BACKGROUND: Europe is currently the most active region in the field of pancreatic islet transplantation, and many of the leading groups are actually achieving similar good outcomes. Further collaborative advances in the field require the standardization of islet cell product isolation processes, and this work aimed to identify differences in the human pancreatic islet isolation processes within European countries. METHODS: A web-based questionnaire about critical steps, including donor selection, pancreas processing, pancreas perfusion and digestion, islet counting and culture, islet quality evaluation, microbiological evaluation, and release criteria of the product, was completed by isolation facilities participating at the Ninth International European Pancreas and Islet Transplant Association (EPITA) Workshop on Islet-Beta Cell Replacement in Milan. RESULTS: Eleven islet isolation facilities completed the questionnaire. The facilities reported 445 and 53 islet isolations per year over the last 3 years from deceased organ donors and pancreatectomized patients, respectively. This activity resulted in 120 and 40 infusions per year in allograft and autograft recipients, respectively. Differences among facilities emerged in donor selection (age, cold ischemia time, intensive care unit length, amylase concentration), pancreas procurement, isolation procedures (brand and concentration of collagenase, additive, maximum acceptable digestion time), quality evaluation, and release criteria for transplantation (glucose-stimulated insulin secretion tests, islet numbers, and purity). Moreover, even when a high concordance about the relevance of one parameter was evident, thresholds for the acceptance were different among facilities. CONCLUSIONS: The result highlighted the presence of a heterogeneity in the islet cell product process and product release criteria.

• MeSH
• časové faktory MeSH
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• kultivované buňky transplantace   MeSH
• Langerhansovy ostrůvky cytologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• novorozenec MeSH
• odběr tkání a orgánů metody   normy   statistika a číselné údaje   MeSH
• perfuze metody   statistika a číselné údaje   MeSH
• počet buněk normy   statistika a číselné údaje   MeSH
• předškolní dítě MeSH
• primární buněčná kultura metody   normy   statistika a číselné údaje   MeSH
• průzkumy a dotazníky statistika a číselné údaje   MeSH
• senioři MeSH
• separace buněk metody   statistika a číselné údaje   MeSH
• směrnice pro lékařskou praxi jako téma MeSH
• studená ischemie normy   statistika a číselné údaje   MeSH
• transplantace Langerhansových ostrůvků metody   normy   MeSH
• věkové faktory MeSH
• výběr dárců metody   normy   statistika a číselné údaje   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• kojenec MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mladý dospělý MeSH
• novorozenec MeSH
• předškolní dítě MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Evropa MeSH

Variability of pancreatic donors may significantly impact the success of islet isolation. The aim of this study was to evaluate donor factors associated with isolation failure and to investigate whether immunohistology could contribute to organ selection. Donor characteristics were evaluated for both successful (n = 61) and failed (n = 98) islet isolations. Samples of donor pancreatic tissue (n = 78) were taken for immunohistochemical examination. Islet isolations with 250000 islet equivalents were considered successful. We confirmed that BMI of less than 25 kg/m(2) (P < 0.001), cold ischemia time more than 8 hours (P < 0.01), hospitalization longer than 96 hours (P < 0.05), higher catecholamine doses (P < 0.05), and edematous pancreases (P < 0.01) all unfavorably affected isolation outcome. Subsequent immunohistochemical examination of donor pancreases confirmed significant differences in insulin-positive areas (P < 0.001). ROC analyses then established that the insulin-positive area in the pancreas could be used to predict the likely success of islet isolation (P < 0.001). At the optimal cutoff point (>1.02%), sensitivity and specificity were 89% and 76%, respectively. To conclude, while the insulin-positive area, determined preislet isolation, as a single variable, is sufficient to predict isolation outcome and helps to improve the success of this procedure, its combination with the established donor scoring system might further improve organ selection.

