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.

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.

Size- and shape-dependent foreign body immune response to materials implanted in rodents and non-human primates.

The efficacy of implanted biomedical devices is often compromised by host recognition and subsequent foreign body responses. Here, we demonstrate the role of the geometry of implanted materials on their biocompatibility in vivo. In rodent and non-human primate animal models, implanted spheres 1.5 mm and above in diameter across a broad spectrum of materials, including hydrogels, ceramics, metals and plastics, significantly abrogated foreign body reactions and fibrosis when compared with smaller spheres. We also show that for encapsulated rat pancreatic islet cells transplanted into streptozotocin-treated diabetic C57BL/6 mice, islets prepared in 1.5-mm alginate capsules were able to restore blood-glucose control for up to 180 days, a period more than five times longer than for transplanted grafts encapsulated within conventionally sized 0.5-mm alginate capsules. Our findings suggest that the in vivo biocompatibility of biomedical devices can be significantly improved simply by tuning their spherical dimensions.

Microfluidic array with integrated oxygenation control for real-time live-cell imaging: effect of hypoxia on physiology of microencapsulated pancreatic islets.

In this article, we present a novel microfluidic islet array based on a hydrodynamic trapping principle. The lab-on-a-chip studies with live-cell multiparametric imaging allow understanding of physiological and pathophysiological changes of microencapsulated islets under hypoxic conditions. Using this microfluidic array and imaging analysis techniques, we demonstrate that hypoxia impairs the function of microencapsulated islets at the single islet level, showing a heterogeneous pattern reflected in intracellular calcium signaling, mitochondrial energetic, and redox activity. Our approach demonstrates an improvement over conventional hypoxia chambers that is able to rapidly equilibrate to true hypoxia levels through the integration of dynamic oxygenation. This work demonstrates the feasibility of array-based cellular analysis and opens up new modality to conduct informative analysis and cell-based screening for microencapsulated pancreatic islets.

Implication of mitochondrial cytoprotection in human islet isolation and transplantation.

Islet transplantation is a promising therapy for type 1 diabetes mellitus; however, success rates in achieving both short- and long-term insulin independence are not consistent, due in part to inconsistent islet quality and quantity caused by the complex nature and multistep process of islet isolation and transplantation. Since the introduction of the Edmonton Protocol in 2000, more attention has been placed on preserving mitochondrial function as increasing evidences suggest that impaired mitochondrial integrity can adversely affect clinical outcomes. Some recent studies have demonstrated that it is possible to achieve islet cytoprotection by maintaining mitochondrial function and subsequently to improve islet transplantation outcomes. However, the benefits of mitoprotection in many cases are controversial and the underlying mechanisms are unclear. This article summarizes the recent progress associated with mitochondrial cytoprotection in each step of the islet isolation and transplantation process, as well as islet potency and viability assays based on the measurement of mitochondrial integrity. In addition, we briefly discuss immunosuppression side effects on islet graft function and how transplant site selection affects islet engraftment and clinical outcomes.

Systematic prevention of bubble formation and accumulation for long-term culture of pancreatic islet cells in microfluidic device.

Reliable long-term cell culture in microfluidic system is limited by air bubble formation and accumulation. In this study, we developed a bubble removal system capable of both trapping and discharging air bubbles in a consistent and reliable manner. Combined with PDMS (Polydimethylsiloxane) hydrophilic surface treatment and vacuum filling, a microfluidic perifusion system equipped with the bubble trap was successfully applied for long-term culture of mouse pancreatic islets with no bubble formation and no flow interruption. In addition to demonstrating normal cell viability and islet morphology, post-cultured islets exhibited normal insulin secretion kinetics, intracellular calcium signaling, and changes in mitochondrial potentials in response to glucose challenge. This design could be easily adapted by other microfluidic systems due to its simple design, ease of fabrication, and portability.

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.

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.