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.

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.

Ubiquitous Luciferase Expression in "Firefly Rats" Does Not Alter the Pancreatic Islet Morphology, Metabolism, and Function.

"Firefly rats" ubiquitously express the luciferase reporter gene under the control of constitutively active ROSA26 promoter in inbred Lewis rats. Due to the minimal immunogenicity of luciferase, wide applications of Firefly rats have been reported in solid organ/cell transplantation studies for in vivo imaging, permitting quantitative and non-invasive tracking of the transplanted graft. ROSA26 is a non-coding gene and generally does not affect the expression of other endogenous genes. However, the effect of ubiquitous luciferase expression on islet morphology and function has not been thoroughly investigated, which is critical for the use of Firefly rats as islet donors in islet transplantation studies. Accordingly, in vivo glucose homeostasis (i.e., islet function in the native pancreas) was compared between age-matched luciferase-expressing Firefly rats and non-luciferase-expressing rats. In vivo assessments demonstrated no statistical difference between these rats in non-fasting blood glucose levels, intraperitoneal glucose tolerance tests, and glucose-stimulated serum C-peptide levels. Furthermore, islets were isolated from both rats to compare the morphology, function, and metabolism in vitro. Isolated islets from both rats exhibited similar in vitro characteristics in post-isolation islet yield, islet size, beta cell populations, insulin content per islet, oxygen consumption rate, and glucose-stimulated insulin secretion. In conclusion, ubiquitous luciferase expression in Firefly rats does not affect their islet morphology, metabolism, and function; this finding is critical and enables the use of isolated islets from Firefly rats for the dual assessment of islet graft function and bioluminescence imaging of islet grafts.

Microwell culture platform maintains viability and mass of human pancreatic islets.

Background: Transplantation of the human pancreatic islets is a promising approach for specific types of diabetes to improve glycemic control. Although effective, there are several issues that limit the clinical expansion of this treatment, including difficulty in maintaining the quality and quantity of isolated human islets prior to transplantation. During the culture, we frequently observe the multiple islets fusing together into large constructs, in which hypoxia-induced cell damage significantly reduces their viability and mass. In this study, we introduce the microwell platform optimized for the human islets to prevent unsolicited fusion, thus maintaining their viability and mass in long-term cultures. Method: Human islets are heterogeneous in size; therefore, two different-sized microwells were prepared in a 35 mm-dish format: 140 µm × 300 µm-microwells for <160 µm-islets and 200 µm × 370 µm-microwells for >160 µm-islets. Human islets (2,000 islet equivalent) were filtered through a 160 µm-mesh to prepare two size categories for subsequent two week-cultures in each microwell dish. Conventional flat-bottomed 35 mm-dishes were used for non-filtered islets (2,000 islet equivalent/2 dishes). Post-cultured islets are collected to combine in each condition (microwells and flat) for the comparisons in viability, islet mass, morphology, function and metabolism. Islets from three donors were independently tested. Results: The microwell platform prevented islet fusion during culture compared to conventional flat bottom dishes, which improved human islet viability and mass. Islet viability and mass on the microwells were well-maintained and comparable to those in pre-culture, while flat bottom dishes significantly reduced islet viability and mass in two weeks. Morphology assessed by histology, insulin-secreting function and metabolism by oxygen consumption did not exhibit the statistical significance among the three different conditions. Conclusion: Microwell-bottomed dishes maintained viability and mass of human islets for two weeks, which is significantly improved when compared to the conventional flat-bottomed dishes.

Micropyramid-patterned, oxygen-permeable bottomed dish for high density culture of pancreatic islets.

The need for maintaining cell-spheroid viability and function within high-density cultures is unmet for various clinical and experimental applications, including cell therapies. One immediate application is for transplantation of pancreatic islets, a clinically recognized treatment option to cure type 1 diabetes; islets are isolated from a donor for subsequent culture prior to transplantation. However, high seeding conditions cause unsolicited fusion of multiple spheroids, thereby limiting oxygen diffusion to induce hypoxic cell death. Here we introduce a culture dish incorporating a micropyramid-patterned surface to prevent the unsolicited fusion and oxygen-permeable bottom for optimal oxygen environment. A 400µm-thick, oxygen-permeable polydimethylsiloxane sheet topped with micropyramid pattern of 400µm-base and 200µm-height was fabricated to apply to the 24-well plate format. The micropyramid pattern separated the individual pancreatic islets to prevent the fusion of multiple islets. This platform supported the high oxygen demand of islets at high seeding density at 260 islet equivalents cm-2, a 2-3-fold higher seeding density compared to the conventional islet culture used in a preparation for the clinical islet transplantations, demonstrating improved islet morphology, metabolism and function in a 4 d-culture. Transplantation of these islets into immunodeficient diabetic mice exhibited significantly improved engraftment to achieve euglycemia compared to islets cultured in the conventional culture wells. Collectively, this simple design modification allows for high-density cultures of three-dimensional cell spheroids to improve the viability and function for an array of investigational and clinical replacement tissues.

