Pancreatic Islet Isolation Research Articles

The effect of different concentrations of Liberase HI used in a non automated method for human adult pancreatic islet isolation

Liberase HI has many advantages over traditional collagenases. Liberase performs the rapid dissociation of human pancreata and allows for the preservation of the morphological and functional integrity of isolated islets. The aim of this study was to investigate the effect of different concentrations of Liberase HI (1.5, 2.5 and 3.5 mg/ml) on the yield, viability and function of islets isolated by a non-automated method. Based on our results, the Liberase HI concentration 1.5 mg/ml is recommended for this method of isolation. .

Activation of human complement by collagenase and ficoll.

Collagenase and ficoll are standard chemicals used for isolation and purification of pancreatic islets. The capacity of these substances to activate human complement was tested by incubation for 45 min at 37 degrees C with fresh or liquid nitrogen-stored human plasma from normal blood donors. Complement-split products were then determined by sensitive enzyme immunoassays from QUIDEL. Collagenase activates both the classical and the alternative pathway, whereas Ficoll activates the alternative pathway only. When present in islet preparations used for islet transplantation, these substances may induce local inflammatory reactions compromising islet viability and engraftment.

Optimization of Human Pancreatic Islet Isolation With a Newly Designed Cooling System for COBE 2991

Islet purification is performed using the cell separator COBE 2991, which allows the purification of large amounts of islets through a continuous density gradient. However, the original cell separator COBE 2991 was not refrigerated, and islets were exposed to inappropriately high temperatures during the purification step of the isolation process. Our aim was to design a cooling system for the purification of human pancreatic islets using COBE 2991, to increase the viability and quality of the preparations. We designed and adapted a cooling system for the COBE 2991 based on a cooling bath connected to a coil containing a recirculation solution with a temperature below 0 degrees C which was placed around the rotor of the COBE 2991. Cell viability was determined by acridine orange/propidium iodide staining, and islet cell function by measuring glucose-stimulated insulin secretion expressed as the insulin stimulation index. Pancreata from 10 consecutive organ donors (mean age, 42.8 +/- 4.3 years) were digested and purified using the newly generated cooling system for COBE 2991. At the end of the purification process, the temperature of the density gradient that contained the islet preparation was reduced by 8 degrees C to 10 degrees C compared with that of a system with no refrigeration. Islet viability increased to 83% +/- 4%, and the insulin stimulation index increased to 11.4 +/- 1.6 (average +/- SEM). This innovative cooling system for COBE 2991 achieved substantial reductions in temperature and improved the quality of human pancreatic islet preparations that were suitable for transplantation.

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37°C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4°C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37 degrees C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4 degrees C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristics, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004 and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al8. As described in Part I: Digestion and Collection of Pancreatic Tissue, human pancreas was trimmed, cannulated, perfused, and digested. After collection and at least 30 minutes of incubation in UW solution, the tissue was loaded in the cell separator (COBE 2991, Cobe, Lakewood, CO) for purification 3. Following purification, islet yield (expressed as islet equivalents, IEQ), tissue volume, and purity was determined according to standard methods 9. Isolated islets were cultured in CMRL-1066 media (Mediatech, Herndon, VA), supplemented with 1.5% human albumin, 0.1% insulin-transferrin-selenium (ITS), 1 ml of Ciprofloxacin, 5 ml o f 1M HEPES, and 14.5 ml of 7.5% Sodium Bicarbonate in T175 flasks at 37°C overnight culture before islets were transplanted or used for research.

PL-304: Pancreatic islet isolation in a rat model shows increased insulin secretion after gastric bypass

Roux-en-Y gastric bypass affords a high remission rate of type 2 diabetes mellitus among morbidly obese diabetic patients. We hypothesize that pancreatic islet secretion is improved after gastric bypass using a rodent model.

Surgical removal of the pancreas with one-step autotransplantation of isolated Langerhans islets into the hepatic portal system in the pig

