Islet Microencapsulation: Strategies and Clinical Status in Diabetes.

PURPOSE OF REVIEW: Type 1 diabetes mellitus (T1DM) is an autoimmune disease that results from the destruction of insulin-producing pancreatic ß cells in the islets of Langerhans. Islet cell transplantation has become a successful therapy for specific patients with T1DM with hypoglycemic unawareness. The reversal of T1DM by islet transplantation is now performed at many major medical facilities throughout the world. However, many challenges must still be overcome in order to achieve continuous, long-term successful transplant outcomes. Two major obstacles to this therapy are a lack of islet cells for transplantation and the need for life-long immunosuppressive treatment. Microencapsulation is seen as a technology that can overcome both these limitations of islet cell transplantation. This review depicts the present state of microencapsulated islet transplantation. RECENT FINDINGS: Microencapsulation can play a significant role in overcoming the need for immunosuppression and lack of donor islet cells. This review focuses on microencapsulation and the clinical status of the technology in combating T1DM.

A pumpless microfluidic device driven by surface tension for pancreatic islet analysis.

We present a novel pumpless microfluidic array driven by surface tension for studying the physiology of pancreatic islets of Langerhans. Efficient fluid flow in the array is achieved by surface tension-generated pressure as a result of inlet and outlet size differences. Flow properties are characterized in numerical simulation and further confirmed by experimental measurements. Using this device, we perform a set of biological assays, which include real-time fluorescent imaging and insulin secretion kinetics for both mouse and human islets. Our results demonstrate that this system not only drastically simplifies previously published experimental protocols for islet study by eliminating the need for external pumps/tubing and reducing the volume of solution consumption, but it also achieves a higher analytical spatiotemporal resolution due to efficient flow exchanges and the extremely small volume of solutions required. Overall, the microfluidic platform presented can be used as a potential powerful tool for understanding islet physiology, antidiabetic drug development, and islet transplantation.

Use of glucagon-like peptide-1 agonists to improve islet graft performance.

Human islet transplantation is an effective and promising therapy for type I diabetes. However, long-term insulin independence is both difficult to achieve and inconsistent. De novo or early administration of incretin-based drugs is being explored for improving islet engraftment. In addition to its glucose-dependent insulinotropic effects, incretins also lower postprandial glucose excursion by inhibiting glucagon secretion, delaying gastric emptying, and can protect beta-cell function. Incretin therapy has so far proven clinically safe and tolerable with little hypoglycemic risk. The present review aims to highlight the new frontiers in research involving incretins from both in vitro and in vivo animal studies in the field of islet transplant. It also provides an overview of the current clinical status of incretin usage in islet transplantation in the management of type I diabetes.

Diazoxide Preconditioning of Nonhuman Primate Pancreas Improves Islet Isolation Outcomes by Mitochondrial Protection.

OBJECTIVES: Previously, we showed that diazoxide (DZ), an effective ischemic preconditioning agent, protected rodent pancreas against ischemia-reperfusion injury. Here, we further investigate whether DZ supplementation to University of Wisconsin (UW) solution during pancreas procurement and islet isolation has similar cytoprotection in a preclinical nonhuman primate model. METHODS: Cynomolgus monkey pancreata were flushed with UW or UW + 150 µM DZ during procurement and preserved for 8 hours before islet isolation. RESULTS: First, a significantly higher islet yield was observed in UW + DZ than in UW (57,887 vs 23,574 IEq/pancreas and 5396 vs 1646 IEq/g). Second, the DZ treated islets had significantly lower apoptotic cells per islet (1.64% vs 9.85%). Third, DZ significantly inhibited ROS surge during reperfusion with a dose-response manner. Fourth, DZ improved in vitro function of isolated islets determined by mitochondrial potentials and calcium influx in responses to glucose and KCI. Fifth, the DZ treated islets had much higher cure rate and better glycemia control in diabetic mice transplant model. CONCLUSIONS: This study showed a strong mitochondrial protection of DZ on nonhuman primate islets against ischemia-reperfusion injury that provides strong evidence for its clinical application in islet and pancreas transplantation.

Structural changes in alginate-based microspheres exposed to in vivo environment as revealed by confocal Raman microscopy.

