Diazoxide, a K(ATP) channel opener, prevents ischemia-reperfusion injury in rodent pancreatic islets.

Diazoxide (DZ) is a pharmacological opener of ATP-sensitive K(+) channels that has been used for mimicking ischemic preconditioning and shows protection against ischemic damage. Here we investigated whether diazoxide supplementation to University of Wisconsin (UW) solution has cellular protection during islet isolation and improves in vivo islet transplant outcomes in a rodent ischemia model. C57/B6 mice pancreata were flushed with UW or UW + DZ solution and cold preserved for 6 or 10 h prior to islet isolation. Islet yield, in vitro and in vivo function, mitochondrial morphology, and apoptosis were evaluated. Significantly higher islet yields were observed in the UW + DZ group than in the UW group (237.5 ± 25.6 vs. 108.7 ± 49.3, p < 0.01). The islets from the UW + DZ group displayed a significantly higher glucose-induced insulin secretion (0.97 ng/ml ± 0.15 vs. 0.758 ng/ml ± 0.21, p = 0.009) and insulin content (60.96 ng/islet ± 13.94 vs. 42.09 ng/islet ± 8.15, p = 0.002). The DZ-treated islets had well-preserved mitochondrial morphology with superior responses of mitochondrial potentials, and calcium influx responded to glucose. A higher number of living cells and less late apoptotic cells were observed in the UW + DZ group (p < 0.05). Additionally, the islets from the UW + DZ group had a significantly higher cure rate and improved glucose tolerance. This study is the first to report mitoprotective effects of DZ for pancreas preservation and islet isolation. In the future, it will be necessary to further understand the underlying mechanism for the mitoprotection and to test this promising approach for pancreas preservation and the islet isolation process in nonhuman primates and ultimately humans.

Five-year follow-up of patients with type 1 diabetes transplanted with allogeneic islets: the UIC experience.

This report summarizes a 5-year phase 1/2 allogeneic islet transplantation clinical trial conducted at the University of Illinois at Chicago (UIC). Ten patients were enrolled in this single center, open label, and prospective trial in which patients received 1-3 transplants. The first four subjects underwent islet transplantation with the Edmonton immunosuppressive regimen and the remaining six subjects received the UIC immunosuppressive protocol (Edmonton plus etanercept and exenatide). All 10 patients achieved insulin independence after 1-3 transplants. At 5 years of follow-up, 6 of the initial 10 patients were free of exogenous insulin. During the follow-up period, 7 of the 10 patients maintained positive C-peptide levels and a composite hypoglycemic score of 0. Most patients maintained HbA1c levels <6.0 % (42.1 mmol/mol) and a significantly improved ß-score. In conclusion, this study demonstrated long-term islet graft function without using T cell depleting induction, with an encouraging outcome that includes 60 % of patients remaining insulin independent after 5 years of initial transplantation.

Implementation of a simplified method of islet isolation for allogeneic islet transplantation in cynomolgus monkeys.

OBJECTIVES: The present study describes a simple and cost-effective islet isolation procedure. Using this method, allogeneic islets reverse diabetes in cynomolgus monkeys. METHODS: Pancreatic tissue from 11 cynomolgus monkeys were digested, collected, and purified using a simplified method. Islet quantification, purity, viability, and glucose static incubation were conducted immediately after isolation. Five streptozotocin-induced monkeys with diabetes were transplanted intrahepatically, and liver biopsies from 3 of these monkeys were taken at different time points for histologic study. RESULTS: The mean (SD) of viability, purity, and static glucose incubation stimulation index were 94.4% (2.3%), 91.8% (3.4%), and 2.6 (1.7), respectively. Monkeys who received a mean (SD) dose of 19,968 (2273) islet equivalent per kilogram (n = 4) from 2 to 3 donors who achieved prolonged normoglycemia (57-232 days), whereas the single monkey who received an islet dose of 8000 islet equivalent per kilogram did not experience diabetes reversal. Immunohistochemical assessment of the liver biopsies taken from the monkeys with normoglycemia revealed an insulin- and glucagon-positive islet graft for up to 6 months with minimal peri-islet inflammatory infiltration. CONCLUSIONS: This study demonstrates that cynomolgus monkey islets can be successfully and efficiently harvested using a simple isolation method, and these islets can restore normoglycemia in monkeys with diabetes.

Reduction in carotid intima-media thickness after pancreatic islet transplantation in patients with type 1 diabetes.

