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.     

Islet transplantation at the Diabetes Research Institute Japan

Since the Edmonton Protocol was announced, more than 600 patients with type 1 diabetes at more than 50 institutions have received islet transplantation to treat their disease. We recently established a new islet isolation protocol, called the Kyoto Islet Isolation Method, based on the Ricordi method. It includes an in-situ cooling system for pancreas procurement, pancreatic ductal protection, a modified two-layer (M-Kyoto /perfluorochemical [PFC]) method of pancreas preservation, and a new islet purification solution (Iodixanol-based solution). Using this islet isolation method, we isolated islets from 19 human pancreata of non-heart-beating donors and transplanted 16 preparations into seven patients with type 1 diabetes between April 7, 2004 and November 18, 2005. The percentage of those meeting the release criteria of the Edmonton Protocol was more than 80%. We also performed living-donor transplantation of islets for unstable diabetes on January 19, 2005. Establishment of this method enables us to make diabetic patients insulin-independent, using islets not only from two or three pancreata of non-heart-beating donors but also using islets from half a pancreas from a living donor.     

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.     

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.     

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.     

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.     

Islet cell transplantation for the treatment of type 1 diabetes in the USA

Islet cell transplantation (ICTx) is one of the most effective treatments for type 1 diabetes and is less invasive compared to whole organ transplantation. The US has been the leader in the research and clinical applications of ICTx for the last 40 years. ICTx requires complex procedures, including pancreas procurement and preservation; pancreas digestion; islet purification; and transplantation. Even with the dramatic progresses in each of the procedures listed above, there are still challenges to make ICTx the standard therapy. These challenges are: (1) obtaining enough islets from a single donor and (2) preventing graft loss due to allogenic rejection and recurrence of autoimmune islet destruction. A new preservation strategy for pancreata and pancreatic ducts using ET-Kyoto solution as well as a new islet purification method using iodixanol has substantially improved islet yields. Continuous research to improve the efficacy of islet isolation will solve the issue of obtaining enough islets from a single donor. Immunological tolerance is an ideal solution for the issue of rejection and autoimmune recurrence and a regulatory T cell strategy seems promising. Moreover, the SUITO index is a simple and powerful tool to assess engrafted islet mass and is, therefore, useful for evaluating the efficacy of new immunosuppressant strategies. Once ICTx becomes a standard treatment, the donor shortage will become the next challenge. Marginal or living donor islet transplantations could help alleviate this issue; however, bio-artificial islet transplantation with animal islets could be the ultimate solution.     

Evaluation of collagenase gold plus BP protease in isolating islets from human pancreata

Selection of enzymes for optimal pancreas digestion is essential for successful human islet isolations. The aim of this study was to evaluate the efficacy and outcome of using Collagenase Gold plus BP protease (VitaCyte) (n = 8) by comparing it to two commercially available enzymes, Liberase MTF C/T (Roche) (n = 48) and Collagenase NB1/NP (Serva) (n = 15). The isolation outcomes were assessed by islet counting, viability, glucose-stimulated oxygen consumption rate (OCR), and successful graft-rate following transplantation in diabetic NOD scid mice. The pancreas donor characteristics were not significantly different between the tested enzyme groups regarding their BMI, pancreas weight, cold ischemia time (CIT) and HbA1c. The results show that digested tissue volume was not statistically significant between the VitaCyte enzyme (34.25 ± 5.4 mL) and the Roche enzyme (55.25 ± 3.42 mL, p = 0.073), however, this was significant with Serva enzyme (64.07 ± 7.95 mL, p = 0.020). Interestingly, the islet yields were not statistically different between all enzyme groups. Moreover, when islets were transplanted into NOD scid mice, the reversal rate of diabetes for the VitaCyte enzyme group was similar to all enzyme groups. In conclusion, the effectiveness of Collagenase Gold plus BP protease is comparable to the MTF C/T and the Collagenase NB1/NP enzymes; the low cost could facilitate the use of more pancreata for islet isolations.     

A new collagenase blend increases the number of islets isolated from mouse pancreas

Diabetes is a predominant metabolic disorder in the industrialized nations. Since pancreatic islets play a key role in type I and type II diabetes, the isolation of islets from pancreatic tissues represents an important step in diabetes research. However, to date, only a small fraction of all islets, resident within any given pancreas, are harvested by using currently available enzyme blends. This holds true for islet isolation from both the mouse and the human pancreas. In the present study, the newly developed Liberase TL Research Grade was compared to the widely used Liberase RI to investigate the effect of increased collagenase purity on islet yield. The study shows that reducing the degradation products of collagenases during Liberase production significantly increases the number of islets isolated from the mouse pancreas by 28%, and, therefore, is expected to lower the numbers of mice and resulting costs for diabetes research accordingly. Furthermore, this study also points to a possibility to increase the number and mass of islets isolated from human pancreases, for which only a limited donor pool exists.     

