A scalable human islet 3D-culture platform maintains cell mass and function long-term for transplantation.

Present-day islet culture methods provide short-term maintenance of cell viability and function, limiting access to islet transplantation. Attempts to lengthen culture intervals remain unsuccessful. A new method was developed to permit the long-term culture of islets. Human islets were embedded in polysaccharide 3D-hydrogel in cell culture inserts or gas-permeable chambers with serum-free CMRL 1066 supplemented media for up to 8 weeks. The long-term cultured islets maintained better morphology, cell mass, and viability at 4 weeks than islets in conventional suspension culture. In fact, islets cultured in the 3D-hydrogel retained ß cell mass and function on par with freshly isolated islets in vitro and, when transplanted into diabetic mice, restored glucose balance similar to fresh islets. Using gas-permeable chambers, the 3D-hydrogel culture method was scaled up over 10-fold and maintained islet viability and function, although the cell mass recovery rate was 50%. Additional optimization of scale-up methods continues. If successful, this technology could afford flexibility and expand access to islet transplantation, especially single-donor islet-after-kidney transplantation.

The CD318/CD6 axis limits type 1 diabetes islet autoantigen-specific human T cell activation.

CD6 is a glycoprotein expressed on CD4 and CD8 T cells involved in immunoregulation. CD318 has been identified as a CD6 ligand. The role of CD318 in T cell immunity is restricted as it has only been investigated in a few mice autoimmune models but not in human diseases. CD318 expression was thought to be limited to mesenchymal-epithelial cells and, therefore, contribute to CD6-mediated T cell activation in the CD318-expressing tissue rather than through interaction with antigen-presenting cells. Here, we report CD318 expression in a subpopulation of CD318+ myeloid dendritic (mDC), whereas the other peripheral blood populations were CD318 negative. However, CD318 can be induced by activation: a subset of monocytes treated with LPS and IFNγ and in vitro monocyte derived DCs were CD318+. We also showed that recombinant CD318 inhibited T cell function. Strikingly, CD318+ DCs suppressed the proliferation of autoreactive T cells specific for GAD65, a well-known targeted self-antigen in Type 1 Diabetes (T1D). Our study provides new insight into the role of the CD318/CD6 axis in the immunopathogenesis of inflammation, suggesting a novel immunoregulatory role of CD318 in T cell-mediated autoimmune diseases and identifying a potential novel immune checkpoint inhibitor as a target for intervention in T1D which is an unmet therapeutic need.

Thrombospondin-1, CD47, and SIRPα display cell-specific molecular signatures in human islets and pancreata.

Thrombospondin-1 (TSP1) is a secreted protein minimally expressed in health but increased in disease and age. TSP1 binds to the cell membrane receptor CD47, which itself engages signal regulatory protein α (SIRPα), and the latter creates a checkpoint for immune activation. Individuals with cancer administered checkpoint-blocking molecules developed insulin-dependent diabetes. Relevant to this, CD47 blocking antibodies and SIRPα fusion proteins are in clinical trials. We characterized the molecular signature of TSP1, CD47, and SIRPα in human islets and pancreata. Fresh islets and pancreatic tissue from nondiabetic individuals were obtained. The expression of THBS1, CD47, and SIRPA was determined using single-cell mRNA sequencing, immunofluorescence microscopy, Western blot, and flow cytometry. Islets were exposed to diabetes-affiliated inflammatory cytokines and changes in protein expression were determined. CD47 mRNA was expressed in all islet cell types. THBS1 mRNA was restricted primarily to endothelial and mesenchymal cells, whereas SIRPA mRNA was found mostly in macrophages. Immunofluorescence staining showed CD47 protein expressed by ß cells and present in the exocrine pancreas. TSP1 and SIRPα proteins were not seen in islets or the exocrine pancreas. Western blot and flow cytometry confirmed immunofluorescent expression patterns. Importantly, human islets produced substantial quantities of secreted TSP1. Human pancreatic exocrine and endocrine tissue expressed CD47, whereas fresh islets displayed cell surface CD47 and secreted TSP1 at baseline and in inflammation. These findings suggest unexpected effects on islets from agents that intersect TSP1-CD47-SIRPα.NEW & NOTEWORTHY CD47 is a cell surface receptor with two primary ligands, soluble thrombospondin-1 (TSP1) and cell surface signal regulatory protein alpha (SIRPα). Both interactions provide checkpoints for immune cell activity. We determined that fresh human islets display CD47 and secrete TSP1. However, human islet endocrine cells lack SIRPα. These gene signatures are likely important given the increasing use of CD47 and SIRPα blocking molecules in individuals with cancer.