A next-generation cure for type 1 diabetes relies on immunoprotection of insulin-producing cells, which can be achieved by their encapsulation in microspheres made of non-covalently crosslinked hydrogels. Treatment success is directly related to the microsphere structure that is characterized by the localization of the polymers constituting the hydrogel material. However, due to the lack of a suitable analytical method, it is presently unknown how the microsphere structure changes in vivo, which complicates evaluation of different encapsulation approaches. Here, confocal Raman microscopy (CRM) imaging was tailored to serve as a powerful new tool for tracking structural changes in two major encapsulation designs, alginate-based microbeads and multi-component microcapsules. CRM analyses before implantation and after explantation from a mouse model revealed complete loss of the original heterogeneous structure in the alginate microbeads, making the intentionally high initial heterogeneity a questionable design choice. On the other hand, the structural heterogeneity was conserved in the microcapsules, which indicates that this design will better retain its immunoprotective properties in vivo. In another application, CRM was used for quantitative mapping of the alginate concentration throughout the microbead volume. Such data provide invaluable information about the microenvironment cells would encounter upon their encapsulation in alginate microbeads.

Reduction of measurement noise in a continuous glucose monitor by coating the sensor with a zwitterionic polymer.

Continuous glucose monitors (CGMs), used by patients with diabetes mellitus, can autonomously track fluctuations in blood glucose over time. However, the signal produced by CGMs during the initial recording period following sensor implantation contains substantial noise, requiring frequent recalibration via fingerprick tests. Here, we show that coating the sensor with a zwitterionic polymer, found via a combinatorial-chemistry approach, significantly reduces signal noise and improves CGM performance. We evaluated the polymer-coated sensors in mice as well as in healthy and diabetic non-human primates, and show that the sensors accurately record glucose levels without the need for recalibration. We also show that the polymer-coated sensors significantly abrogated immune responses to the sensor, as indicated by histology, fluorescent whole-body imaging of inflammation-associated protease activity, and gene expression of inflammation markers. The polymer coating may allow CGMs to become standalone measuring devices.

Alginate encapsulation as long-term immune protection of allogeneic pancreatic islet cells transplanted into the omental bursa of macaques.

The transplantation of pancreatic islet cells could restore glycaemic control in patients with type-I diabetes. Microspheres for islet encapsulation have enabled long-term glycaemic control in diabetic rodent models; yet human patients transplanted with equivalent microsphere formulations have experienced only transient islet-graft function, owing to a vigorous foreign-body reaction (FBR), to pericapsular fibrotic overgrowth (PFO) and, in upright bipedal species, to the sedimentation of the microspheres within the peritoneal cavity. Here, we report the results of the testing, in non-human primate (NHP) models, of seven alginate formulations that were efficacious in rodents, including three that led to transient islet-graft function in clinical trials. Although one month post-implantation all formulations elicited significant FBR and PFO, three chemically modified, immune-modulating alginate formulations elicited reduced FBR. In conjunction with a minimally invasive transplantation technique into the bursa omentalis of NHPs, the most promising chemically modified alginate derivative (Z1-Y15) protected viable and glucose-responsive allogeneic islets for 4 months without the need for immunosuppression. Chemically modified alginate formulations may enable the long-term transplantation of islets for the correction of insulin deficiency.

Effect of Manufacturing Procedures on Human Islet Isolation From Donor Pancreata Standardized by the North American Islet Donor Score.

This study investigates manufacturing procedures that affect islet isolation outcomes from donor pancreata standardized by the North American Islet Donor Score (NAIDS). Islet isolations performed at the University of Illinois, Chicago, from pancreata with NAIDS ≥65 were investigated. The research cohort was categorized into two groups based on a postpurification yield either greater than (group A) or less than (group B) 400,000 IEQ. Associations between manufacturing procedures and islet isolation outcomes were analyzed using multivariate logistic or linear regressions. A total of 119 cases were retrieved from 630 islet isolations performed since 2003. Group A is composed of 40 cases with an average postpurified yield of 570,098 IEQ, whereas group B comprised 79 cases with an average yield of 235,987 IEQ. One third of 119 cases were considered successful islet isolations that yielded >400,000 IEQ. The prepurified and postpurified islet product outcome parameters were detailed for future reference. The NAIDS (>80 vs. 65-80) [odds ratio (OR): 2.91, 95% confidence interval (CI): 1.27-6.70], cold ischemic time (≤10 vs. >10 h) (OR: 3.68, 95% CI: 1.61-8.39), and enzyme perfusion method (mechanical vs. manual) (OR: 2.38, 95% CI: 1.01-5.56) were independent determinants for postpurified islet yield ≥400,000 IEQ. The NAIDS (>80, p < 0.001), cold ischemic time (≤10 h, p < 0.05), increased unit of collagenase (p < 0.01), and pancreatic duct cannulation time (<30 min, p < 0.01) all independently correlated with better islet quantity parameters. Furthermore, cold ischemic time (≤10 h, p < 0.05), liberase MTF (p < 0.001), increased unit of collagenase (p < 0.05), duct cannulation time (<30 min, p < 0.05), and mechanical enzyme perfusion (p < 0.05) were independently associated with better islet morphology score. Analysis of islet manufacturing procedures from the pancreata with standardized quality is essential in identifying technical issues within islet isolation. Adequate processing duration in each step of islet isolation, using liberase MTF, and mechanical enzyme perfusion all affect isolation outcomes.