A new collagenase blend increases the number of islets isolated from mouse pancreas

Diabetes is a predominant metabolic disorder in the industrialized nations. Since pancreatic islets play a key role in type I and type II diabetes, the isolation of islets from pancreatic tissues represents an important step in diabetes research. However, to date, only a small fraction of all islets, resident within any given pancreas, are harvested by using currently available enzyme blends. This holds true for islet isolation from both the mouse and the human pancreas. In the present study, the newly developed Liberase TL Research Grade was compared to the widely used Liberase RI to investigate the effect of increased collagenase purity on islet yield. The study shows that reducing the degradation products of collagenases during Liberase production significantly increases the number of islets isolated from the mouse pancreas by 28%, and, therefore, is expected to lower the numbers of mice and resulting costs for diabetes research accordingly. Furthermore, this study also points to a possibility to increase the number and mass of islets isolated from human pancreases, for which only a limited donor pool exists.     

Augmented damage of islets by impaired exocrine acinar cells undergoing apoptosis that is possibly converted to necrosis during isolation

Islet damage attributed to impaired exocrine cells during pancreas preservation and isolation procedure remains elusive, although released exocrine enzymes could directly damage islets. The aim of this study is to investigate the cellular mechanisms associated with exocrine cells and their possible impact on the islet cell survival and function after isolation. Mouse pancreata were stored in cold University of Wisconsin preservation solution for 0, 24 and 48 h and incubated with or without collagenase at 37°C for 15 min. During preservation, the percentage of exocrine cells with necrosis, which means impaired cellular membrane that allows intracellular enzymes to be released, remains low (< 10%) regardless of preservation time; whereas the percentage of exocrine cells with apoptosis, which means impaired nucleus and possible intact cellular membrane, increases over time of preservation. After collagenase-free incubation, however, the percentage of exocrine cells with necrosis became higher in longer preservation time, and more than 60% of the necrotic exocrine cells contained apoptosis as well. Islet cells located in pancreata with intact structure are almost kept away either from necrotic or apoptotic changes even after 48 h preservation followed by collagenase-free incubation. However, when islets are isolated after collagenase-containing incubation, the percentage of islet cells with necrosis increases over time of preservation up to approximately 40%. This study suggests that exocrine cells with necrosis could cause damage of isolated islets when the pancreas is dissociated and that the necrosis in exocrine cells might be induced mainly as the conversion from apoptosis that has already existed during preservation.     

Augmented damage of islets by impaired exocrine acinar cells undergoing apoptosis that is possibly converted to necrosis during isolation

Islet damage attributed to impaired exocrine cells during pancreas preservation and isolation procedure remains elusive, although released exocrine enzymes could directly damage islets. The aim of this study is to investigate the cellular mechanisms associated with exocrine cells and their possible impact on the islet cell survival and function after isolation. Mouse pancreata were stored in cold University of Wisconsin preservation solution for 0, 24 and 48 h and incubated with or without collagenase at 37℃ for 15 min. During preservation, the percentage of exocrine cells with necrosis, which means impaired cellular membrane that allows intracellular enzymes to be released, remains low (     

Improved quality and yield of islets isolated from human pancreata using a two-step digestion method

A new approach, involving a two-step digestion process and Los Angeles preservation solution #1 (LAP-1), a cold storage solution, was developed for isolation of high-quality islets from human pancreata for transplantation. This approach markedly improves the islet yield, purity and viability, and the isolation success rate. In this method, the pancreas was digested first in warm collagenase solution for up to 20 minutes. After decanting the enzyme solution, partially digested tissue was dissociated by gentle agitation in cold LAP-1 solution without additional collagenase. The digested tissues were stored in cold LAP-1 solution until islet purification on Euro-Ficoll. Forty-six islet isolations were performed consecutively by the new method (group 1). These results were compared to those obtained earlier with 46 consecutive isolations, using our previous method that had been used before development of the new method (group 2). Our old method was a modification of Ricordi's method involving only warm collagenase digestion and the storage of digested tissues in cold Hanks balanced salt solution. All pancreata were partial, containing the body and tail. There were no significant differences in both groups with regard to the donor age, cold ischemic time, harvesting conditions, and pancreatic weight. Pancreas digestion was completed in approximately 1 hour in both groups. The isolation success rate as determined by viable islets after 2 days in culture was 93.5% (43 of 46 cases) in group 1, and 56.5% (26 of the 46) in group 2. Immediately after isolation, the new method yielded a total of 335,739 +/- 36,244 islets equivalent to 150 microm (IEQ) and 6,233 +/- 681 IEQ/g of pancreas with 83 +/- 2.5% purity, whereas the old method yielded a total of 195,587 +/- 25,242 IEQ and 3,763 +/- 5,509 IEQ/g with 69.2 +/- 4.7% purity. Isolated islets in group 1 maintained a good three-dimensional structure, displayed normal insulin release to high glucose stimulation in vitro, and restored euglycemia after transplantation into streptozotocin-diabetic athymic mice. The two-step digestion method provides a sufficient number of islets for transplantation from a single pancreas.     

