Influence of donor and pancreatic characteristics on the success rate of islet isolation / 中华内分泌代谢杂志

Objective:To explore the selection criteria of the donor for islet transplantation of Chinese people by analyzing the correlation between pancreas characteristics and success rate of islets isolation.Methods:Data from 113 cases of human islet isolation were collected. According to the result of islet isolation, the donors were divided into two groups, the success group(IEQ≥250 000, purification≥30%, and viability≥80%), and the failure group(IEQ<250 000, or purification<30%, or viability<80%). The modified Ricordi method was used to digest pancreas tissue, and the continuous density gradient method was performed to purify islets. The islets were identified by staining with the Dithizone(DTZ), the islets were analyzed for cell viability and purity.Results:The donor age in success group was significantly younger than failure group in the range of age eligible for this study( t=2.479, P=0.015). Pearson correlation showed that donor age was positively corelated with islet yield( r=-0.214, P=0.047). There was more fat on the pancreas surface in the successful islet isolation group( z=-2.007, P=0.045). The digestibility( t=2.133, P=0.035) and recovery rate( t=5.912, P=0.001) were elevated in success group. Conclusion:The pancreases from younger donors could obtain the higher-yielding islet, the pancreas with more surface fat or with higher weight was associated with islet isolation success in the scope covered by the inclusion criteria of this study.

Comparison of two techniques for assessing pancreatic islet viability: flow cytometry and fluorescein diacetate/propidium iodine staining

Introduction: The success of islet transplantation for patients with unstable type 1 diabetes mellitus depends, in part, on the number of isolated islets and their quality, which is assessed by functional and viability tests. The test currently employed to evaluate islet viability, used by the Collaborative Islet Transplant Registry to release products for transplantation, is fluorescein diacetate/propidium iodide (FDA/PI) staining. However, the efficacy of this method relies on researcher experience; in this context, a quantitative method may be useful. The aim of this study was to compare islet viability as assessed by flow cytometry and the FDA/PI assay. Methods: Viability was analyzed in islets isolated from 10 male Wistar rats. Upon FDA/PI staining, 50 islets from each animal were analyzed under fluorescence microscopy by two well-trained researchers. For flow cytometry, islets were dispersed and 100 000 single cells were incubated with the 7-amino-actinomycin D (7AAD) fluorophore (dyes necrotic and late apoptotic cells) and the Annexin V-APC antibody (marks early apoptotic cells). Results: A moderate correlation was found between techniques (r = 0.6; p = 0.047). The mean islet viability measured by flow cytometry was higher than that estimated using FDA/PI staining (95.5 ± 1.4% vs 89.5 ± 5.0%; p = 0.002). Conclusions: Although flow cytometry is more expensive and time-consuming than FDA/PI staining, it is a quantitative technique with greater reproducibility that is less subject to inter-observer variability than FDA/PI. Therefore, flow cytometry appears to be the technique of choice when aiming for a more precise determination of islet viability. (AU)

Comparison of different collagenases in islet isolation from Chinese organ donors / 中华器官移植杂志

Objective To retrospectively compare the efficacy of Serva NB1 collagenase with Vitacyte GOLD collagenase on islet isolation of pancreas.Methods All the human pancreata were obtained from Chinese organ donors.In GMP laboratory,the pancreata were trimmed and distended with Serva NB1 collagenase (Serva NB1,n =12) or Vitacyte GOLD collagenase (Vitacyte GOLD,n =5) and digested according to a modified Ricordi semi-automatic protocol,and the digestion duration was recorded.The digested islets were then collected and washed,followed by the continuous density purification in a Cobe 2991 cell separator.The islet yield,purity,viability and glucose-stimulated insulin release (GSI) were determined each time after purification.Quantity and quality of isolated islets were determined by digestion efficacy.Results The digestion duration in Vitacyte GOLD collagenase group was significantly shorter than in Serva NB1 collagenase group to achieve the same digestion endpoint (P＜ 0.05).The islets yields of different sizes were variable between the two groups.The Vitacyte GOLD collagenase digestion produced more islets with a diameter range of 50-100 μm than the ServaNB1 collagenase digestion (P＜0.05),but the latter yielded more islets with a diameter range of 251-300 μm and 301-350μm (P＜0.05).There was no significant difference in total islets yields,viability,and GSI between two collagenase digestions (P＞0.05).Conclusion Both Vitacyte GOLD collagenase and Serva NB1 collagenase can be used for the clinical islet isolation in China.