A microfluidic array for real-time live-cell imaging of human and rodent pancreatic islets.

In this study, we present a microfluidic array for high-resolution imaging of individual pancreatic islets. The device is based on hydrodynamic trapping principle and enables real-time analysis of islet cellular responses to insulin secretagogues. This device has significant advantages over our previously published perifusion chamber device including significantly increased analytical power and assay sensitivity, as well as improved spatiotemporal resolution. The islet array, with live-cell multiparametric imaging integration, provides a better tool to understand the physiological and pathophysiological changes of pancreatic islets through the analysis of single islet responses. This platform demonstrates the feasibility of array-based islet cellular analysis and opens up a new modality to conduct informative and quantitive evaluation of islets and cell-based screening for new diabetes treatments.

Long-term glycemic control using polymer-encapsulated human stem cell-derived beta cells in immune-competent mice.

The transplantation of glucose-responsive, insulin-producing cells offers the potential for restoring glycemic control in individuals with diabetes. Pancreas transplantation and the infusion of cadaveric islets are currently implemented clinically, but these approaches are limited by the adverse effects of immunosuppressive therapy over the lifetime of the recipient and the limited supply of donor tissue. The latter concern may be addressed by recently described glucose-responsive mature beta cells that are derived from human embryonic stem cells (referred to as SC-ß cells), which may represent an unlimited source of human cells for pancreas replacement therapy. Strategies to address the immunosuppression concerns include immunoisolation of insulin-producing cells with porous biomaterials that function as an immune barrier. However, clinical implementation has been challenging because of host immune responses to the implant materials. Here we report the first long-term glycemic correction of a diabetic, immunocompetent animal model using human SC-ß cells. SC-ß cells were encapsulated with alginate derivatives capable of mitigating foreign-body responses in vivo and implanted into the intraperitoneal space of C57BL/6J mice treated with streptozotocin, which is an animal model for chemically induced type 1 diabetes. These implants induced glycemic correction without any immunosuppression until their removal at 174 d after implantation. Human C-peptide concentrations and in vivo glucose responsiveness demonstrated therapeutically relevant glycemic control. Implants retrieved after 174 d contained viable insulin-producing cells.

Neurogenin 3 Expressing Cells in the Human Exocrine Pancreas Have the Capacity for Endocrine Cell Fate.

Neurogenin 3 (NGN3) is necessary and sufficient for endocrine differentiation during pancreatic development and is expressed by a population of progenitor cells that give rise exclusively to hormone-secreting cells within islets. NGN3 protein can be detected in the adult rodent pancreas only following certain types of injury, when it is transiently expressed by exocrine cells undergoing reprogramming to an endocrine cell fate. Here, NGN3 protein can be detected in 2% of acinar and duct cells in living biopsies of histologically normal adult human pancreata and 10% in cadaveric biopsies of organ donor pancreata. The percentage and total number of NGN3+ cells increase during culture without evidence of proliferation or selective cell death. Isolation of highly purified and viable NGN3+ cell populations can be achieved based on coexpression of the cell surface glycoprotein CD133. Transcriptome and targeted expression analyses of isolated CD133+ / NGN3+ cells indicate that they are distinct from surrounding exocrine tissue with respect to expression phenotype and Notch signaling activity, but retain high level mRNA expression of genes indicative of acinar and duct cell function. NGN3+ cells have an mRNA expression profile that resembles that of mouse early endocrine progenitor cells. During in vitro differentiation, NGN3+ cells express genes in a pattern characteristic of endocrine development and result in cells that resemble beta cells on the basis of coexpression of insulin C-peptide, chromogranin A and pancreatic and duodenal homeobox 1. NGN3 expression in the adult human exocrine pancreas marks a dedifferentiating cell population with the capacity to take on an endocrine cell fate. These cells represent a potential source for the treatment of diabetes either through ex vivo manipulation, or in vivo by targeting mechanisms controlling their population size and endocrine cell fate commitment.

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.

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.