Colony stimulating factor-1 receptor is a central component of the foreign body response to biomaterial implants in rodents and non-human primates.

Host recognition and immune-mediated foreign body response to biomaterials can compromise the performance of implanted medical devices. To identify key cell and cytokine targets, here we perform in-depth systems analysis of innate and adaptive immune system responses to implanted biomaterials in rodents and non-human primates. While macrophages are indispensable to the fibrotic cascade, surprisingly neutrophils and complement are not. Macrophages, via CXCL13, lead to downstream B cell recruitment, which further potentiated fibrosis, as confirmed by B cell knockout and CXCL13 neutralization. Interestingly, colony stimulating factor-1 receptor (CSF1R) is significantly increased following implantation of multiple biomaterial classes: ceramic, polymer and hydrogel. Its inhibition, like macrophage depletion, leads to complete loss of fibrosis, but spares other macrophage functions such as wound healing, reactive oxygen species production and phagocytosis. Our results indicate that targeting CSF1R may allow for a more selective method of fibrosis inhibition, and improve biomaterial biocompatibility without the need for broad immunosuppression.

Encompassing ATP, DNA, insulin, and protein content for quantification and assessment of human pancreatic islets.

Islet transplantation has made major progress to treat patients with type 1 diabetes. Islet mass and quality are critically important to ensure successful transplantation. Currently, islet status is evaluated using insulin secretion, oxygen consumption rate, or adenosine triphosphate (ATP) measurement. These parameters are evaluated independently and do not effectively predict islet status post-transplant. Therefore, assessing human pancreatic islets by encompassing ATP, DNA, insulin, and protein content from a single tissue sample would serve as a better predictor for islet status. In this study, a single step procedure for extracting ATP, DNA, insulin, and protein content from human pancreatic islets was described and the biomolecule contents were quantified. Additionally, different mathematical calculations integrating total ATP, DNA, insulin, and protein content were randomly tested under various conditions to predict islet status. The results demonstrated that the ATP assay was efficient and the biomolecules were effectively quantified. Furthermore, the mathematical formula we developed could be optimized to predict islet status. In conclusion, our results indicate a proof-of-concept that a simple logarithmic formula can predict overall islet status for various conditions when total islet ATP, DNA, insulin, and protein content are simultaneously assessed from a single tissue sample.

Sodium levels of human pancreatic donors are a critical factor for determination of islet efficacy and survival.

Organs from hypernatremia (elevated Na+) donors when used for transplantation have had dismal outcomes. However, islet isolation from hypernatremic donors for both transplantation and research applications has not yet been investigated. A retrospective analysis of in vivo and in vitro islet function studies was performed on islets isolated from hypernatremic (serum sodium levels≥160 meq/l) and normal control (serum sodium levels≤155 meq/l) donors. Twelve isolations from 32 hypernatremic and 53 isolations from 222 normal donors were randomly transplanted into diabetic NOD Scid mice. Sodium levels upon pancreas procurement were significantly elevated in the hypernatremia group (163.5±0.6 meq/l) compared with the normal control group (145.9±0.4 meq/l) (P<0.001). The postculture islet recovery rate was significantly lower in the hypernatremia (59.1±3.8%) group compared with the normal (73.6±1.8%) group (P=0.005). The duration of hypernatremia was inversely correlated with the recovery rate (r2=0.370, P<0.001). Furthermore, the percentage of successful graft function when transplanted into diabetic NOD Scid mice was significantly lower in the hypernatremia (42%) group compared with the normal control (85%) group (P<0.001). The ability to predict islet graft function posttransplantation using donor sodium levels and duration of hypernatremia was significant (ROC analysis, P=0.022 and 0.042, respectively). In conclusion, duration of donor hypernatremia is associated with reduced islet recovery postculture. The efficacy of islets from hypernatremia donors diminished when transplanted into diabetic recipients.