Autotransplantation of isolated Langerhans islets is regarded as the only way to prevent iatrogenic diabetes in patients who had been scheduled for pancreatectomy due to painful chronic pancreatitis. A sufficient number of Langerhans islets capable of secretory activity need to be transplanted to maintain normoglycemia after the surgical procedure. In order to optimize all stages, including collection, storage, isolation and transplantation of pancreatic islets, a reproducible animal-based experimental model should be developed before a new method is introduced into clinical practice. The aim of the present study was to develop a reproducible autogenic model-based method for collection, conservation and isolation of porcine pancreas so that transplantation of isolated pancreatic islets could be performed and postoperative normoglycemia achieved. Pigs were subjected to total pancreatectomy with simultaneous splenectomy and without removal of the duodenum. The collected pancreas was stored in the University of Wisconsin solution with the addition of pentoxifylline (PTX) until the isolation procedure (<4 hours). Efficacy of isolation was evaluated based on the number, quality and viability of obtained islets. Following autotransplantation into the liver, secretory activity of the islets was assessed intravitally by serum glucose monitoring. The islet yield per gram of pancreas was 1452 (standard deviation [SD] +/- 125) for the PTX group and 384 (SD +/- 115) for the control non-PTX group (p<0.01). Viability of islets for individual isolations did not reveal any statistically significant differences between groups and was estimated at 85-93%. Three out of five animals demonstrated normoglycemia with features of neoangiogenesis in the islets transplanted into the liver, which was confirmed by histological examination. One animal developed hyperglycemia up to 430 mg/dl, and histological image showed intensive apoptosis and degranulation in the transplanted islets. Efficacy of the isolation method was confirmed by achieving normoglycemia after autotransplantation of pancreatic islets into the liver, while histological examination showed hepatic vascularization to be the most appropriate location for an autogenic graft. PTX presence in the preserving solution for the pancreas storage produced the cytoprotective effect, which directly correlated with the islet yield.

Assessment of six different collagenase-based methods to isolate feline pancreatic islets

Isolation of pancreatic islets is necessary to study the molecular mechanisms underlying β-cell demise in diabetic cats. Six collagenase-based methods of isolation were compared in 10 cat pancreata, including single and double course of collagenase, followed or not by Ficoll centrifugation or accutase, and collagenase plus accutase. Morphometric analysis was performed to measure the relative area of islet and exocrine tissue. Islet specific mRNA transcripts were quantified in isolates by real-time PCR. The single and double course of collagenase digestion was successful in each cat and provided similar islet-to-exocrine tissue ratio. Quantities of insulin mRNA did not differ between the two methods. However, on histological examination either method yielded only approximately 2% of pure islets. The other methods provided disrupted islets or insufficient samples in 1–7 cats. Although pancreas digestion with single and double course of collagenase was superior, further studies are needed to improve islet isolation in cats.

Predicting the outcome of islet isolation in large mammals. Reply to Hubert T, Gmyr V, Vantyghem MC et al. [letter

To the Editor: We are grateful to Hubert et al. [1] for their comment on our recent publication in Diabetologia [2]. Much effort has been made to identify factors or variables that improve pancreatic islet yield and promote successful islet isolation [3, 4]. Adequate in vivo functional islet mass in the pancreas donor is one of many factors that can have an impact on the outcome of pancreatic islet isolation. Therefore, we agree with Hubert et al. [1] that a reliable method to screen functional donor islet mass in vivo has the potential to improve successful islet isolation outcomes. Other tests, such as glucose tolerance tests, beta cell secretory capacity [5], positron-emission tomography [6], and glucoseor arginineinduced acute insulin release (AIR), have been used to assess functional islet mass. Of these, arginine-induced AIR (AIRarg) is a logical choice for this purpose because it is simple and non-invasive. AIR has been used to measure functional beta cell mass in normal individuals as well as in pancreas/ islet transplant recipients [7]. The preliminary data presented by Hubert et al. demonstrating that AIRarg permitted prediction of successful islet isolation are encouraging [8, 9]. However, we caution that further testing in humans is necessary because the reported data were derived from a relatively limited number of subjects (n=29). As far as we know, there is no published standardised procedure for the AIRarg test that correlates AIRarg with functional beta cell mass for nonhuman primates (NHP). Clearly it will be useful to establish a standard AIRarg test for NHP to evaluate whether the test can be used to predict successful islet isolation in these species. Since a standardised test will allow results from different laboratories to be compared, the AIRarg test has the potential to predict successful islet isolation and functional transplantable islet mass. Pancreatic islet transplantation remains a motivating challenge for scientists and a hopeful dream for diabetic patients. Rather than a useless sacrifice of NHP, these valuable animals have given us the wonderful opportunity to understand further the complexity of pancreatic islet isolation and transplantation. A single variable might have a significant impact on the final result, but when weighed in conjunction with all the other variables involved, its effect may change dramatically. This is clearly demonstrated in the univariate and multivariate analysis performed in our study [2]. We will have to wait for further improvements in donor selection, islet isolation and implantation in order to have a reliable procedure to offer to our patients.