Human pancreatic islet transplantation: an update and description of the establishment of a pancreatic islet isolation laboratory

Type 1 diabetes mellitus (T1DM) is associated with chronic complications that lead to high morbidity and mortality rates in young adults of productive age. Intensive insulin therapy has been able to reduce the likelihood of the development of chronic diabetes complications. However, this treatment is still associated with an increased incidence of hypoglycemia. In patients with “brittle T1DM”, who have severe hypoglycemia without adrenergic symptoms (hypoglycemia unawareness), islet transplantation may be a therapeutic option to restore both insulin secretion and hypoglycemic perception. The Edmonton group demonstrated that most patients who received islet infusions from more than one donor and were treated with steroid-free immunosuppressive drugs displayed a considerable decline in the initial insulin independence rates at eight years following the transplantation, but showed permanent C-peptide secretion, which facilitated glycemic control and protected patients against hypoglycemic episodes. Recently, data published by the Collaborative Islet Transplant Registry (CITR) has revealed that approximately 50% of the patients who undergo islet transplantation are insulin independent after a 3-year follow-up. Therefore, islet transplantation is able to successfully decrease plasma glucose and HbA1c levels, the occurrence of severe hypoglycemia, and improve patient quality of life. The goal of this paper was to review the human islet isolation and transplantation processes, and to describe the establishment of a human islet isolation laboratory at the Endocrine Division of the Hospital de Clínicas de Porto Alegre – Rio Grande do Sul, Brazil.

Transplante de ilhotas pancreáticas humanas: revisão da literatura e implantação de um laboratório de isolamento de ilhotas pancreáticas / Human pancreatic islet transplantation: literature review and establishment of a pancreatic islet isolation laboratory

O diabetes melito tipo 1 (DM1) está associado ao desenvolvimento de complicações crônicas de elevada morbi-mortalidade em indivíduos jovens em idade produtiva. A terapia intensiva com insulina comprovadamente diminui o aparecimento das complicações crônicas da doença. Entretanto, essa terapia ainda está associada ao aumento da incidência de hipoglicemia. Em pacientes com “DM1 lábil”, os quais apresentam hipoglicemias graves sem sintomas de alerta, o transplante de ilhotas pancreáticas humanas é uma das melhores alternativas para restaurar a secreção de insulina e a percepção da hipoglicemia. Cerca de 80% dos pacientes que receberam transplante de ilhotas de mais de um doador, submetidos ao tratamento imunossupressor do protocolo de Edmonton, adquiriram independência de insulina após 1 ano do transplante. Porém, apenas 10% destes pacientes permaneceram livres de insulina após 5 anos. Entretanto, mesmo aqueles pacientes que necessitaram utilizar novamente insulina tiveram a normalização da homeostase glicêmica e da percepção da hipoglicemia, com prevenção da hipoglicemia grave. Sendo assim, o transplante de ilhotas é capaz de diminuir os níveis de glicose plasmática e HbA1c, reduzir a ocorrência de hipoglicemias graves e melhorar a qualidade de vida dos pacientes. O objetivo deste artigo foi fazer uma breve revisão da literatura sobre o isolamento e transplante de ilhotas pancreáticas humanas e relatar a implantação de um laboratório de isolamento de ilhotas humanas no Serviço de Endocrinologia do Hospital de Clínicas de Porto Alegre.

Type 1 diabetes mellitus (DM1) is associated with chronic complications of high morbidity and mortality in young adults in a productive age. Insulin therapy has proved to reduce the chronic complications of diabetes. However, this therapy is still associated to an increased incidence of hypoglycemia. In patients with “brittle DM1”, who have severe hypoglycemia without any symptoms (hypoglycemia unawareness), the pancreatic islet transplantation is one of the best alternatives for restoring insulin secretion and hypoglycemia perception. About 80% of the patients who received islet transplantation from more than one donor, on immunosuppressive treatment with the Edmonton’s protocol, maintained insulin independence 1 year after transplantation. Nevertheless, only 10% of these patients remained free of insulin after 5 years post-transplantation. However, even those patients who returned to insulin treatment had a normalization of the glucose homeostasis and hypoglycemia perception. Therefore, islet transplantation is able to diminish plasmatic glucose and HbA1c levels, to reduce the occurrence of severe hypoglycemia, and to improve the quality of life of the patients. The purpose of this paper is to briefly review islet isolation and transplantation process, and report the establishing of a human islet isolation laboratory in the Endocrine Service at Hospital de Clínicas de Porto Alegre.