Autologous islet cell transplantation to prevent surgical diabetes

Autologous islet transplantation (AIT) is performed to prevent surgical diabetes after total or semi-total pancreatectomy for the treatment of chronic pancreatitis with severe abdominal pain. In addition, AIT is used in cases of benign pancreatic tumors and pancreatic trauma. It has been shown that AIT results in better outcomes in terms of glycemic control compared with allogeneic islet transplantation. The reasons for the favorable outcomes of AIT are thought to be: (i) patients have no autoimmune diseases; (ii) the transplanted islets do not suffer allogeneic rejection; (iii) diabetogenic antirejection drugs are not required; (iv) pancreata do not undergo a cytokine storm as a result of periods of brain death; (v) the period of cold preservation of retrieved pancreata is short; (vi) the isolated islets are immediately transplanted without culture; and (vii) pancreata with pancreatitis may contain more progenitor cells. Further research into AIT would help improve the results of allogeneic islet transplantation. Conversely, the technical difficulties associated with islet isolation appear to be the largest hurdle for AIT; therefore, remote center islet isolation may prove to be key in the promotion of this treatment.     

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 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.     

Pancreatic islet isolation variables in non-human primates (rhesus macaques)

BACKGROUND: Non-human primates (NHPs) are important preclinical models for pancreatic islet transplantation (PIT) because of their close phylogenetic and immunological relationship with humans. However, low availability of NHP tissue, long learning curves and prohibitive expenses constrain the consistency of isolated NHP islets for PIT studies. To advance preclinical studies, we attempted to identify key variables that consistently influence the quantity and quality of NHP islets. METHODS: Seventy-two consecutive pancreatic islet isolations from rhesus macaques were reviewed retrospectively. A scaled down, semi-automated islet isolation method was used, and monkeys with streptozotocin-induced diabetes, weighing 3-7 kg, served as recipients for allotransplantation. We analysed the effects of 22 independent variables grouped as donor factors, surgical factors and isolation technique factors. Islet yields, success of isolation and transplantation results were used as quantitative and qualitative outcomes. RESULTS: In the multivariate analysis, variables that significantly affected islet yield were the type of monkey, pancreas preservation, enzyme lot and volume of enzyme delivered. The variables associated with successful isolation were the enzyme lot and volume delivered. The transplant result was correlated with pancreas preservation, enzyme lot, endotoxin levels and COBE collection method. CONCLUSIONS: Islet quantity and quality are highly variable between isolations. The data reviewed suggest that future NHP isolations should use bilayer preservation, infuse more than 80 ml of Liberase into the pancreas, collect non-fractioned tissue from the COBE, and strictly monitor for infection.     

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.     

Neonatal rat islets of Langerhans and fetal rat pancreas Isolation,immunohistochemical, functional,and autoradiographic evaluation

The aim of this study was to develop an optimal isolation technique for neonatal rat islets of Langerhans, to perform functional evaluation in vitro, to evaluate immunohistochemically isolated rat islets and fetal rat pancreata after a variable period of culture, and to study growth potentials by means of autoradiography. The islets were isolated using minor modifications of standard procedures including collagenase and DNase. Islets were separated on a discontinuous Percoll gradient. The maximum yield of islets amounted to 240 per pancreas. Fetal pancreata from rats were cultured under similar conditions as neonatal islets to compare their insulin secretory capacity after different periods of culture. The insulin secretion increased gradually, and isolated islets achieved a similar secretion potential to adult rat islets. The mitotic activity of both islets and fetal pancreata was confirmed using tritiated thymidine. The isolation procedure was found suitable for producing well-functioning islets, which could be kept in culture for a period of about 1 month without deterioration in their insulin secretory capacity. The gradual increase in insulin secretory capacity of islets and fetal pancreata was due, in part, to hyperplasia and not just hypertrophia. Autoradiographical evaluation revealed a high mitotic activity after culture, in particular of fetal pancreata. Fetal pancreata cultured for about 10 days showed a phenomenon of budding endocrine cells at the organ surface. A high mitotic activity was found in these buds.