Critical Considerations in Bioluminescence Imaging of Transplanted Islets: Dynamic Signal Change in Early Posttransplant Phase and Signal Absorption by Tissues.

OBJECTIVES: In pancreatic islet transplantation studies, bioluminescence imaging enables quantitative and noninvasive tracking of graft survival. Amid the recent heightened interest in extrahepatic sites for islet and stem cell-derived beta-like cell transplantations, proper understanding the nature of bioluminescence imaging in these sites is important. METHODS: Islets isolated from Firefly rats ubiquitously expressing luciferase reporter gene in Lewis rats were transplanted into subcutaneous or kidney capsule sites of wild-type Lewis rats or immunodeficient mice. Posttransplant changes of bioluminescence signal curves and absorption of bioluminescence signal in transplantation sites were examined. RESULTS: The bioluminescence signal curve dynamically changed in the early posttransplantation phase; the signal was low within the first 5 days after transplantation. A substantial amount of bioluminescence signal was absorbed by tissues surrounding islet grafts, correlating to the depth of the transplanted site from the skin surface. Grafts in kidney capsules were harder to image than those in the subcutaneous site. Within the kidney capsule, locations that minimized depth from the skin surface improved the graft detectability. CONCLUSIONS: Posttransplant phase and graft location/depth critically impact the bioluminescence images captured in islet transplantation studies. Understanding these parameters is critical for reducing experimental biases and proper interpretation of data.

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.

Early-Phase Luciferase Signals of Islet Grafts Predicts Successful Subcutaneous Site Transplantation in Rats.

PURPOSE: The transplantation of pancreatic islets is a promising cell replacement therapy for type 1 diabetes. Subcutaneous islet transplantation is currently under investigation as a means to circumvent problems associated with standard intra-hepatic islet transplantation. As modifications are being developed to improve the efficacy of subcutaneous islet transplantation, it is important to have robust methods to assess engraftment. Experimentally, ATP-dependent bioluminescence imaging using luciferase reporter genes has been effective for non-invasively tracking engraftment. However, it was heretofore unknown if the bioluminescence of subcutaneously transplanted luciferase-expressing islet grafts correlates with diabetes reversal, a primary outcome of transplantation. PROCEDURES: A retrospective analysis was conducted using data obtained from subcutaneous islet transplantations in Lewis rats. The analysis included transplantations from our laboratory in which islet donors were transgenic rats ubiquitously expressing luciferase and recipients were wild type, streptozotocin-induced diabetic rats. Data from 79 bioluminescence scans were obtained from 27 islet transplantations during the post-transplant observation period (up to 6 weeks). The bioluminescence intensity of the subcutaneously transplanted grafts, captured after the intravenous administration of luciferin, was correlated with diabetes reversal. RESULTS: After subcutaneous transplantation, islet bioluminescence decreased over time, dropping > 50 % from 1 to 3 weeks post-transplant. Bioluminescence intensity in the early post-transplant phase (1-2 weeks) correlated with the subsequent reversal of diabetes; based on optimized bioluminescence cutoff values, the bioluminescence intensity of islets at 1 and 2 weeks predicted successful transplantations. However, intensity in the late post-transplant phase (≥ 4 weeks) did not reflect transplantation outcomes. CONCLUSIONS: Early-phase bioluminescence imaging of luciferase-expressing islets could serve as a useful tool to predict the success of subcutaneous islet transplantations by preceding changes in glucose homeostasis.

Intermittent normobaric oxygen inhalation enhances subcutaneous prevascularization for cell transplantation.