OBJECTIVE: Determine the impact of islet transplantation on carotid intima-media thickness (CIMT), a marker for atherosclerosis, in type 1 diabetes without kidney disease. RESEARCH DESIGN AND METHODS: Consecutive case series of 15 adults (mean age [SD], 49 years [10 years]; 87% female) with type 1 diabetes for ≥5 years (mean duration [SD], 30 years [12 years]; mean HbA(1c) [SD], 7.2% [0.9%]), without kidney disease, presenting with severe hypoglycemic unawareness to undergo allogeneic pancreatic islet transplant(s) (one to three each) in a phase 1/2 and 3 clinical trial. Current follow-up ranges from 1 to 5 years (2005-2011). CIMT of the common and internal carotid arteries was measured before and every 12-16 months after the first transplant (two to six CIMTs each) by one ultrasonographer and one blinded reader. CIMT was analyzed as change from baseline to 12- and 50-month follow-up; a combined CIMT score was calculated as the sum of the standardized IMT scores (SD units [SDs]) of both arteries. RESULTS: All patients achieved insulin independence after one to three transplants. CIMT decreased at 12 months (n = 15) for the common carotid (-0.058 mm; P = 0.006) and combined score (-1.28 SDs; P = 0.004). In those with 50-month follow-up (n = 7), the decrease in the combined score continued from 12 (-1.59 SDs; P = 0.04) to 50 months (-0.77 SDs; P = 0.04). During follow-up, the decreasing slope of change in CIMT was associated with decreasing slopes of change in HbA(1c), lipoproteins, and cardiovascular/inflammatory markers. CONCLUSIONS: Islet transplantation may ameliorate diabetes-related atherosclerosis through improved glycemic control consequent to restoring endogenous insulin secretion, and optimal lipid management posttransplant also contributes.

Histidine-tryptophan-ketoglutarate and University of Wisconsin solution demonstrate equal effectiveness in the preservation of human pancreata intended for islet isolation: a large-scale, single-center experience.

We previously reported a small-scale study on the efficacy of histidine-tryptophan-ketoglutarate (HTK) solution versus University of Wisconsin (UW) solution on pancreas preservation for islet isolation. In this large-scale, retrospective analysis (n = 252), we extend our initial description of the impact of HTK on islet isolation outcomes and include pancreatic digestion efficacy, purification outcomes, and islet size distribution. Multivariable linear regression analysis, adjusted for donor age, sex, BMI, cold ischemia time, and enzyme, demonstrated similar results for the HTK group (n = 95) and the UW group (n = 157), including postpurification islet yields (HTK: 289,702 IEQ vs. UW: 283,036 IEQ; p = 0.76), percentage of digested pancreatic tissue (HTK: 66.9% vs. UW: 64.1%; p = 0.18), and islet loss from postdigestion to postpurification (HTK: 24,972 IEQ vs. UW: 39,551 IEQ; p = 0.38). Changes in islet size between the postdigestion and postpurification stages were comparable within each islet size category for HTK and UW (p = 0.14-0.99). Tissue volume distribution across purification fractions and islet purity in the top fractions were similar between the groups; however, the HTK group had significantly higher islet purity in the middle fractions (p = 0.003-0.008). Islet viability and stimulation indices were also similar between the HTK and the UW groups. In addition, we analyzed a small sample of patients transplanted either with HTK (n = 7) or UW (n = 8) preserved islets and found no significant differences in posttransplant HbA1c, ß-score, and frequency of insulin independence. This study demonstrates that HTK and UW solutions offer comparable pancreas preservation for islet transplantation. More in vivo islet outcome data are needed for a complete analysis of the effects of HTK on islet transplantation.

Survival of human islets in microbeads containing high guluronic acid alginate crosslinked with Ca2+ and Ba2+.

BACKGROUND: The main hurdles to the widespread use of islet transplantation for the treatment of type 1 diabetes continue to be the insufficient number of appropriate donors and the need for immunosuppression. Microencapsulation has been proposed as a means to protect transplanted islets from the host's immune system. METHODS: This study investigated the function of human pancreatic islets encapsulated in Ca(2+) /Ba(2+) -alginate microbeads intraperitoneally transplanted in diabetic Balb/c mice. RESULTS: All mice transplanted with encapsulated human islets (n = 29), at a quantity of 3000 islet equivalent (IEQ), achieved normoglycemia 1 day after transplantation and retained normoglycemia for extended periods of time (mean graft survival 134 ± 17 days). In comparison, diabetic Balb/c mice transplanted with an equal amount of non-encapsulated human islets rejected the islets within 2 to 7 days after transplantation (n = 5). Microbeads retrieved after 232 days (n = 3) were found with little to no fibrotic overgrowth and contained viable insulin-positive islets. Immunofluorescent staining on the retrieved microbeads showed F4/80-positive macrophages and alpha smooth muscle actin-positive fibroblasts but no CD3-positive T lymphocytes. CONCLUSIONS: The Ca(2+) /Ba(2+) -alginate microbeads can protect human islets from xenogeneic rejection in immunocompetent mice without immunosuppression. However, grafts ultimately failed likely secondary to a macrophage-mediated foreign body reaction.