Analysis of technique about pancreatic islet isolation and transplantation in rats

目的 探索高效的大鼠胰岛分离移植技术方法.方法 应用Wistai-Furth大鼠,于体内或体外胶原酶经胰管灌注膨化胰腺,联合不同密度Ficoll液或Histopaque液纯化胰岛细胞,评估胰岛的数量、纯度、胰岛当量以及肾被膜下胰岛移植的有效性.结果 体外经胰管灌注膨化胰腺结合Histopaque液纯化提取胰岛的数量、纯度和胰岛当量值均显著高于体内灌注组各数值(P＜0.01),其提取时间无显著差别.1000个胰岛细胞移植进入左肾被膜下,有效的逆转了糖尿病大鼠高血糖,其远期糖耐受结果优于500和800个胰岛细胞移植组.结论 体外灌注膨化消化胰腺结合Histopaque液纯化胰岛的分离方法是一种满意的分离技术.1000个胰岛细胞是保证肾被膜下胰岛移植成功的最低有效数量。

Trasplante experimental de islotes de páncreas en el tracto genitourinario asociado al trasplante renal

Simultaneous kidney and pancreas transplant is a good treatment for both renal and pancreas insufficiency. Experimental apply of genitourinary tract for pancreas implantation is reported in this work. Twenty animals aged as average 5.5 monts (SD 1.1) and an average weight of 53 kgr were submitted to this protocol. In the day 1 a left nephrectomy is completed and the graft is perfused with University of Wisconsin solution. A partial pancreatectomy is completed at following, isolation of pancreatic islets by colagenase enzymatic digestion. Islets are dryed with Ditizone and culptured for 24 hours at 37 degrees C and 5% CO2. Day-2 a right nephrectomy is performed and orthotopic renal autotransplant using the left kidney is completed. Pancreatic islets are transplanted in 4 different locations of the genitourinary tract: renal subcapsular space, bladder submucosae, testis parenchyma and vas deferens. Day-7, all the animals were sacrifized to complete pathological study. Viable islets were isolated in bladder submucosae and testis after transdeferential injection.

Islet Specific Wnt Activation in Human Type II Diabetes

The Wnt pathway effector gene TCF7L2 has been linked to type II diabetes, making it important to study the role of Wnt signaling in diabetes pathogenesis. We examined the expression of multiple Wnt pathway components in pancreases from normal individuals and type II diabetic individuals. Multiple members of the Wnt signaling pathway, including TCF7L2, Wnt2b, β-catenin, pGSK3β, TCF3, cyclinD1, and c-myc, were undetectable or expressed at low levels in islets from nondiabetic individuals, but were also upregulated specifically in islets of type II diabetic patients. Culture of pancreatic tissue and islet isolation led to Wnt activation that was reversed by the Wnt antagonist sFRP, demonstrating that Wnt activation in that setting was due to soluble Wnt factors. These data support a model in which the Wnt pathway plays a dynamic role in the pathogenesis of type II diabetes and suggest manipulation of Wnt signaling as a new approach to β-cell-directed diabetes therapy.

Murine Pancreatic Islet Isolation

Murine Pancreatic Islet Isolation

Murine Pancreatic Islet Isolation

Murine Pancreatic Islet Isolation

A New Preservation Solution Increases Islet Yield and Reduces Graft Immunogenicity in Pancreatic Islet Transplantation

The aim of the study was to test a new preservation solution containing polyethylene glycol (S.C.O.T. solution) as pancreatic islet isolation medium both to increase the islet yield and to prolong the allograft survival. In a model of islet transplantation in diabetic mouse, islets were isolated with S.C.O.T. in experimental groups and with Hank's balanced salt solution (HBSS) in control groups. The use of S.C.O.T. solution improved the islet yield (596+/-27 IEQ/pancreas) as compared to HBSS (456+/-11 IEQ/pancreas) (P<0.001). Allograft survival was prolonged in experimental group (17.3+/-4.3 days) versus controls (7.3+/-3.6 days) in a full mismatch combination (P<0.001) and in absence of recipient immunosuppression. The same prolongation (10 days) was also found in a strongly alloreactive transgenic combination. It is hypothesized that a transitory phenomenon of immunocamouflage of the graft surface antigens occurs, as shown by immunofluorescence studies. The use of this new solution could improve the results of islet transplantation in humans.

The Use of Iodixanol for the Purification of Rat Pancreatic Islets

Transplantation of pancreatic islets is a promising therapeutic treatment for type 1 diabetes mellitus. For clinical and experimental transplantation, a large number of pure pancreatic islets are required for transplantation. Thus, the improvement of islet isolation and purification techniques are crucial. In this context, iodixanol-based solution, successfully used for the purification of porcine islets, seems to be a possible alternative to Ficoll for purification of islets. The aim of this study was to test the efficacy of iodixanol compared with Ficoll density gradients for the purification of rat pancreatic islets. Twelve Wistar rats were used for isolation and purification of pancreatic islets. Pancreata were digested with Liberase R1 and islets purified by two gradients: Ficoll or iodixanol gradient. The number and the purity of the pancreatic islets were assessed. To analyze the response of isolated pancreatic islet to glucose challenge, in vitro experiments were performed by measuring the insulin concentration in the Supernatant. The results demonstrated that the iodixanol gradient provided a higher purity of pancreatic islets compared to the Ficoll gradient. In addition, the rat islet yield by iodixanol gradient was significantly higher compared to a Ficoll gradient (751 ± 16 versus 464 ± 19 pancreatic islets, respectively; P < .001). The viability of pancreatic islets isolated by an iodixanol gradient was confirmed by high glucose challenge, with more than twofold higher increase in insulin secretion. The present study demonstrated that iodixanol density gradient overcomes Ficoll density gradient, providing a greater number of pure and functional rat pancreatic islets.

Chronic fetal hypoglycemia inhibits the later steps of stimulus-secretion coupling in pancreatic β-cells

We measured the impact of chronic late gestation hypoglycemia on pancreatic islet structure and function to determine the cause of decreased insulin secretion in this sheep model of fetal nutrient deprivation. Late gestation hypoglycemia did not decrease pancreas weight, insulin content, beta-cell area, beta-cell mass, or islet size. The pancreatic islet isolation procedure selected a group of islets that were larger and had an increased proportion of beta-cells compared with islets measured in pancreatic sections, but there were no morphologic differences between islets isolated from control and hypoglycemic fetuses. The rates of glucose-stimulated pancreatic islet glucose utilization (126.2 +/- 25.3 pmol glucose.islet(-1).h(-1), hypoglycemic, vs. 93.5 +/- 5.5 pmol glucose.islet(-1).h(-1), control, P = 0.47) and oxidation (10.5 +/- 1.7 pmol glucose.islet(-1).h(-1), hypoglycemic, vs. 10.6 +/- 1.6 pmol glucose.islet(-1).h(-1), control) were not different in hypoglycemic fetuses compared with control fetuses. Chronic late gestation hypoglycemia decreased insulin secretion in isolated pancreatic islets by almost 70% in response to direct nonnutrient membrane depolarization and in response to increased extracellular calcium entry. beta-Cell ultrastructure was abnormal with markedly distended rough endoplasmic reticulum in three of the seven hypoglycemic fetuses studied, but in vitro analysis of hypoglycemic control islets showed no evidence that these changes represented endoplasmic reticulum stress, as measured by transcription of glucose regulatory protein-78 and processing of X-box binding protein-1. In conclusion, these studies show that chronic hypoglycemia in late gestation decreases insulin secretion by inhibiting the later steps of stimulus-secretion coupling after glucose metabolism, membrane depolarization, and calcium entry.

Islet Yield After Different Methods of Pancreatic Liberase Delivery

Objective Enzymatic digestion of the pancreas is a fundamental step in islet isolation and there are many ways to administer the enzyme during procurement. The aim of this study was to evaluate the influence of different methods of Liberase delivery during pancreas harvest on the quality and quantity of islets. Methods Depending on the type of Liberase delivery, 4 groups were created. Group 1 was intraductal, Group 2 was interstitial, Group 3 was intragallbladder, and Group 4 was no infusion of enzyme. After injection, the pancreata were harvested, digested in Liberase solution, mechanically disrupted, and purified using discontinuous gradient centrifugation. After 24-hour culture, the number, purity, and viability of the isolated islets were determined. Results Intraductal injection of the enzyme yielded statistically significantly more islets per mouse when compared with interstitial, intragallbladder, and no injection administration. Although there was a trend toward better islet purity and viability for Group 1, this was not statistically significant. Conclusion Intraductal administration is the best enzyme delivery method for pancreatic islet isolation. The pancreatic ducts are the most anatomic and physiological way to transport the enzyme uniformly inside the pancreas, determining an adequate digestion and better islet quantity and quality when compared with other delivery methods.