Mapping Endothelial-Macrophage Interactions in Diabetic Vasculature: Role of TREM2 in Vascular Inflammation and Ischemic Response.

Vasculopathies occur 15 years earlier in individuals with diabetes mellitus (DM) as compared to those without, but the underlying mechanisms driving diabetic vasculopathy remain incompletely understood. Endothelial cells (ECs) and macrophages (MΦ) are critical players in vascular wall and their crosstalk is crucial in diabetic vasculopathy. In diabetes, EC activation enables monocyte recruitment, which transmigrate into the intima and differentiate into macrophages (MΦ). Beyond this established model of diapedesis, EC-MΦ interplay is highly intricate and heterogenous. To capture these highly context dependent EC-MΦ interactions, we leveraged single-cell (sc)RNA-seq in conjunction with spatial transcriptome (ST)-seq profiling to analyze human mesenteric arteries from non-diabetic (ND) and type 2 diabetic (T2D) donors. We provide in this study a transcriptomic map encompassing major arterial vascular cells, e.g., EC, mononuclear phagocyte (MP), and T cells, and their interactions associated with human T2D. Furthermore, we identified Triggering Receptor Expressed on Myeloid Cells 2 ( TREM2) as a top T2D-induced gene in MP, with concomitant increase of TREM2 ligands in ECs. TREM2 induction was confirmed in mouse models of T2D and monocyte/MΦ subjected to DM-mimicking stimuli. Perturbing TREM2 with either an antibody or silencing RNA in MPs led to decreased pro-inflammatory responses in MPs and ECs and increased EC migration in vitro . In a mouse model of diabetes, TREM2 expression and its interaction with ECs are increased in the ischemic, as compared to non-ischemic muscles. Importantly, neutralization of TREM2 using a neutralizing antibody enhanced ischemic recovery and flow reperfusion in the diabetic mice, suggesting a role of TREM2 in promoting diabetic PAD. Finally, we verified that both TREM2 expression and the TREM2-EC-interaction are increased in human patients with DM-PAD. Collectively, our study presents the first atlas of human diabetic vessels with a focus on EC-MP interactions. Exemplified by TREM2, our study provides valuable insights into EC-MΦ interactions, key processes contributing to diabetic vasculopathies and the potential of targeting these interactions for therapeutic development.

Biological hypoxia in pre-transplant human pancreatic islets induces transplant failure in diabetic mice.

Evaluating the quality of isolated human islets before transplantation is crucial for predicting the success in treating Type 1 diabetes. The current gold standard involves time-intensive in vivo transplantation into diabetic immunodeficient mice. Given the susceptibility of isolated islets to hypoxia, we hypothesized that hypoxia present in islets before transplantation could indicate compromised islet quality, potentially leading to unfavorable outcomes. To test this hypothesis, we analyzed expression of 39 hypoxia-related genes in human islets from 85 deceased donors. We correlated gene expression profiles with transplantation outcomes in 327 diabetic mice, each receiving 1200 islet equivalents grafted into the kidney capsule. Transplantation outcome was post-transplant glycemic control based on area under the curve of blood glucose over 4 weeks. In linear regression analysis, DDIT4 (R = 0.4971, P < 0.0001), SLC2A8 (R = 0.3531, P = 0.0009) and HK1 (R = 0.3444, P = 0.0012) had the highest correlation with transplantation outcome. A multiple regression model of 11 genes increased the correlation (R = 0.6117, P < 0.0001). We conclude that assessing pre-transplant hypoxia in human islets via gene expression analysis is a rapid, viable alternative to conventional in vivo assessments. This approach also underscores the importance of mitigating pre-transplant hypoxia in isolated islets to improve the success rate of islet transplantation.

Physiomimetic Fluidic Culture Platform on Microwell-Patterned Porous Collagen Scaffold for Human Pancreatic Islets.

