Automated Method for Isolation of Human Pancreatic Islets

Ricordi, Camillo; Lacy, Paul E.; Finke, Edward H.; Olack, Barbara; Scharp, David W.

doi:10.2337/diab.37.4.413

Cited by 1,188 publications

(727 citation statements)

References 7 publications

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“…Islets were isolated and purified using Ricordi's automated method [22] with local modifications [23]. After isolation, islets were suspended in Connaught Medical Research Laboratory (CMRL) medium containing 5.6 mmol/l glucose and 10% FCS, and incubated for 24 h at 37°C and at 24°C thereafter.…”

Section: Methodsmentioning

confidence: 99%

Insulin secretion from human beta cells is heterogeneous and dependent on cell-to-cell contacts

Wojtusciszyn

Armanet²,

Morel³

et al. 2008

Diabetologia

133

102

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Aims/hypothesis We assessed the heterogeneity of insulin secretion from human isolated beta cells and its regulation by cell-to-cell contacts. Methods Insulin secretion from single and paired cells was assessed by a reverse haemolytic plaque assay. The percentage of plaque-forming cells, the mean plaque area and the total plaque development were evaluated after 1 h of stimulation with different secretagogues. Results Not all beta cells were surrounded by a haemolytic plaque under all conditions tested. A small fraction of the beta cell population (20%) secreted more than 90% and 70% of total insulin at 2.2 and 22.2 mmol/l glucose, respectively. Plaque-forming cells, mean plaque area and total plaque development were increased at 12.2 and 22.2 compared with 2.2 mmol/l glucose. Insulin secretion of single beta cells was similar at 12.2 and 22.2 mmol/l glucose. Insulin secretion of beta cell pairs was increased compared with that of single beta cells and was higher at 22.2 than at 12.2 mmol/l glucose. Insulin secretion of beta cells in contact with alpha cells was also increased compared with single beta cells, but was similar at 22.2 compared with 12.2 mmol/l glucose. Delta and other non-beta cells did not increase insulin secretion of contacting beta cells compared with that of single beta cells. Differences in insulin secretion between 22.2 and 12.2 mmol/l glucose were observed in murine but not in human islets. Conclusions/interpretation Human beta cells are highly heterogeneous in terms of insulin secretion so that a small fraction of beta cells contributes to the majority of insulin secreted. Homologous and heterologous intercellular contacts have a significant impact on insulin secretion and this could be related to the particular architecture of human islets.

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Section: Methodsmentioning

confidence: 99%

Insulin secretion from human beta cells is heterogeneous and dependent on cell-to-cell contacts

Wojtusciszyn

Armanet²,

Morel³

et al. 2008

Diabetologia

133

102

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“…Islet isolation Islets were isolated using the automated method described by Ricordi [9]. Two types of enzymes were used: collagenase type P (1-3 mg/ml) and liberase (0.5-1.4 mg/ml) (Roche, Indianapolis, IN, USA).…”

Section: Subjects Materials and Methodsmentioning

confidence: 99%

Islet isolation for allotransplantation: variables associated with successful islet yield and graft function

et al. 2005

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Aims/hypothesis: Efficient islet isolation is an important prerequisite for successful clinical islet transplantation. Although progressively improved, islet yield and quality are, however, unpredictable and variable and require standardisation. Methods: Since 1989 we have processed 437 pancreases using the automated method. The donor characteristics, pancreas procurement, and digestion and purification procedures including a wide enzyme characterisation of these pancreases were analysed and correlated with islet yield and transplant outcome. Results: By univariate analysis, islet yield was significantly associated with donor age (r=0.16; p=0.0009), BMI (r=0.19; p= 0.0004), good pancreas condition (p=0.0031) and weight (r=0.15; p=0.0056), total collagenase activity (r=0.22; p= 0.0001), adjusted collagenase activity/mg (r=0.18; p=0.0002), collagenase activity/solution volume (r=0.18; p=0.0002) and neutral protease activity/solution volume (r=0.14; p= 0.0029). A statistically significant contribution to the variability of islet yield in a multivariate analysis performed on donor variables was found for donor BMI (p=0.0008). In a multivariate analysis performed on pancreas variables a contribution was found for pancreas weight (p=0.0064), and for a multivariate analysis performed on digestion variables we found a contribution for digestion time (p= 0.0048) and total collagenase activity (p=0.0001). Twenty-four patients with type 1 diabetes received single islet preparations from single donors. In these patients, multivariate analyses showed that the reduction in insulin requirement was significantly associated with morphological aspects of islets (p= 0.0010) and that 1-month C-peptide values were associated with islet purity (p=0.0071). Conclusions/interpretation: These data provide baseline donor, digestion and purification selection criteria for islet isolation using the automated method and indicate that the morphological aspect may be a clinically relevant measure of islets on which the decision for transplant can be based.