Standardization of Isolation Procedure and Analysis of Variables on Successful Isolation of Islet from the Human Pancreas / 대한내분비학회지

BACKGROUND: Identifying the donor and isolation-related factors during the islet isolation would be greatly helpful to improve the result of human islet isolation for successful clinical islet transplantation. METHODS: Sixty-nine pancreata from cadaveric donors were isolated with standard protocol and analyzed to identify the donor factors and isolation variables for successful isolation. Islet isolations recovered > or = 100,000 Islet Equivalent (IEQ, n=53) were compared to islet mass less than 100,000 IEQ (n=16). RESULTS: The mean islet recovery was 216.0 x 10(3) +/- 173.7 x 10(3) (IEQ) before purification and 130.6 x 10(3) +/- 140.2 x 10(3) (IEQ) after purification. Mean purity was 54 +/- 31%. Mean age of donor was 31.2 +/- 13.2 year and mean cold ischemic time was 6.9 +/- 6.2 hour. Quality of isolated islets was acceptable in terms of bacterial culture, viability and secretory function in vitro and in vivo. In univariate analysis on successful isolation, status of pancreas was the only significant factor and sex, duration of collagenase expansion and digestion time were marginal factors. Stepwise multivariate logistic regression analysis showed donor sex, status of pancreas and digestion time were significant factors for the successful islet isolation. CONCLUSION: This study confirms some donor factors and variables in isolation process can influence the ability to obtain the successful isolation of human islet. Enough experiences and pertinent review of donor and isolation factors can make islet isolation successful, supporting the clinical islet transplantation without spending of cost.

In vitro study of insulin secretion from mouse pancreatic islets-siriraj experiences.

Pancreatic islet isolation and culture technique are tools for direct investigation of the effects of substances on insulin secretion. Glucose is a well known insulin stimulating substance from the pancreatic islet. This study aims to demonstrate the first success (in Thailand) in islet isolation with intact insulin secretion from a mouse pancreas using collagenase P enzyme and histopaque separation. Isolated islets were cultured in RPMI 1640 for 24 hours before undergoing a glucose stimulation test. Glucose at five different concentrations (2.8, 5.6, 10, 15 and 20 mM glucose was higher than with 2.8mM basal glucose concentration. Insulin secretion increased about 1.7 to 3.5 fold from a basal level of 2.8 mM glucose without any difference in insulin content at any glucose concentrations used. To our knowledge, these data demonstrate the first success in mouse pancreatic islet isolation and culture in Thailand. This technique can be used as a tool for further investigation of the in vitro effects of substances such as plants or new drugs on insulin secretion.

Experimental Study on Cryopreservation of Pancreas Islet Cell in Rat / 대한이식학회지

PURPOSE: Cryopreservation of pancreas islet cells can facilitate the clinical islet transplantation by giving a means of storage of islets and immunomodulation on pancreatic islet preparations. METHODS: Pancreatic islets were isolated by standard technique using collagenase in rat. Cryopreservation was performed by using DMSO as a cryoprotectant after one day or 48 hr culture. Recovery rate, viability and insulin release in assay in vitro and vivo were checked under the various conditions, such as concentration of DMSO (1.5 M or 2.0 M), culture condition (1 day or 2 day), and taurine treatment. RESULTS: Percentage of recovery of cryopreserved islet was 56.8+/-10% after thawing. Viability was decreased from 97.2+/-1.1% before cryopreservation to 82.7+/-9.9% after thawing. Glucose stimulation index was reduced from 1.7+/-0.2 before cryopreservation to 1.2+/-0.8 after thawing. Nucleation method by metal rod showed better viability than control (no nucleation) or chamber nucleation method. Mean viability and glucose stimulation index was 81% and 1.5 in one day culture, and 84.2% and 1.2 in 2 day culture before cryopreservtion. Islet treated with taurine showed better insulin release and intracellular insulin content compared with non treated islets before and after cryopreservation. When 4000 IEQ (Islet Equivalent) of islets treated with taurine and non treated cryopreserved islets were transplanted into syngenic streptozotocin induced diabetic rat, all showed normoglycemia over 60 days. CONCLUSION: Cryopreservation of islets could give a tool of storage with preservation of islet secretion function. However pertinent effort to improve the recovery is needed in order to be used in the clinical islet transplantation.

Effect of Prophylactic Administration of Trypsin Inhibitors on Pancreatic Islet Isolation of Adult Porcine / 中国普外基础与临床杂志

Objective To develop a method of adult porcine pancreatic islet isolation.Methods The tails of adult porcine pancreas were perfused through the pancreatic duct with 0.1% cold collagenase(type Ⅺ) and incubated at 38.5 ℃.The digested tissue was dispersed in 4 ℃ Hanks balanced salt solution(HBSS).The tissue suspensions were filtered through a 600 ?m mesh.The residual tissue was resuspended in cold HBSS,and put in the Ricordi’s chamber and shaken for 5 minutes,then filtered again.The isolated islets were divided into three groups: control group( n =14),Pefabloc(trypsin inhibitor, n =8) group and FOY(trypsin inhibitor, n =5) group.The collagenase solution of the Pefabloc and FOY group was supplemented with 1.0 mmol/L Pefabloc and FOY respectively. Results The islet yields of the Pefabloc group and FOY group 〔(11 848?3 530) islet/g pancreas and (14 496?3 693) islet/g pancreas〕 were significantly higher than that of the control group 〔(8 505?3 349) islet/g pancreas〕, P