• MeSH
• dárci tkání statistika a číselné údaje   MeSH
• délka pobytu statistika a číselné údaje   MeSH
• diabetes mellitus 1. typu chirurgie   MeSH
• edém epidemiologie   MeSH
• hospitalizace MeSH
• imunohistochemie MeSH
• index tělesné hmotnosti MeSH
• inzulin metabolismus   MeSH
• Langerhansovy ostrůvky metabolismus   chirurgie   MeSH
• lidé MeSH
• pankreas MeSH
• retrospektivní studie MeSH
• studená ischemie statistika a číselné údaje   MeSH
• transplantace Langerhansových ostrůvků metody   MeSH
• vazokonstriktory terapeutické užití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Successful isolation of Langerhans islets is a crucial prerequisite for their experimental or possible clinical use such as transplantation. Centrifugation in a Ficoll gradient is a common step used for separation of Langerhans islets from exocrine tissue. However, islets have been reported to be negatively affected by employing Ficoll gradients. Therefore, the aim of this study was to modify the isolation procedure by excluding Ficoll gradient centrifugation to obtain a similar or better yield of viable, functional islets. In our modification of the isolation procedure, the separation of islets from exocrine tissue was based on their sedimentation rate combined with their differential ability to attach to the surface of culture dishes for suspension cells. The resulting purity of islets facilitated their handpicking from the suspension. The mean yield was 900 viable, insulin-producing islets per mouse, which was comparable to or even higher than the yield in commonly used protocols. Our modification of the isolation method may be useful when centrifugation in Ficoll gradient is undesirable due to potential toxicity.

• MeSH
• buněčná adheze MeSH
• Langerhansovy ostrůvky cytologie   MeSH
• myši inbrední BALB C MeSH
• myši inbrední ICR MeSH
• myši MeSH
• pankreas MeSH
• počet buněk MeSH
• separace buněk metody   MeSH
• transplantace Langerhansových ostrůvků metody   MeSH
• viabilita buněk MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

• MeSH
• diabetes mellitus 1. typu terapie   MeSH
• kolagenasy MeSH
• myši MeSH
• psi MeSH
• transplantace Langerhansových ostrůvků metody   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• psi MeSH
• zvířata MeSH

Organ pancreas transplantation represents the only method enabling long-term normalization of glucose metabolism in type-1 diabetic subjects so far. Unfortunately, surgical complications of this kind of therapy are still frequent. As a safer alternative, transplantation of isolated pancreatic islets was introduced at the Institute for Clinical and Experimental Medicine as a clinical experiment in the year 2005. METHODS AND RESULTS: We isolated the islets from pancreases of cadaveric donors which did not fulfil criteria to perform organ pancreas transplantation. Altogether, 36 islet implantations were performed in 28 C-peptide negative subjects suffering from type-1 diabetes by August 2010. In 15 subjects (21 implantations) the main indication was extremely instable course of diabetes due to the hypoglycaemia unawareness syndrome. In 5 and 3 cases, combined islet and kidney and islet and liver transplants were performed, respectively. In addition, islet autotransplantation was performed in 5 subjects undergoing total pancreatectomy. No patient died during the study period. In all but 1 patient with primary islet afunction, islet transplantation led to a complete cure of the hypoglycemia unawareness syndrome. Out of 15 patients, 11 subjects in this group showed a significant C-peptide production (> 0.2 pmol/ml) after 1 year. The mean insulin dose after allotransplantation decreased from 37 to 14 units per day and in 3 subjects, insulin therapy could be withdrawn. Serious technical complications occurred in 6 subjects, which only in 2 cases required surgical revision and did not cause long-term sequels. CONCLUSIONS: In comparison with organ pancreas transplantation, pancreatic islet transplantation represents a substantially safer method for restitution of endogenous insulin production. Though it eliminates serious hypoglycemic episodes in labile diabetes, complete insulin withdrawal is still often not possible. However, due to continuing progress in the laboratory techniques as well as in the transplant procedure itself, the results are steadily improving.

• Klíčová slova
• slinivka břišní, imunosuprese,
• MeSH
• diabetes mellitus 1. typu farmakoterapie   chirurgie   krev   MeSH
• dospělí MeSH
• hypoglykemika terapeutické užití   MeSH
• inzulin terapeutické užití   MeSH
• komplikace diabetu MeSH
• krevní glukóza analýza   MeSH
• lidé středního věku MeSH
• lidé MeSH
• transplantace Langerhansových ostrůvků metody   přístrojové vybavení   škodlivé účinky   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• kazuistiky MeSH

We previously developed a deep learning-based web service (IsletNet) for an automated counting of isolated pancreatic islets. The neural network training is limited by the absent consensus on the ground truth annotations. Here, we present a platform (IsletSwipe) for an exchange of graphical opinions among experts to facilitate the consensus formation. The platform consists of a web interface and a mobile application. In a small pilot study, we demonstrate the functionalities and the use case scenarios of the platform. Nine experts from three centers validated the drawing tools, tested precision and consistency of the expert contour drawing, and evaluated user experience. Eight experts from two centers proceeded to evaluate additional images to demonstrate the following two use case scenarios. The Validation scenario involves an automated selection of images and islets for the expert scrutiny. It is scalable (more experts, images, and islets may readily be added) and can be applied to independent validation of islet contours from various sources. The Inquiry scenario serves the ground truth generating expert in seeking assistance from peers to achieve consensus on challenging cases during the preparation for IsletNet training. This scenario is limited to a small number of manually selected images and islets. The experts gained an opportunity to influence IsletNet training and to compare other experts' opinions with their own. The ground truth-generating expert obtained feedback for future IsletNet training. IsletSwipe is a suitable tool for the consensus finding. Experts from additional centers are welcome to participate.

