined criteria. Definition of parameters that would predict the outcome of islet isolation from local pancreas donors would be useful for defining allocation priority in Brazil. Objective: To analyze the relationship between multiple donor-related and islet isolation variables with the total number of isolated pancreatic islet equivalents (IEQ) in a Brazilian sample of pancreas donors. Methods: Several variables were analyzed in 74 pancreata relative to the outcome of total IEQs obtained at the end of the process. Results: In univariate analysis, body mass index (BMI) (p = 0.003), the presence of fatty infiltrates in the pancreas as observed during harvesting (p = 0.042) and pancreas digestion time (p = 0.046) were identified as variables related to a greater IEQ yield. In a multivariate analysis a statistically significant contribution to the variability of islet yield was found only for the BMI (p = 0.017). A ROC curve defined a BMI = 30 as a cut-off point, with pancreata from donors with BMI > 30 yielding more islets than donors with BMI < 30 (p < 0.001). Conclusion: These data reinforces the importance of the donor BMI as a defining parameter for successful islet isolation and establishes this variable as a potential pancreas allocation criterion in Brazil, where there is an unequal competition for good quality organs between WOPT and PIT.     

An effective purification method using large bottles for human pancreatic islet isolation

The purification process is one of the most difficult procedures in pancreatic islet isolation. It was demonstrated that the standard purification method using a COBE 2991 cell processor with Ficoll density gradient solution harmed islets mechanically by high shear force. We reported that purification using large bottles with a lower viscosity gradient solution could improve the efficacy of porcine islet purification. In this study, we examined whether the new bottle purification method could improve the purification of human islets. Nine human pancreata from brain-dead donors were used. After pancreas digestion, the digested tissue was divided into three groups. Each group was purified by continuous density gradient using ET-Kyoto and iodixanol gradient solution with either the standard COBE method (COBE group) or the top loading (top group) or bottom loading (bottom group) bottle purification methods. Islet yield, purity, recovery rate after purification, and in vitro and in vivo viability were compared. Islet yield per pancreas weight (IE/g) and the recovery rate in the top group were significantly higher than in the COBE and bottom groups. Furthermore, the average size of purified islets in the top group was significantly larger than in the COBE group, which indicated that the bottle method could reduce the shear force to the islets. In vivo viability was also significantly higher in the top group compared with the COBE group. In conclusion, the top-loading bottle method could improve the quality and quantity of human islets after purification.     

An effective purification method using large bottles for human pancreatic islet isolation

The purification process is one of the most difficult procedures in pancreatic islet isolation. It was demonstrated that the standard purification method using a COBE 2991 cell processor with Ficoll density gradient solution harmed islets mechanically by high shear force. We reported that purification using large bottles with a lower viscosity gradient solution could improve the efficacy of porcine islet purification. In this study, we examined whether the new bottle purification method could improve the purification of human islets. Nine human pancreata from brain-dead donors were used. After pancreas digestion, the digested tissue was divided into three groups. Each group was purified by continuous density gradient using ET-Kyoto and iodixanol gradient solution with either the standard COBE method (COBE group) or the top loading (top group) or bottom loading (bottom group) bottle purification methods. Islet yield, purity, recovery rate after purification, and in vitro and in vivo viability were compared. Islet yield per pancreas weight (IE/g) and the recovery rate in the top group were significantly higher than in the COBE and bottom groups. Furthermore, the average size of purified islets in the top group was significantly larger than in the COBE group, which indicated that the bottle method could reduce the shear force to the islets. In vivo viability was also significantly higher in the top group compared with the COBE group. In conclusion, the top-loading bottle method could improve the quality and quantity of human islets after purification.     

Quality control for clinical islet transplantation: organ procurement and preservation, the islet processing facility, isolation, and potency tests

Pancreatic islet transplantation has become one of the ideal treatments for patients with type 1 diabetes mellitus due to improvements in isolation techniques and immunosuppression regimens. In order to ensure the safety and rights of patients, isolated islets need to meet the criteria for regulation as both a biological product and a drug product. For the constant success of transplantation, therefore, all investigators involved in clinical islet transplantation must strive to ensure the safety, purity, and potency of islets in all the phases of clinical islet isolation and transplantation. In this review, we summarize the quality control for clinical islet isolation and transplantation, and the latest topics of pre-transplant islet assessment.     

