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.     

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.     

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.     

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.     

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.     

Different digestion enzymes used for human pancreatic islet isolation: A mixed treatment comparison (MTC) meta-analysis

Collagenases are critical reagents determining yield and quality of isolated human pancreatic islets and may affect islet transplantation outcome. Some islet transplantation centers have compared 2 or more collagenase blends; however, the results regarding differences in quantity and quality of islets are conflicting. Thus, for the first time, a mixed treatment comparison (MTC) meta-analysis was carried out to compile data about the effect of different collagenases used for human pancreas digestion on islet yield, purity, viability and stimulation index (SI). Pubmed, Embase and Cochrane libraries were searched. Of 755 articles retrieved, a total of 15 articles fulfilled the eligibility criteria and were included in the MTC meta-analysis. Our results revealed that Vitacyte and Liberase MTF were associated with a small increase in islet yield (islet equivalent number/g pancreas) when compared with Sevac enzyme [standardized mean difference (95% credible interval – CrI) = −2.19 (−4.25 to −0.21) and −2.28 (−4.49 to −0.23), respectively]. However, all other enzyme comparisons did not show any significant difference regarding islet yield. Purity and viability percentages were not significantly different among any of the analyzed digestion enzymes. Interestingly, Vitacyte and Serva NB1 were associated with increased SI when compared with Liberase MTF enzyme [unstandardized weighted mean difference (95% CrI) = −1.69 (−2.87 to −0.51) and −1.07 (−1.79 to −0.39), respectively]. In conclusion, our MTC meta-analysis suggests that the digestion enzymes currently being used for islet isolation works with similar efficiency regarding islet yield, purity and viability; however, Vitacyte and Serva NB1 enzymes seem to be associated with an improved SI as compared with Liberase MTF.     

Different digestion enzymes used for human pancreatic islet isolation: A mixed treatment comparison (MTC) meta-analysis

Collagenases are critical reagents determining yield and quality of isolated human pancreatic islets and may affect islet transplantation outcome. Some islet transplantation centers have compared 2 or more collagenase blends; however, the results regarding differences in quantity and quality of islets are conflicting. Thus, for the first time, a mixed treatment comparison (MTC) meta-analysis was carried out to compile data about the effect of different collagenases used for human pancreas digestion on islet yield, purity, viability and stimulation index (SI). Pubmed, Embase and Cochrane libraries were searched. Of 755 articles retrieved, a total of 15 articles fulfilled the eligibility criteria and were included in the MTC meta-analysis. Our results revealed that Vitacyte and Liberase MTF were associated with a small increase in islet yield (islet equivalent number/g pancreas) when compared with Sevac enzyme [standardized mean difference (95% credible interval – CrI) = ?2.19 (?4.25 to ?0.21) and ?2.28 (?4.49 to ?0.23), respectively]. However, all other enzyme comparisons did not show any significant difference regarding islet yield. Purity and viability percentages were not significantly different among any of the analyzed digestion enzymes. Interestingly, Vitacyte and Serva NB1 were associated with increased SI when compared with Liberase MTF enzyme [unstandardized weighted mean difference (95% CrI) = ?1.69 (?2.87 to ?0.51) and ?1.07 (?1.79 to ?0.39), respectively]. In conclusion, our MTC meta-analysis suggests that the digestion enzymes currently being used for islet isolation works with similar efficiency regarding islet yield, purity and viability; however, Vitacyte and Serva NB1 enzymes seem to be associated with an improved SI as compared with Liberase MTF.     

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.     

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.     

胰岛移植治疗糖尿病的现状和进展

胰岛移植是治疗糖尿病尤其是1型糖尿病的一种简单有效的方法,相较与胰腺移植,它较为简单和方便,但存在组织来源匮乏和免疫移植排斥等障碍。新的胰岛分离纯化方法提高了供移植的胰岛的纯度和活性。成体干细胞研究、异种移植研究,有望解决移植的供源问题。Edmonton方案在胰岛移植的临床应用中具有里程碑意义。新型的免疫抑制剂和免疫诱导剂的研究可以提高临床胰岛移植的成功率。     