PURPOSE: Subcutaneous tissue is a promising site for cell transplantation; advantages include minimally invasive procedures and easy post-transplant monitoring. However, limited vascularity is the major known challenge. To address this challenge, a prevascularized graft bed is prepared in recipients. We aimed to establish an improved, clinically applicable approach to promote prevascularization of the subcutaneous graft bed prior to cell transplantation. METHODS: We applied a conventional prevascularization approach by subcutaneously implanting nylon discs into the backs of Lewis rats. After disc implantation, we treated rats with or without intermittent normobaric 100% oxygen inhalation (1 h, twice a day, for consecutive 7 days). We used histology to compare vascular density between the oxygen-treated or control groups. To assess the functional effects of prevascularization, we transplanted three hundred islets isolated from luciferase-transgenic Lewis rats into the oxygen-treated or control wild type Lewis recipients, then used bioluminescence imaging to track engraftment for 4 weeks. RESULTS: Oxygen treatment significantly augmented prevascularization in the subcutaneous site compared to controls. Islet transplantation into prevascularized graft beds demonstrated significant improvement in engraftment efficiency in oxygen-treated recipients compared to controls at 2-4 weeks post-transplantation. CONCLUSION: Combining intermittent normobaric 100% oxygen inhalation with a conventional vascularization approach promotes a functional vasculature within a week. A simple approach using normobaric oxygen has the potential for translation into clinical application in subcutaneous site cell transplantations.

Semi-Automated Assessment of Human Islet Viability Predicts Transplantation Outcomes in a Diabetic Mouse Model.

In clinical and experimental human pancreatic islet transplantations, establishing pretransplant assessments that accurately predict transplantation outcomes is crucial. Conventional in vitro viability assessment that relies on manual counting of viable islets is a routine pretransplant assessment. However, this method does not correlate with transplantation outcomes; to improve the method, we recently introduced a semi-automated method using imaging software to objectively determine area-based viability. The goal of the present study was to correlate semi-automated viability assessment with posttransplantation outcomes of human islet transplantations in diabetic immunodeficient mice, the gold standard for in vivo functional assessment of isolated human islets. We collected data from 61 human islet isolations and 188 subsequent in vivo mouse transplantations. We assessed islet viability by fluorescein diacetate and propidium iodide staining using both the conventional and semi-automated method. Transplantations of 1,200 islet equivalents under the kidney capsule were performed in streptozotocin-induced diabetic immunodeficient mice. Among the pretransplant variables, including donor factors and post-isolation assessments, viability measured using the semi-automated method demonstrated a strong influence on in vivo islet transplantation outcomes in multivariate analysis. We calculated an optimized cutoff value (96.1%) for viability measured using the semi-automated method and showed a significant difference in diabetes reversal rate for islets with viability above this cutoff (77% reversal) vs. below this cutoff (49% reversal). We performed a detailed analysis to show that both the objective measurement and the improved area-based scoring system, which distinguished between small and large islets, were key features of the semi-automated method that allowed for precise evaluation of viability. Taken together, our results suggest that semi-automated viability assessment offers a promising alternative pretransplant assessment over conventional manual assessment to predict human islet transplantation outcomes.

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.

A subcutaneous pancreatic islet transplantation platform using a clinically applicable, biodegradable Vicryl mesh scaffold - an experimental study.

Pancreatic islet transplantation into the liver is an effective treatment for type 1 diabetes but has some critical limitations. The subcutaneous site is a potential alternative transplant site, requiring minimally invasive procedures and allowing frequent graft monitoring; however, hypoxia is a major drawback. Our previous study without scaffolding demonstrated post-transplant graft aggregation in the subcutaneous site, which theoretically exacerbates lethal intra-graft hypoxia. In this study, we introduce a clinically applicable subcutaneous islet transplantation platform using a biodegradable Vicryl mesh scaffold to prevent aggregation in a diabetic rat model. Islets were sandwiched between layers of clinically proven Vicryl mesh within thrombin-fibrin gel. In vitro, the mesh prevented islet aggregation and intra-islet hypoxia, which significantly improved islet viability. In vivo rat syngeneic islet transplantations into a prevascularized subcutaneous pocket demonstrated that the mesh significantly enhanced engraftment, as measured by assays for graft survival and function. Histological examination at 6 weeks showed well-vascularized grafts sandwiched in a flat shape between the mesh layers. The biodegradable mesh was fully absorbed by three months, which alleviated chronic foreign body reaction and fibrosis, and supported long-term graft maintenance. This simple graft shape modification approach is an effective and clinically applicable strategy for improved subcutaneous islet transplantation.

Inflammatory biomarkers in the blood and pancreatic tissue of organ donors that predict human islet isolation success and function.