Transgenic and other experimental models of pancreas and islet damage

To unravel the cellular and molecular mechanisms involved in β-cell renewal and expansion throughout life, several different experimental models were devised in the past. A number of experimental approaches and transgenic models have been engineered to trigger specifically pancreatic injury and thus explore regeneration. Globally, three main strategies are followed to induce pancreas damage: surgical, chemical and genetic. Some of the most relevant studies regarding these three approaches are briefly summarized in this short overview. Although significant progress has been achieved in recent years, there is much room for improving our understanding of many fundamental processes regulating β-cell mass maintenance.     

Risks and benefits of transplantation in the cure of type 1 diabetes: whole pancreas versus islet transplantation. A single center study

BACKGROUND: Pancreas and islet transplantation are the only available options to replace beta-cell function in patients with type 1 diabetes. Great variability in terms of rate of success for both approaches is reported in the literature and it is difficult to compare the respective risks and benefits. OBJECTIVES: The aim of this study was to analyze risks and benefits of pancreas transplantation alone (PTA) and islet transplantation alone (ITA) by making use of the long-term experience of a single center where both transplantations are performed. We focused on the risks and benefits of both procedures, with the objective of better defining indications and providing evidence to support the decision-making process. The outcomes of 33 PTA and 33 ITA were analyzed, and pancreas and islet function (i.e., insulin independence), perioperative events, and long-term adverse events were recorded. RESULTS: We observed a higher rate of insulin independence in PTA (75%) versus ITA (59%), with the longer insulin independence among PTA patients receiving tacrolimus. The occurrence of adverse events was higher for PTA patients in terms of hospitalization length and frequency, re-intervention for surgical and immunological acute complications, CMV reactivation, and other infections. CONCLUSIONS: In conclusion, these results support the practice of listing patients for PTA when the metabolic control and the progression of chronic complications require a rapid normalization of glucose levels, with the exception of patients with cardiovascular disease, because of the high surgical risks. ITA is indicated when replacement of beta-cell mass is needed in patients with a high surgical risk.     

Islet separation and islet cell culture in vitro from human embryo pancreas

INTRODUCTlONDiabetesmellitusisthemostc0mmondiseaseanditsdeathrateranksthe8thintheworld.Upt0n0w,itsincidencehasatendencytoincrease[11.Sincedisorderofsugarmetabolismmightresultinmicrovasculardegenerati0nandinjuryofimportanthumanorgans,itwillendangerhumanhealthseri0usly-Inl969,Younszaifirstreportedthemethodthatcoulddecreasediabetessymptomsbyislettissuetransplantation.In1981,ourcountrybegantotreatdiabetestype1bytransplantingculturedislettissues.Inourstudy,wedigestedthepiecesofpancreaswithcol…     

The IKEM pancreas and islet transplant program as part of healthcare for type 1 diabetes patients: retrospective analysis of outcome from 1983 to 2010

Currently, 25-30 pancreas transplantations per year are carried out in type 1 diabetes (T1D) recipients residing in Czech Republic. Most of the recipients are transplanted together with kidney allografts, but pancreas is also transplanted alone in selected patients with brittle diabetes. Since 2005, the Institute for Clinical and Experimental Medicine (IKEM) islet transplant program was initiated as complementary therapeutic modality. The aim of this paper was to analyze the transplant program at our clinical center, and to examine the survival of recipients, and their pancreas, kidney, and islet grafts. Patient and graft survival rates were evaluated in the following three categories using Kaplan-Meier test: simultaneous pancreas and kidney transplantation (SPKTx), pancreas transplantation alone (PTA), and islet transplantation (ITx). Three hundred and ninety SPKTx, 34 PTA and 44 ITx were carried out between 1983 and 2010. One- and 5-year patient survival rates were 92 % and 81% in SPKTx, respectively. In SPKTx, the 1-year survival rate of pancreas grafts was 78%, and the 5-year rate was 66%. Kidney graft survival rates were 89% and 79%, respectively, after the same follow-up periods. In the PTA category, recipient survivals were 100% after 1 year, and 92% after 3 years. 70% and 65% of pancreatic grafts were working properly at 1 and 3-year follow-ups, respectively. To date, we have carried out 44 islet transplantations in 31 recipients. Islet function (C-peptide ≥ 0.2 ng/ml) was documented in 60% of recipients after 12 months. So far, only 3 patients remained free of exogenous insulin. While SPKTx is a well established treatment for uremic T1D patients, ITx represents an emerging complementary treatment modality. The latter is especially suitable for high-risk recipients, but routine clinical application is still hampered by the limited availability of usable organ transplants and viability of transplanted islets.     