Current status of islet cell transplantation

Despite substantial advances in islet isolation methods and immunosuppressive protocol, pancreatic islet cell transplantation remains an experimental procedure currently limited to the most severe cases of type 1 diabetes mellitus. The objectives of this treatment are to prevent severe hypoglycemic episodes in patients with hypoglycemia unawareness and to achieve a more physiological metabolic control. Insulin independence and long term-graft function with improvement of quality of life have been obtained in several international islet transplant centers. However, experimental trials of islet transplantation clearly highlighted several obstacles that remain to be overcome before the procedure could be proposed to a much larger patient population. This review provides a brief historical perspective of islet transplantation, islet isolation techniques, the transplant procedure, immunosuppressive therapy, and outlines current challenges and future directions in clinical islet transplantation.     

Use of additives,scaffolds and extracellular matrix components for improvement of human pancreatic islet outcomes in vitro: A systematic review

Pancreatic islet transplantation is an established treatment to restore insulin independence in type 1 diabetic patients. Its success rates have increased lately based on improvements in immunosuppressive therapies and on islet isolation and culture. It is known that the quality and quantity of viable transplanted islets are crucial for the achievement of insulin independence and some studies have shown that a significant number of islets are lost during culture time. Thus, in an effort to improve islet yield during culture period, researchers have tested a variety of additives in culture media as well as alternative culture devices, such as scaffolds. However, due to the use of different categories of additives or devices, it is difficult to draw a conclusion on the benefits of these strategies. Therefore, the aim of this systematic review was to summarize the results of studies that described the use of medium additives, scaffolds or extracellular matrix (ECM) components during human pancreatic islets culture. PubMed and Embase repositories were searched. Of 5083 articles retrieved, a total of 37 articles fulfilled the eligibility criteria and were included in the review. After data extraction, articles were grouped as follows: 1) “antiapoptotic/anti-inflammatory/antioxidant,” 2) “hormone,” 3) “sulphonylureas,” 4) “serum supplements,” and 5) “scaffolds or ECM components.” The effects of the reviewed additives, ECM or scaffolds on islet viability, apoptosis and function (glucose-stimulated insulin secretion - GSIS) were heterogeneous, making any major conclusion hard to sustain. Overall, some “antiapoptotic/anti-inflammatory/antioxidant” additives decreased apoptosis and improved GSIS. Moreover, islet culture with ECM components or scaffolds increased GSIS. More studies are needed to define the real impact of these strategies in improving islet transplantation outcomes.     

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.     

The use of continuous density gradients for the assessment of islet and exocrine tissue densities and islet purification

The purification of large numbers of human pancreatic islets remains one of the limiting factors in islet transplantation. This paper describes and validates a method for accurately and reproducibly determining the density of islets and exocrine tissue in pancreatic digest on the basis of their isopycnic distribution on linear continuous density gradients. The use of this data to analyse and compare the purity of a standard 60% islet yield is described. The results obtained using such gradients will enable factors responsible for the variation in yield between pancreases to be determined and optimized, improving the results and reliability of islet purification.     

Islet amyloid deposition limits the viability of human islet grafts but not porcine islet grafts

Islet transplantation is a promising treatment for diabetes but long-term success is limited by progressive graft loss. Aggregates of the beta cell peptide islet amyloid polypeptide (IAPP) promote beta cell apoptosis and rapid amyloid formation occurs in transplanted islets. Porcine islets are an attractive alternative islet source as they demonstrate long-term graft survival. We compared the capacity of transplanted human and porcine islets to form amyloid as an explanation for differences in graft survival. Human islets were transplanted into streptozotocin-diabetic immune-deficient mice. Amyloid deposition was detectable at 4 weeks posttransplantation and was associated with islet graft failure. More extensive amyloid deposition was observed after 8 weeks. By contrast, no amyloid was detected in transplanted neonatal or adult porcine islets that had maintained normoglycemia for up to 195 days. To determine whether differences in IAPP sequence between humans and pigs could explain differences in amyloid formation and transplant viability, we sequenced porcine IAPP. Porcine IAPP differs from the human sequence at 10 positions and includes substitutions predicted to reduce its amyloidogenicity. Synthetic porcine IAPP was considerably less amyloidogenic than human IAPP as determined by transmission electron microscopy, circular dichroism, and thioflavin T binding. Viability assays indicated that porcine IAPP is significantly less toxic to INS-1 beta cells than human IAPP. Our findings demonstrate that species differences in IAPP sequence can explain the lack of amyloid formation and improved survival of transplanted porcine islets. These data highlight the potential of porcine islet transplantation as a therapeutic approach for human diabetes.     