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“…Islet preparations were used for research purpose only if they could not be used for clinical islet transplantation. The islets were isolated at Giessen Islet Isolation and Transplantation Center according to a modified semi-automated digestion-filtration method, followed by purification on a continuous density gradient on a refrigerated COBE 2991 centrifuge [14,15]. Subsequently, islet preparations were placed in untreated culture flasks and maintained in suspension culture at 24°C (5% CO 2 in humidified air) for 4 to 7 days.…”

Section: Methodsmentioning

confidence: 99%

Long-Chain CoA esters activate human pancreatic beta-cell K ATP channels: potential role in Type 2 diabetes

et al. 2004

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Aims/hypothesis. The ATP-regulated potassium (K ATP ) channel in the pancreatic beta cell couples the metabolic state to electrical activity. The primary regulator of the K ATP channel is generally accepted to be changes in ATP/ADP ratio, where ATP inhibits and ADP activates channel activity. Recently, we showed that long-chain CoA (LC-CoA) esters form a new class of potent K ATP channel activators in rodents, as studied in inside-out patches. Methods. In this study we have investigated the effects of LC-CoA esters in human pancreatic beta cells using the inside-out and whole-cell configurations of the patch clamp technique. Results. Human K ATP channels were potently activated by acyl-CoA esters with a chain length exceeding 12 carbons. Activation by LC-CoA esters did not require the presence of Mg 2+ or adenine nucleotides. A detailed characterization of the concentration-dependent relationship showed an EC 50 of 0.7±0.1 µmol/l. Furthermore, in the presence of an ATP/ADP ratio of 10 (1.1 mmol/l total adenine nucleotides), whole-cell K ATP channel currents increased approximately sixfold following addition of 1 µmol/l LC-CoA ester. The presence of 1 µmol/l LC-CoA in the recording pipette solution increased beta-cell input conductance, from 0.5±0.2 nS to 2.5±1.3 nS. Conclusion/interpretation. Taken together, these results show that LC-CoA esters are potent activators of the K ATP channel in human pancreatic beta cells. The fact that LC-CoA esters also stimulate K ATP channel activity recorded in the whole-cell configuration, points to the ability of these compounds to have an important modulatory role of human beta-cell electrical activity under both physiological and pathophysiological conditions. [Diabetologia (2004) essential function has been included in the model of glucose-induced insulin secretion. In this model, increases in blood glucose levels lead to metabolism of glucose which generates an increase in the ATP/ADP ratio. The consensus view is that increases in the ATP/ADP ratio promote closure of the beta-cell K ATP channel, thereby depolarizing the cell membrane resulting in opening of voltage-dependent Ca 2+ channels. The subsequent rise in cytoplasmic free Ca 2+ concentration triggers a series of events leading to exocytosis of insulin. Hence, the K ATP channel provides a critical link between glucose metabolism, electrical activity and insulin secretion in the pancreatic beta cell.The most important molecular regulators of K ATP channel activity are believed to be adenine nucleo-ATP-sensitive potassium (K ATP ) channels play a key role in the coupling between cellular metabolism and electrical activity in a wide range of tissues. Since the discovery of these channels in the beta cell [1], their

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Automated Method for Isolation of Human Pancreatic Islets

Cited by 1,188 publications

References 7 publications

Insulin secretion from human beta cells is heterogeneous and dependent on cell-to-cell contacts

Insulin secretion from human beta cells is heterogeneous and dependent on cell-to-cell contacts

Islet isolation for allotransplantation: variables associated with successful islet yield and graft function

Long-Chain CoA esters activate human pancreatic beta-cell K ATP channels: potential role in Type 2 diabetes

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