Robot-assisted pancreatoduodenectomy with preservation of the vascular supply for autologous islet cell isolation and transplantation: a case report.

INTRODUCTION: For patients with chronic pancreatitis presenting with medically intractable abdominal pain, surgical intervention may be the only treatment option. However, extensive pancreatic resections are typically performed open and are associated with a substantial amount of postoperative pain, wound complications and long recovery time. Minimally invasive surgery offers an avenue to improve results; however, current limitations of laparoscopic surgery render its application in the setting of chronic pancreatitis technically demanding. Additionally, pancreatic resections are associated with a high incidence of diabetes. Transplantation of islets isolated from the resected pancreas portion offers a way to prevent post-surgical diabetes; however, preservation of the vascular supply during pancreatic resection, which determines islet cell viability, is technically difficult using current laparoscopic approaches. With recent advances in the surgical field, robotic surgery now provides a means to overcome these obstacles to achieve the end goals of pain relief and preserved endocrine function. We present the first report of a novel, minimally invasive robotic approach for resection of the pancreatic head that preserves vascular supply and enables the isolation of a high yield of viable islets for transplantation. CASE PRESENTATION: A 35-year old Caucasian woman presented with intractable chronic abdominal pain secondary to chronic pancreatitis, with a stricture of her main pancreatic duct at the level of the ampulla of Vater and distal dilatation. She was offered a robotic-assisted pylorus-preserving pancreatoduodenectomy and subsequent islet transplantation, to both provide pain relief and preserve insulin-secretory reserves. CONCLUSION: We present a novel, minimally invasive robotic approach for resection of the pancreatic head with complete preservation of the vascular supply, minimal warm ischemia time (less than three minutes) and excellent islet recovery (134,727 islet equivalent). Our patient is currently pain-free with normal glycemic control. Robot-assisted pylorus-preserving pancreatoduodenectomy and autologous islet transplantation can be safely performed and has the potential to minimize operative traumas as well as to partially preserve endocrine function. Results from this case report suggest that this dual procedure should be considered as a treatment option for patients with chronic pancreatitis at earlier stages of the disease, before irreversible islet loss occurs.

Dual microfluidic perifusion networks for concurrent islet perifusion and optical imaging.

This study explores a new class of duplex microfluidic device which utilizes a dual perifusion network to simultaneously perform live-cell optical imaging of physiological activities and study insulin release kinetics on two islet populations. This device also incorporates on-chip staggered herringbone mixers (SHMs) to increase mixing efficiency and facilitate the generation of user-defined chemical gradients. Mouse islets are used to simultaneously measure dynamic insulin release, changes in mitochondrial potentials, and calcium influx in response to insulin secretagogues (glucose and tolbutamide), and show a high signal-to-noise ratio and spatiotemporal resolution of all measured parameters for both perifusion chambers. This system has many potential applications for studying ß-cell physiology and pathophysiology, as well as for therapeutic drug screening. This dual perifusion device is not limited to islet studies and could easily be applied to other tissues and cells without major modifications.

Long-term metabolic and immunological follow-up of nonimmunosuppressed patients with type 1 diabetes treated with microencapsulated islet allografts: four cases.

OBJECTIVE: To assess long-term metabolic and immunological follow-up of microencapsulated human islet allografts in nonimmunosuppressed patients with type 1 diabetes (T1DM). RESEARCH DESIGN AND METHODS: Four nonimmunosuppressed patients, with long-standing T1DM, received intraperitoneal transplant (TX) of microencapsulated human islets. Anti-major histocompatibility complex (MHC) class I-II, GAD65, and islet cell antibodies were measured before and long term after TX. RESULTS: All patients turned positive for serum C-peptide response, both in basal and after stimulation, throughout 3 years of posttransplant follow-up. Daily mean blood glucose, as well as HbA(1c) levels, significantly improved after TX, with daily exogenous insulin consumption declining in all cases and being discontinued, just transiently, only in patient 4. Anti-MHC class I-II and GAD65 antibodies all tested negative at 3 years after TX. CONCLUSIONS: The grafts did not elicit any immune response, even in the cases where more than one preparation was transplanted, as a unique finding, compatible with encapsulation-driven "bioinvisibility" of the grafted islets. This result had never been achieved with the recipient's general immunosuppression.