Establishment of a pig model of combined pancreas-kidney transplantation

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Experimental research on the mechanism of improved rat pancreas preservation by ligustrazine

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Combined analysis of islet cell antibodies which cross-react with mouse pancreas,antibodies to the Mr 64,000 islet protein,and antibodies to glutamate decarboxylase in subjects at risk for IDDM

Summary With regard to progression to diabetes, ICA cross-reactive with mouse pancreas, antibodies to the M_r 64,000 islet antigen (64K), antibodies immunotrapping brain GAD activity, and IAA were analysed in 53 ICA-positive first-degree relatives of IDDM patients and 18 ICA-positive schoolchildren without a family history of diabetes. Sera from 29 (55 %) relatives did not bind to mouse pancreas, whereas 24 (45 %) displayed cross-species reaction. ICA titres on human and mouse pancreas were weakly correlated in the overall population (p <0.05) but more strongly (p <0.01) in only those subjects who displayed antibodies on tissues from both species. GAD and 64K antibodies were detected in 31 % and 35 % of relatives. In schoolchildren, the frequencies of cross-species reactive ICA (22 %), GAD antibodies (6 %), 64K antibodies (22 %), and IAA (6 %), were lower (p <0.05) than in relatives. A strong correlation (p <0.0001) was observed between GAD and 64K antibodies. GAD or 64K antibodies were strongly correlated with ICA on human pancreas (p <0.0001) but poorly with ICA on mouse pancreas (p =0.05). After pre-incubation of sera with brain homogenate, ICA titres were unaffected on mouse pancreas but reduced on human pancreas. ICA-positive subjects who displayed neither cross-species reactive ICA nor GAD or 64K antibodies were more frequent (p <0.05) among schoolchildren than relatives, whereas subjects who displayed all antibody specificities were more numerous (p <0.04) in relatives. All relatives with ICA binding only to human pancreas, as well as all schoolchildren, permanently displayed an AIRG higher than the first control percentile and remained non-diabetic. Five of ten relatives with cross-species reactive ICA, GAD and 64K antibodies at the same time displayed acute insulin response to glucose which fell below the first control percentile and developed the disease. The cross-species heterogeneity of ICA was thus confirmed in a large series of relatives and revealed in the general population. Detection of cross-species reactive ICA, GAD antibodies, or 64K antibodies enhances the prognostic significance after conventional ICA screening. The combination of these antibodies is more indicative of diabetes development than any antibody alone. Correlations between tests and absorption experiments indicate that GAD 64 is an ICA antigen on human but not on mouse pancreas, and that ICA which recognize GAD 64K coexist with others which react with mouse pancreas but not with GAD. A third ICA subset might have been revealed by high-titred ICA without either cross-species reactivity or GAD or 64K antibodies. This latter state was more frequent in the general population than in relatives and might typify an early immune response which may or may not progress. [Diabetologia (1994) 37: 491–499] Received: 29 July 1993 and in revised form: 25 November 1993     

Prolonging warm ischemia reduces the cold preservation limits of liver grafts in swine

Introduction Liver transplantation (LT) is widely accepted as an effective treatment for end-stage liver disease, but a serious shortage of donor organs limits itsclinical application. To increase the number of livers available for transplantation, graft procurement from non-heart-beating donors (NHBDs) has again become a focus of attention.[1-3] It is suggested that warm ischemia (WI), cold ischemia (CI) or ischemia/reperfusion injury of the graft are risk factors for postoperative graft …