Pancreatic islet transplantation is one of the clinical options for certain types of diabetes. However, difficulty in maintaining islets prior to transplantation limits the clinical expansion of islet transplantations. Our study introduces a dynamic culture platform developed specifically for primary human islets by mimicking the physiological microenvironment, including tissue fluidics and extracellular matrix support. We engineered the dynamic culture system by incorporating our distinctive microwell-patterned porous collagen scaffolds for loading isolated human islets, enabling vertical medium flow through the scaffolds. The dynamic culture system featured four 12 mm diameter islet culture chambers, each capable of accommodating 500 islet equivalents (IEQ) per chamber. This configuration calculates > five-fold higher seeding density than the conventional islet culture in flasks prior to the clinical transplantations (442 vs 86 IEQ/cm2). We tested our culture platform with three separate batches of human islets isolated from deceased donors for an extended period of 2 weeks, exceeding the limits of conventional culture methods for preserving islet quality. Static cultures served as controls. The computational simulation revealed that the dynamic culture reduced the islet volume exposed to the lethal hypoxia (< 10 mmHg) to ~1/3 of the static culture. Dynamic culture ameliorated the morphological islet degradation in long-term culture and maintained islet viability, with reduced expressions of hypoxia markers. Furthermore, dynamic culture maintained the islet metabolism and insulin-secreting function over static culture in a long-term culture. Collectively, the physiological microenvironment-mimetic culture platform supported the viability and quality of isolated human islets at high-seeding density. Such a platform has a high potential for broad applications in cell therapies and tissue engineering, including extended islet culture prior to clinical islet transplantations and extended culture of stem cell-derived islets for maturation.

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.

Reassessing the Abundance of miRNAs in the Human Pancreas and Rodent Cell Lines and Its Implication.

miRNAs are critical for pancreas development and function. However, we found that there are discrepancies regarding pancreatic miRNA abundance in published datasets. To obtain a more relevant profile that is closer to the true profile, we profiled small RNAs from human islets cells, acini, and four rodent pancreatic cell lines routinely used in diabetes and pancreatic research using a bias reduction protocol for small RNA sequencing. In contrast to the previous notion that miR-375-3p is the most abundant pancreatic miRNA, we found that miR-148a-3p and miR-7-5p were also abundant in islets. In silico studies using predicted and validated targets of these three miRNAs revealed that they may work cooperatively in endocrine and exocrine cells. Our results also suggest, compared to the most-studied miR-375, that both miR-148a-3p and miR-7-5p may play more critical roles in the human pancreas. Moreover, according to in silico-predicted targets, we found that miR-375-3p had a much broader target spectrum by targeting the coding sequence and the 5' untranslated region, rather than the conventional 3' untranslated region, suggesting additional unexplored roles of miR-375-3p beyond the pancreas. Our study provides a valuable new resource for studying miRNAs in pancreata.

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.

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.

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.

Effect of prolonged gelling time on the intrinsic properties of barium alginate microcapsules and its biocompatibility.

Pericapsular fibrotic overgrowth (PFO) may be attributed to an immune response against microcapsules themselves or to antigen shedding through microcapsule pores from encapsulated islet tissue. Modification of microcapsules aimed at reducing pore size should prevent PFO and improve graft survival. This study investigated the effect of increased gelling time (20 vs. 2 min) in barium chloride on intrinsic properties of alginate microcapsules and tested their biocompatibility in vivo. Prolonged gelling time affected neither permeability nor size of the microcapsules. However, prolonged gelling time for 20 min produced brittle microcapsules compared to 2 min during compression test. Encapsulation of human islets in both types of microcapsules affected neither islet viability nor function. The presence of PFO when transplanted into a large animal model such as baboon and its absence in small animal models such as rodents suggest that the host immune response towards alginate microcapsules is species rather than alginate specific.

Chronic marijuana usage by human pancreas donors is associated with impaired islet function.