Highly purified versus filtered crude collagenase: comparable human islet isolation outcomes.

This study was designed to retrospectively compare the impact of crude Sigma V collagenase (Sigma V, n = 52) with high-purified Serva NB1 collagenase (Serva NB1, n = 42) on human islet isolation outcomes. A three-step filtration was applied to the crude Sigma V to remove endotoxin contamination and impurities; in addition, this process was used as a lot prescreening tool. Isolation outcomes were determined by digestion efficacy, islet yields, purity, viability, glucose-stimulated insulin release, and endotoxin content. The digestion efficacy between Sigma V and Serva NB1 was statistically significant (Sigma V: 64.71% vs. Serva NB1: 69.71%, p = 0.0014). However, the islet yields were similar (Sigma V: 23422.58 vs. Serva NB1: 271097 IEq, p = 0.23) between groups. There was no significant purity difference observed in fractions with purities greater than 75%. Viability (Sigma V: 93.3% vs. Serva NB1: 94.8%, p = 0.061) and stimulation indexes (Sigma V: 3.41 vs. Serva NB1: 2.74, p = 0.187) were also similar between the two groups. The impact of cold ischemia and age on the isolation outcome in the Sigma V group was comparable to the Serva NB1 group. The endotoxin content of the final products in the filtered Sigma V group was significantly less than that in the high-purified Serva NB1 group (0.022 vs. 0.052 EU/ml, p = 0.003). Additionally, in the Sigma V group there was minimal lot to lot variation and no significant loss of enzymatic activity after filtration. These findings indicate that the use of Sigma V or other crude enzyme blends for research pancreata is warranted to reduce isolation costs and increase the amount of islets available for critical islet research. These findings also validate the need for a systematic enzyme analysis to resolve these inconsistencies in overall enzyme quality once and for all.

A recommended laparoscopic procedure for implantation of microcapsules in the peritoneal cavity of non-human primates.

BACKGROUND: The anatomical spatial distribution of microencapsulated islets transplanted into the peritoneal cavity of large animals remains a relatively unexplored area of study. In this study, we developed a new implantation approach using laparoscopy in order to avoid microcapsule amalgamation. This approach constitutes a clinically relevant method, which can be used to evaluate the distribution and in vivo biocompatibility of various types of transplanted microcapsules in the future. MATERIALS AND METHODS: Two healthy baboons were implanted intraperitoneally with microencapsulated islets through mini-laparotomy and observed at 76 d after implantation. Nine baboons underwent laparoscopic implantation of approximately 80,000 empty microcapsules. Microcapsule distribution was observed by laparoscopic camera during and after implantation at 1, 2, and 4 wk. At each time point, microcapsules were retrieved and evaluated with brightfield microscopy and histologic analysis. RESULTS: Mini-laparotomic implantation resulted in microcapusle aggregation in both baboons. In contrast, laparoscopic implantation resulted in even distribution of microcapsules throughout the peritoneum without sedimentation to the Douglas space in all animals. In eight out of nine animals, retrieved microcapsules were evenly distributed in the peritoneal cavity and presented with no pericapsular overgrowth and easily washed out during laparoscopic procedure. The one exception was attributed to microcapsule contamination with blood from the abdominal wall following trocar insertion. CONCLUSIONS: Laparoscopic implantation of microcapsules in non-human primates can be successfully performed and prevents microcapsule aggregation. Given the current widespread clinical application of laparoscopy, we propose that this presented laparoscopy technique could be applied in future clinical trials of microencapsulated islet transplantation.

Application of microfluidic technology to pancreatic islet research: first decade of endeavor.

ß-cells respond to blood glucose by secreting insulin to maintain glucose homeostasis. Perifusion enables manipulation of biological and chemical cues in elucidating the mechanisms of ß-cell physiology. Recently, microfluidic devices made of polydimethylsiloxane and Borofloat glass have been developed as miniaturized perifusion setups and demonstrated distinct advantages over conventional techniques in resolving rapid secretory and metabolic waveforms intrinsic to ß-cells. In order to enhance sensing and monitoring capabilities, these devices have been integrated with analytical tools to increase assay throughput. The spatio-temporal resolutions of these analyses have been improved through enhanced flow control, valves and compartmentalization. For the first time, this review provides an overview of current devices used in islet studies and analyzes their strengths and experimental suitability. To realize the potential of microfluidic islet applications, it is essential to bridge the gap in design and application between engineers and biologists through the creation of standardized bioassays and user-friendly interfaces.