Systematic review of islet cryopreservation

Pancreatic islet transplantation is being extensively researched as an alternative treatment for type 1 diabetic patients. This treatment is currently limited by temporal mismatch, between the availability of pancreas and isolated islets from deceased organ donor, and the recipient's need for freshly isolated islets. To solve this issue, cryopreservation of islets may offer the potential to bank islets for transplant on demand. Cryopreservation, however, introduces an overwhelmingly harsh environment to the ever-so-fragile islets. After exposure to the freezing and thawing, islets are usually either apoptotic, non-functional, or non-viable. Several studies have proposed various techniques that could lead to increased cell survival and function following a deep freeze. The purpose of this article is to critically review the techniques of islet cryopreservation, with the goal of highlighting optimization parameters that can lead to the most viable and functional islet upon recovery and/or transplant.     

Overexpression of pancreatitis-associated protein (PAP) in human pancreatic ductal adenocarcinoma

Pancreatitis-associated protein (PAP) is almost absent in normal pancreas, but is strongly induced in acute pancreatitis. PAP mRNA is also expressed in cancer cells, including pancreatic ductal adenocarcinoma. However, the clinicopathological significance of PAP in human pancreatic cancer is not clear. We examined PAP expression in pancreatic tissues from individuals with pancreatic ductal adenocarcinoma using immunohistochemistry. PAP was overexpressed in 79% (30 of 38) of pancreatic ductal adenocarcinoma, 19% (7 of 36) of chronic pancreatitis, and 29% (2 of 7) of mucinous cystadenoma. PAP was found in malignant ductular structures in pancreatic carcinomas as well as in benign proliferating ductules and acinar cells in chronic pancreatitis. It was not expressed in normal pancreas. The incidence of PAP overexpression was significantly higher in pancreatic cancer than in the other pancreatic diseases (P < 0.01). PAP overexpression was significantly correlated with nodal involvement, distant metastasis (P < 0.05), and short survival (P < 0.01) in pancreatic cancer. These results suggest that overexpression of PAP in human pancreatic ductal adenocarcinoma indicates tumor aggressiveness.     

Neurotensin receptors: a new marker for human ductal pancreatic adenocarcinoma

Background/Aims—New imaging possibilities forearly diagnosis of the devastating exocrine pancreatic adenocarcinomaswould be highly welcome. Recently, pancreatic neuroendocrine tumours have been successfully visualised in vivo on the basis of their highdensity of receptors for the regulatory peptide somatostatin. Unfortunately, exocrine pancreatic tumours do not express sufficient amounts of somatostatin receptors. Therefore overexpression of other regulatory peptide receptors in these tumours needs to be found.
Methods—Receptors for the regulatory peptideneurotensin were evaluated in vitro by receptor autoradiography in 24 ductal pancreatic adenocarcinomas, 20 endocrine pancreatic cancers, 18cases of chronic pancreatitis, and 10 normal pancreatic glands.
Results—Some 75% of all ductal pancreaticadenocarcinomas, most of them differentiated, were neurotensin receptorpositive, whereas endocrine pancreatic cancers did not expressneurotensin receptors. No neurotensin receptors were found in chronicpancreatitis or normal pancreatic tissues, including pancreatic acini,ducts, and islets.
Conclusions—The selective and high expression ofneurotensin receptors in ductal pancreatic adenocarcinomas could formthe molecular basis for potential clinical applications, such as in vivo neurotensin receptor scintigraphy for early tumour diagnosis, radiotherapy with radiolabelled neurotensin analogues, and chemotherapy with neurotensin receptor antagonists.

Keywords:neurotensin receptors; ductal pancreatic carcinomas; chronic pancreatitis; endocrine pancreatic tumours; in vivoscintigraphy; regulatory peptides