• MeSH
• Langerhansovy ostrůvky * MeSH
• neuronové sítě MeSH
• pilotní projekty MeSH
• transplantace Langerhansových ostrůvků * metody   MeSH
• znalecký posudek MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• diabetes mellitus 1. typu * MeSH
• transplantace Langerhansových ostrůvků * MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• diabetologie
• NLK Publikační typ
• kolektivní monografie

In order to assess the quality of freshly isolated and cultivated pancreatic islets designed for experimental transplantation in rats we combined the vitality staining test, in vitro measurement of insulin secretion capacity, and assessment of islet respiration. Oxygen consumption was measured using the respirometer Oxygraph 2K equipped with polarographic oxygen sensors. The results of oxymetry demonstrated a linear correlation between islet number and oxygen consumption. Respiration per unit of viable islet tissue was constant. Oxygen consumption tests were in good correlation with the results of insulin release assays, with a correlation coefficient of 0.82. We found no significant differences in all three vitality-testing methods performed with fresh and 24-hour cultivated islets (P > .05). We conclude that polarographic oxymetry provides a fast and easy evaluation test of islet quality. After appropriate standardization, the oxymetric technique can be used for routine clinical pretransplant islet quality testing. In addition, cell membrane integrity and mitochondrial function could be assessed after addition of specific respiration inhibitors or stimulators.

• MeSH
• inzulin sekrece   MeSH
• krysa rodu rattus MeSH
• kultivované buňky MeSH
• Langerhansovy ostrůvky * cytologie   fyziologie   sekrece   MeSH
• oxymetrie metody   MeSH
• potkani Wistar MeSH
• spotřeba kyslíku * fyziologie   MeSH
• viabilita buněk MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

• MeSH
• finanční podpora výzkumu jako téma MeSH
• krysa rodu rattus MeSH
• Langerhansovy ostrůvky cytologie   metabolismus   MeSH
• počet buněk MeSH
• počítačové zpracování obrazu metody   MeSH
• separace buněk MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH

BACKGROUND: Diabetic Goto Kakizaki (GK) rats represent an established model of type 2 diabetes that exhibit an onset of pancreatic islet (PI) pathology characterized by islet hypertrophy with a decreased number of insulin-secreting beta-cells. Among the remaining beta-cells, oxidative phosphorylation (OXPHOS) and consequently glucose-stimulated insulin secretion (GSIS) are impaired, perhaps owing to a deficit in mitochondrial DNA (mtDNA). We sought to identify this abnormality. METHODS: beta-Cells were obtained from Accutase-dissolved PI isolated from GK or Wistar rats and sorted based on the positive Zn(2+) signal of Newport Green. The mtDNA copy number per cell was quantified as the amplicon ratio by polymerase chain reaction using specific primers against the rat ND5 mt gene and UCP2 nuclear gene. RESULTS: The 12-month-old GK rats exhibited drastically reduced copy numbers per remaining beta-cell, from 7,400 + 600 in 12-month old Wistar rats (100%) to 24 + 4%; mtDNA content in heart and liver was 70 + 25% and 60 + 20%, respectively. Versus age-paired Wistar rats, 6- and 4-month-old GK rats showed reductions to 60 + 15% and 50 + 20%, respectively. CONCLUSIONS: OXPHOS of remnant beta-cells in diabetic GK was drastically impaired due to the lack of sufficient mtDNA levels. We suggest the use of mtDNA quantification to quickly assess PI quality before transplantation. Copyright 2011 Elsevier Inc. All rights reserved.

• MeSH
• buněčné kultury metody   MeSH
• experimentální diabetes mellitus * metabolismus   MeSH
• genová dávka MeSH
• glukosa metabolismus   MeSH
• inzulin metabolismus   MeSH
• krysa rodu rattus MeSH
• Langerhansovy ostrůvky * cytologie   MeSH
• mitochondriální DNA * genetika   metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• oxidativní fosforylace MeSH
• separace buněk MeSH
• spotřeba kyslíku MeSH
• zinek metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH