Islet Purification Research Articles

Culture of Impure Human Islet Fractions in the Presence of Alpha-1 Antitrypsin Prevents Insulin Cleavage and Improves Islet Recovery

Background Exocrine tissue is commonly cotransplanted with islets in autografting and allotransplantation of impure preparations. Proteases and insulin are released by acinar cells and islets, respectively, during pretransplantation culture and also systemically after transplantation. We hypothesized that released proteases could cleave insulin molecules and that addition of alpha-1 antitrypsin (A1AT) to impure islet cultures would block this cleavage, improving islet recovery and function. Methods Trypsin, chymotrypsin, and elastase (TCE) activity and insulin levels were measured in culture supernates of pure (n = 5) and impure (n = 5) islet fractions, which were isolated from deceased donors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect insulin after incubation with proteases. We assessed the effects of A1AT supplementation (0.5 mg/mL; n = 4] on TCE activity, insulin levels, culture recovery, and islet quality. The ultrastructure of islets exposed to TCE versus control medium was examined using electron microscopy (EM). Results Protease (TCE) activity in culture supernatants was indirectly proportional to the percentage purity of islets: pure, impure, or highly impure. Increasingly lower levels of insulin were detected in culture supernatants when higher protease activity levels were present. Insulin levels measured from supernatants of impure and highly impure islet preparations were 61 ± 23.7% and 34 ± 33% of that in pure preparations, respectively. Incubation with commercially available proteases (TCE) or exocrine acinar cell supernatant cleaved insulin molecules as assessed using SDS-PAGE. Addition of A1AT to impure islet preparations reduced protease activity and restored normal insulin levels as detected using enzyme-linked immunosorbent assay (ELISA) and SDS-PAGE of culture supernates. A1AT improved insulin levels to 98% ± 1.3% in impure and 78% ± 34.2% in highly impure fractions compared with pure islet fractions. A1AT supplementation improved postculture recovery of islets in impure preparations compared with nontreated controls (72% ± 9% vs 47% ± 15%). Islet viability as measured using membrane integrity assays was similar in both the control (98% ± 2%) and the A1AT-treated groups (99% ± 1%). EM results revealed a reduction or absence of secretory granules after exposure to proteases (TCE). Conclusion Culture of impure human islet fractions in the presence of A1AT prevented insulin cleavage and improved islet recovery. A1AT supplementation of islet culture media, therefore, may increase the proportion of human islet products that meet release criteria for transplantation.

Assessment of Islet Quality following International Shipping of more than 10,000 km

Islet transplantation is an attractive therapy for type 1 diabetes, although some issues remain. One of them is the severe donor shortage in some countries. In this study, we investigated the possibility of international islet shipping beyond 10,000 km to supply islets to countries with donor shortages. Human islets were isolated from six cadaver donors and cultured until shipment. Islets were packed in either gas-permeable bags or in non-gas-permeable bags and shipped from Baylor Research Institute (Dallas, TX, USA) to Fukuoka University (Fukuoka, Japan). Pre- and postshipment islet number, purity, viability, and stimulation index (by glucose stimulation test) were assessed. Shipped 1,500 IE islets were transplanted into streptozotocin-induced diabetic nude mice for in vivo assay. The distance of our shipment was 11,148.4 km, and the mean duration of the shipments was 48.2 ± 8.2 h. The islet number recovery rate (postshipment/preshipment) was significantly higher in gas-permeable bags (56.4 ± 10.1% vs. 20.5 ± 20.6%, p < 0.01). Islet purity was significantly reduced during shipment in non-gas-permeable bags (from 47.7 ± 18.6% to 40.2 ± 28.2 in gas-permeable bags vs. from 50.4 ± 6.4% to 25.9 ± 15.6% in non-gas-permeable bags, p < 0.05). Islet viability and stimulation index did not change significantly between pre- and postshipping, in either gas-permeable bags or in non-gas-permeable bags. One of three diabetic nude mice (33.3%) converted to normoglycemia. It is feasible to ship human islet cells internationally in gas-permeable bags. This strategy would promote basic and preclinical research for countries with donor shortages, even though the research centers are remote (over 10,000 km from the islet isolation center).

Superiority of Visipaque (Iodixanol)-Controlled Density Gradient Over Ficoll-400 in Adult Porcine Islet Purification

Objective Sufficient and favorable biological functions of islets are major problems hindering xenotransplantation. The aim of the present study was to evaluate the effects on harvesting, purity, viability, and function of using improved Visipaque (iodixanol) and Ficoll-400 for adult porcine islet purification. Methods Twelve adult porcine pancreata were randomly divided into an Iodixanol-University of Wisconsin (UW) group and a Ficoll-400-UW group according to the purification method. Porcine pancreata were isolated by collagenase digestion. After isolation and purification, the islet yield and purity were evaluated by dithizone staining, and islet function assessed by in vitro insulin release assays and in vivo islet xenotransplantation. Results There were no marked differences in the islet yield before purification (5254.67 ± 189.44 IEQ/g vs 5092.67 ± 178.94 IEQ/g, P > .05). After purification, there were significantly more islets harvested in Iodixanol-UW group than in the Ficoll-400-UW group: 4222.00 ± 228.84 IEQ/g vs 3036.83 ± 79.60 IEQ/g ( P < .05). Islets from the two groups showed satisfactory insulin secretory ability. There were no significant differences in islet survival times between the two groups in diabetic rats: 8.2 ± 1.619 days vs 6.9 ± 1.197 days ( P > .05). Conclusion The improved iodixanol-UW density gradient method was superior to Ficoll-400 method to improve the number, viability, and insulin secret of purified adult porcine islets although the benefits did not improve in vivo survival.

Islet Isolation from Juvenile Porcine Pancreas after 24-h Hypothermic Machine Perfusion Preservation

Pancreas procurement for islet isolation and transplantation is limited by concerns for the detrimental effects of postmortem ischemia. Hypothermic machine perfusion (HMP) preservation technology has had a major impact in circumventing ischemic injury in clinical kidney transplantation and is applied here to the preservation and procurement of viable islets after hypothermic perfusion preservation of porcine pancreata because pigs are now considered the donor species of choice for xenogeneic islet transplantation. Pancreases were surgically removed from young (<6 months) domestic Yorkshire pigs (25-32 kg), either before or after 30 min of warm ischemia time (WIT), and cannulated for perfusion. Each pancreas was assigned to one of six preservation treatment groups: fresh controls-processed immediately (cold ischemia <1 h) (G1, n = 7); static cold storage-flushed with cold UW-Viaspan and stored in UW-Viaspan at 2-4 degrees C for 24 h with no prior WIT (G2, n = 9); HMP perfused on a LifePort(R) machine at 4-6 degrees C and low pressure (10 mmHg) for 24 h with either KPS1 solution (G3, n = 7) or Unisol-UHK (G4, n = 7). Additional treatment groups to evaluate the effects of prior warm ischemia examined islet isolation after 30 min WIT in situ without (G5, n = 6) or with subsequent 24-h HMP with KPS1 (G6, n = 7). The pancreas was intraductally distended with Liberase PI enzyme and normothermically digested. The isolated islets were purified by a continuous density-gradient centrifugation. Perfusion-induced glandular edema was G3 = 138 +/- 19%, G4 = 160 +/- 16%, and G6 = 127 +/- 22%. Islet yield (IEQ/g of pancreas) varied between the groups: G1 = 1,425 +/- 610, G2 = 1,002 +/- 262, G3 = 2,242 +/- 449 (p < 0.05 vs. G2), G4 = 1,901 +/- 420 (p < 0.05 vs. G2), G5 = 1,756 +/- 329, and G6 = 1,396 +/- 243. Islet stimulation indices were equivalent between the groups and similar to controls (G1). Insulin content (ng/IE) was different between the treatment groups with the highest insulin content in islets harvested from HMP pancreata. Dithizone staining for islets consistently showed more uniform digestion of the perfused organs, with greater separation of the tissue, less entrapped islets, and higher islet yield and purity. The salutary effects of HMP for 24 h were also manifest after 30-min prior warm ischemia. We conclude that 24 h of HMP is well tolerated, leading to moderate edema but no loss of function of the harvested islets. The edema appears to aid in enzymatic digestion, producing a greater yield and purity of islets compared with pancreas subjected to 24 h of static cold storage.

A New Oxygen Carrier for Improved Long-Term Storage of Human Pancreata Before Islet Isolation

Pancreas oxygenation during cold storage has been established in islet isolation and transplantation to prevent ischemic tissue damage using perfluorodecalin (PFD) as hyperoxygen carrier. However, studies in humans and pigs provided conflicting results about the efficiency of PFD for pancreas oxygenation. The aim of this study was to compare PFD with a newly developed oxygen carrier composed of perfluorohexyloctane and polydimethylsiloxane 5 (F6H8S5) for long-term storage of human pancreata. After 24-hr storage in preoxygenated PFD or F6H8S5, pancreata were processed using Liberase HI for pancreas dissociation and a Ficoll gradient for islet purification. Islet quality assessment was performed measuring glucose-stimulated insulin release, viability, islet ATP content, and posttransplant function in diabetic nude mice. Compared with PFD, F6H8S5 significantly increased the intrapancreatic partial oxygen pressure and islet ATP content. This corresponded to an increase of islet yield, recovery after culture, glucose stimulation index, viability, and improved graft function in diabetic nude mice. The present findings indicate clearly that F6H8S5 improves isolation outcome after prolonged ischemia compared with PFD. This observation seems to be related to the significant lipophilicity and almost pancreas-specific density of F6H8S5. Moreover, these characteristics facilitate pancreas shipment without using custom-made transport vessels as required for PFD.

In vivo and in vitro characterization of [18F]-FE-(+)-DTBZ as a tracer for beta-cell mass

The positron emission tomography (PET) tracer 9-[(18)F]fluoroethyl-(+)-dihydrotetrabenazine ([(18)F]-FE-(+)-DTBZ) is a potential candidate for quantifying beta-cell mass in vivo. The purpose was to investigate in vitro and in vivo utility of this tracer for the assessment of beta-cell mass. Three pigs were intravenously administered [(18)F]-FE-(+)-DTBZ and examined by PET/computed tomography. Binding parameters were estimated by kinetic modeling. In vitro k(D) and B(max) were determined by saturation binding studies of endocrine and exocrine human tissue homogenates. In vitro pancreatic uptake was determined by tissue autoradiography with pancreases from patients with types 1 (T1DM) and 2 diabetes mellitus (T2DM) and healthy controls. [(18)F]-FE-(+)-DTBZ had a k(D) of 3.5+/-1.0 nM, a B(max) of 382+/-108 fmol/mg protein and a specificity of 89+/-1.8% in islet homogenates. The total exocrine uptake was lower and 65% was nondisplaceable. No uptake difference was observed in pancreatic tissue slices from patients with T1DM, T2DM or healthy controls. The in vivo porcine pancreatic uptake reached a peak of standardized uptake value (SUV) of 2.8 with a low distribution volume ratio in all animals. Moderate to high tracer uptake was identified in the bile system and in bone. [(18)F]-FE-(+)-DTBZ binds to vesicular monoamine transporter 2 (VMAT2) with high specificity in pure islet tissue in vitro. However, there is high nondisplaceable binding to exocrine tissue. In addition, in vivo tracer metabolism and dehalogenation result in severe underestimation of porcine pancreatic VMAT2 expression and BCM. The results do not support [(18)F]-FE-(+)-DTBZ as a suitable tracer for in vivo beta-cell imaging.

Influence of the purification of human adult pancreatic islets on insulin secretion

The most effective method for human adult pancreatic islets purification is density-gradient centrifugation. The aim of this study was to analyze the effects of non-automated purification on preservation of functional capacity of human adult pancreatic islet cells. Human pancreata were obtained after pancreatectomy in the patients with chronic pancreatitis or benign tumors. Pancreatic islets were purified by non-automated method in discontinuous Ficoll density gradient. The samples were divided in 2 fractions: purified (P) and non-purified (NP) cultures. Islets were stained with diphenyl-thiocarbazone. The efficiency of separation was determined by comparing percentage of stained cells in P and NP cultures on day 1, 3 and 7 of shortterm cultivation. Glucose-stimulated insulin secretion was expressed as stimulation index (SI). The results obtained showed a statistically significant difference (p < 0.01) between P and NP cultures. P cultures had higher percentages of stained cells (70.43 +/- 3.97%, 73.77 +/- 4.22% and 71.34 +/- 4.69% on the first, third and seventh day of cultivation, respectively) than NP cultures (53.68 +/- 1.71%, 57.14 +/- 3.94% and 43.97 +/- 4.56%, respectively). P cultures had higher values of SI for the first, third and seventh day of cultivation than NP cultures (0.45 +/- 0.08, 0.80 +/- 0.21, 1.28 +/- 0.15 and 0.46 +/- 0.10, 0.752 +/- .0.16, 0.76 +/- 0.11 for P and NP cultures respectively). The difference was statistically significant on day seven (p = 0.01). Although during purification process islets were exposed to a number of insults that might result in cellular damage and functional impairment, our assessments showed that islets in P cultures preserved their functional capacity better than islets in NP cultures, since they had greater insulin secretion.

A Practical Guide to Rodent Islet Isolation and Assessment

Pancreatic islets of Langerhans secrete hormones that are vital to the regulation of blood glucose and are, therefore, a key focus of diabetes research. Purifying viable and functional islets from the pancreas for study is an intricate process. This review highlights the key elements involved with mouse and rat islet isolation, including choices of collagenase, the collagenase digestion process, purification of islets using a density gradient, and islet culture conditions. In addition, this paper reviews commonly used techniques for assessing islet viability and function, including visual assessment, fluorescent markers of cell death, glucose-stimulated insulin secretion, and intracellular calcium measurements. A detailed protocol is also included that describes a common method for rodent islet isolation that our laboratory uses to obtain viable and functional mouse islets for in vitro study of islet function, beta-cell physiology, and in vivo rodent islet transplantation. The purpose of this review is to serve as a resource and foundation for successfully procuring and purifying high-quality islets for research purposes.

Improved, Low-Cost Methods for Pancreatic Islet Purification in Rats

Objective Density gradient separation of islets from exocrine tissue is usually performed using Ficoll. However, this reagent significantly increases the cost of isolation. The aim of the present study was to investigate the effects on islet preparations of purification methods using Lymphoprep and Iodixanol (OptiPrep) density gradients. Methods Pancreata were procured from 46 Wistar rats, loaded with collagenase V (Sigma), and mechanically dissociated using standard procedures. After the digestion phase, the islets purified by 3 methods—Ficoll, Lymphoprep, and Iodixanol (OptiPrep)—were assessed for yields, purity, morphology, and in vitro function. Result We expressed the yields as islet equivalents (IEQ, diameter standardizing to 150 μm), showing no significant differences. Compared with the Ficoll group, the purity was significantly higher in the Lymphoprep ( P = .005) and Iodixanol (OptiPrep) groups ( P = .011). While the viability was >90% in all 3 groups, the viability in the Lymphoprep Group and OptiPrep groups was significantly higher than that of the Ficoll group ( P < .001). In vitro islet function did not differ among the 3 experimental groups. Conclusion Lymphoprep and Iodixanol were as effective as Ficoll in terms of islet yield and in vitro function. High-purity and high-viability islet cells were obtained using improved Lymphoprep-based or Iodixanol (OptiPrep)-based density gradient methods, potential low-cost substitutes for Ficoll.

A protocol for islet isolation from mouse pancreas

Mouse islets are commonly used in diabetes-related studies. Adequate amounts of good quality islets are prerequisites for a reliable investigation. We describe a protocol for islet isolation from mouse pancreas. Three major manipulations are employed in the islet isolation procedure: in situ pancreas perfusion with collagenase, pancreas digestion and islet purification. The whole procedure takes 30-45 min for each individual mouse. By using this protocol, a reasonable number of islets can be obtained in a relatively short period of time. This protocol has been proven to be practicable and reproducible. It can be easily followed by individuals who do not have previous experience in the related research field.

Islet isolation from the pancreas of large mammals and humans: 10 years of experience.

Despite experience, that has been obtained in the field of islet isolation in large mammals for about the past 20 years, allo- and autotransplantation of both human and porcine islets are still not routine procedures. The reasons for islet isolation and purification associated problems, which prevent a continuous isolation success and regular islet transplantation, can be categorized to variables related to 1) pancreas donor; 2) pancreas procurement; 3) isolation-techniques and 4) purification techniques. The development of porcine and human islet isolation was carried out by the authors since 1986. During this period several techniques for the isolation and purification of human and porcine islets were compared with regard to their influence on islet isolation outcome, integrity of islet morphology, islet in-vitro and in-vivo function: sequential vs continuous digestion-filtration, counterflow elutriation vs neutral density separation, conventional density gradient centrifugation vs Cobe cell separation and discontinuous vs continuous gradient purification. Furthermore, we analysed the influence of donor factors and variables related to organ procurement on islet isolation success. From this experience we concluded that successful porcine islet isolation is possible if islets of adult donors are isolated utilizing the continuous digestion-filtration following distension with UW-solution prior to a neutral density gradient purification on a Cobe. Human islets can be successfully isolated by digestion-filtration and purified utilizing a continuous Ficoll-sodium-diatrizoate gradient on a Cobe when intact pancreata are harvested after a secondary pancreatic warm ischemia time < 20 min from adult donors > 30 years.

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.

Islet transplantation. The connection of experiment and clinic exemplified by the transplantation of islets of Langerhans.

Since approximately 25 years islet transplantation as a mean for the treatment of diabetes has been developed from early attempts in rodents to first clinical applications in man in recent years. The review describes the results obtained in diabetic rats including metabolic effects as well as prevention of late complications. In the syngeneic system (Lewis-rats) the intraportal transplantation of 1,000-2,000 isolated islets may cure streptozotocin-induced diabetes for the whole lifespan of the animal. In the allogeneic system (across a major histocompatibility barrier), however, isolated islets are highly immunogenic. According to present knowledge, mainly class-II antigen bearing cells are responsible while the role of the endocrine cells seems to be less important. By various in vitro methods (e.g. low temperature culture at 24 degrees C) it was possible to induce immunoalteration of allogeneic islets which was followed by long-term acceptance by the host in rodents. Whether the same technique is effective in larger animals and in man remains open. New technologies for the isolation of high numbers of pure islets from human pancreatic glands have been the key for the use of islet transplantation in man in several centers. The review describes the details regarding institutions as well as results in patients on the basis of the International Islet Transplant Registry which is located at Giessen University. Although longer lasting insulin independency (longest lasting effect 2 1/2 years) has been observed only in a few patients, islet transplantation has been proven to be a very safe procedure for the patient. It is hoped that with increasing experiences regarding the factors which at present limit the function as number and quality of islets, immunosuppressive protocols etc. islet transplantation will develop to a definite alternative to whole-organ transplantation. One of the most important challenges for modern medicine is the fact of a worldwide increase of diabetic long-term complications such as kidney failure, coronary and peripheral vascular disease, blindness and neuropathy. Although intensified insulin treatment in a small percentage of patients may moderate morbidity and mortality, from experimental work there is sufficient evidence suggesting that only the restoration of normoglycemia early in the course of the disease may prevent complications. This, however, can only be achieved by the replacement of the destroyed islets of Langerhans either by transplantation of an intact vascularised pancreas or by isolated islets.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Iodixanol-Controlled Density Gradient During Islet Purification Improves Recovery Rate in Human Islet Isolation

For pancreatic islet transplantation, islet purification minimizes the risks associated with islet infusion through the portal vein by reducing the amount of transplanted tissue. However, the purification step may result in decreased numbers of islets recovered from digested tissue and be traumatic to the islets. In this study, we evaluated the effectiveness of iodixanol-controlled density gradients on the islet purification step. For 14.3% of the isolations, the density was 1.085 g/cm3, 32.1% were 1.090 g/cm3, 46.4% were 1.095 g/cm3, 3.6% were 1.100 g/cm3, and 3.6% were 1.105 g/cm3, indicating that the density varies with each isolation. This has profound implications for the difficulty of islet purification. According to the density of digested tissue before purification, the density of the purification solutions was controlled by changing the volumetric ratio of iodixanol and the purification solutions (iodixanol-Kyoto [IK] solutions). Islet yield after purification and rate of postpurification recovery were significantly higher in the IK group than with standard continuous gradient purification by Ficoll solutions (islet yield=Ficoll group: 377,230+/-50,207 islet equivalents, IK group: 594,136+/-50,570 islet equivalents, P less than 0.01; percentage of recovery=Ficoll group: 55.6%+/-5.8%, IK group: 84.9%+/-4.2%, P less than 0.01). In vitro and in vivo assays suggest that the quality of islets was similar between the two groups. Our data suggest that using an iodixanol-controlled density gradient improves the islet recovery rate in human islet isolation. On the basis of these data, we now use this purification method for clinical islet 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.

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.