We investigated the effect of chronic marijuana use, defined as 4 times weekly for more than 3 years, on human pancreatic islets. Pancreata from deceased donors who chronically used marijuana were compared to those from age, sex and ethnicity matched non-users. The islets from marijuana-users displayed reduced insulin secretion as compared to islets from non-users upon stimulation with high glucose (AUC, 3.41 ± 0.62 versus 5.14 ±0.47, p<0.05) and high glucose plus KCl (AUC, 4.48 ± 0.41 versus 7.69 ± 0.58, p<0.001). When human islets from chronic marijuana-users were transplanted into diabetic mice, the mean reversal rate of diabetes was 35% versus 77% in animals receiving islets from non-users (p<0.01). Immunofluorescent staining for cannabinoid receptor type 1 (CB1R) was shown to be colocalized with insulin and enhanced significantly in beta cells from marijuana-users vs. non-users (CB1R intensity/islet area, 14.95 ± 2.71 vs. 3.23 ± 0.87, p<0.001). In contrast, CB1R expression was not co-localized with glucagon or somatostatin. Furthermore, isolated islets from chronic marijuana-users appeared hypertrophic. In conclusion, excessive marijuana use affects islet endocrine phenotype and function in vitro and in vivo. Given the increasing use of marijuana, our results underline the importance of including lifestyle when evaluating human islets for transplantation or research.

A Multiparametric Assessment of Human Islets Predicts Transplant Outcomes in Diabetic Mice.

Prior to transplantation into individuals with type 1 diabetes, in vitro assays are used to evaluate the quality, function and survival of isolated human islets. In addition to the assessments of these parameters in islet, they can be evaluated by multiparametric morphological scoring (0-10 points) and grading (A, B, C, D, and F) based on islet characteristics (shape, border, integrity, single cells, and diameter). However, correlation between the multiparametric assessment and transplantation outcome has not been fully elucidated. In this study, 55 human islet isolations were scored using this multiparametric assessment. The results were correlated with outcomes after transplantation into immunodeficient diabetic mice. In addition, the multiparametric assessment was compared with oxygen consumption rate of isolated islets as a potential prediction factor for successful transplantations. All islet batches were assessed and found to score: 9 points (n = 18, Grade A), 8 points (n = 19, Grade B), and 7 points (n = 18, Grade B). Islets that scored 9 (Grade A), scored 8 (Grade B) and scored 7 (Grade B) were transplanted into NOD/SCID mice and reversed diabetes in 81.2%, 59.4%, and 33.3% of animals, respectively (P < 0.0001). Islet scoring and grading correlated well with glycemic control post-transplantation (P < 0.0001) and reversal rate of diabetes (P < 0.05). Notably, islet scoring and grading showed stronger correlation with transplantation outcome compared to oxygen consumption rate. Taken together, a multiparametric assessment of isolated human islets was highly predictive of transplantation outcome in diabetic mice.

High Fractions of Large Islets in Human Islet Preparations Detrimentally Affect Posttransplant Outcomes in Streptozotocin-Induced Diabetic Immunodeficient Mice.

OBJECTIVES: The aim of this study was to determine whether the size of islets isolated from human donors-measured pretransplant-impacts transplantation outcomes in diabetic mice. METHODS: Human islets (1200 islet equivalents) were transplanted into the kidney capsules of streptozotocin-induced diabetic immunodeficient mice. Data from a total of 174 mice that received islets from 45 isolations were analyzed to evaluate the correlation between pretransplant islet size and posttransplant diabetes reversal. Fluorescent images of islet clusters were used to categorize individual islets by size (small, 50-150 µm; medium, 150-250 µm; large, >250 µm), and the fractions of islets in each category were calculated. RESULTS: The fraction of large islets negatively correlated with diabetes reversal rates. Mice that received islet grafts containing 0% to 5%, 5% to 10%, and more than 10% large islets had diabetes reversal rates of 75%, 61%, and 45%, respectively (P = 0.0112). Furthermore, mice that exhibited diabetes reversal received smaller fractions of large islets than mice that did not (5.5% vs 8.0%, P = 0.0003). Intriguingly, the fractions of medium and small islets did not correlate with diabetes reversal outcomes. CONCLUSIONS: The fraction of large islets is a sensitive predictor of human islet transplantation outcomes in diabetic mice.

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.