The pancreas of brain-dead donors is the primary source of islets for transplantation. However, brain death mediates systemic inflammation, which may affect the quantity and quality of isolated islets. Our aim was to identify inflammatory biomarkers in donor blood and/or pancreatic tissue capable of predicting islet isolation success. Blood samples were collected from 21 pancreas donors and 14 healthy volunteers. Pancreatic tissue samples were also collected from the corresponding donor during organ procurement. Six serum cytokines were measured by a fluorescent bead-based immunoassay, and the expression of fifteen inflammatory target genes was quantified by quantitative reverse transcription polymerase chain reaction (RT-qPCR). There was no correlation between serum inflammatory cytokines and mRNA expression of the corresponding genes in peripheral blood mononuclear cells (PBMCs) or pancreatic tissue. The IL6 expression in pancreatic tissue correlated negatively with post-isolation islet yield. Islets isolated from donors highly expressing IFNG in PBMCs and MAC1 in pancreatic tissue functioned poorly in vivo when transplanted in diabetic NODscid mice. Furthermore, the increased MAC1 in pancreatic tissue was positively correlated with donor hospitalization time. Brain death duration positively correlated with higher expression of IL1B in PBMCs and TNF in both PBMCs and pancreatic tissue but failed to show a significant correlation with islet yield and in vivo function. The study indicates that the increased inflammatory genes in donor pancreatic tissues may be considered as biomarkers associated with poor islet isolation outcome.

Isolated pancreatic islet yield and quality is inversely related to organ donor age in rats.

Pancreatic islets consist of several endocrine cell types that maintain glucose homeostasis. Type 1 diabetes (T1D) results from autoimmune-mediated destruction of insulin producing beta cells in pancreatic islets. Islet transplantation is a treatment for certain individuals with T1D. Islet transplantation in rodents, as an experimental model of the clinical scenario, requires consistency of islet quantity and quality to obtain reproducible results. In this study, we investigated the yield and function of the isolated islets from rats of different ages. Pancreata were harvested from young (10-20 week-old), intermediate (21-40 week-old) and old (>41 week-old) male rats and islets were isolated using a standard protocol. Islet number, morphometry, viability, function, and metabolism were characterized. Islet yield, normalized to body weight, decreased as a function of increasing donor age. Islets from pancreata from young animals were larger and less fragmented compared to islets from organs from intermediate and older animals. Islet viability following overnight culture was the same for islets derived from young and intermediate aged donors but less for islets from old donors. Glucose-stimulated insulin secretion was decreased in islets from older donors. Islet metabolism following glucose challenge, as measured by oxygen consumption, revealed that islets from old donors were metabolically slower and lagged in response to glucose-stimuli. These data demonstrate that increasing donor age has a negative impact on isolated islet yield and quality.

Optimizing Temperature and Oxygen Supports Long-term Culture of Human Islets.

Islet transplantation is a promising treatment for type-1 diabetes; however, donor shortage is a concern. Even when a pancreas is available, low islet yield limits the success of transplantation. Islet culture enables pooling of multiple low-yield isolations into an effective islet mass, but isolated islets rapidly deteriorate under conventional culture conditions. Oxygen (O2) depletion in the islet core, which leads to central necrosis and volume loss, is one of the major reasons for this deterioration. METHODS: To promote long-term culture of human islets in PIM-R medium (used for islet research), we adjusted temperature (12°C, 22°C, and 37°C) and O2 concentration (21% and 50%). We simulated the O2 distribution in islets based on islet O2 consumption rate and dissolved O2 in the medium. We determined the optimal conditions for O2 distribution and volume maintenance in a 2-week culture and assessed viability and insulin secretion compared to noncultured islets. In vivo islet engraftment was assessed by transplantation into diabetic nonobese diabetic-severe combined immunodeficiency mouse kidneys. We validated our results using CMRL 1066 medium (used for clinical islet transplantation). RESULTS: Simulation revealed that 12°C of 50% O2 PIM-R culture supplied O2 effectively into the islet core. This condition maintained islet volume at greater than 90% for 2 weeks. There were no significant differences in viability and function in vitro or diabetic reversal rate in vivo between 2-week cultured and noncultured islets. Similar results were obtained using CMRL 1066. CONCLUSIONS: By optimizing temperature and O2 concentration, we cultured human islets for 2 weeks with minimal loss of volume and function.

Oxygen transporter for the hypoxic transplantation site.

Cell transplantation is a promising treatment for complementing lost function by replacing new cells with a desired function, e.g. pancreatic islet transplantation for diabetics. To prevent cell obliteration, oxygen supply is critical after transplantation, especially until the graft is sufficiently re-vascularized. To supply oxygen during this period, we developed a chemical-/electrical-free implantable oxygen transporter that delivers oxygen to the hypoxic graft site from ambient air by diffusion potential. This device is simply structured using a biocompatible silicone-based body that holds islets, connected to a tube that opens outside the body. In computational simulations, the oxygen transporter increased the oxygen level to >120 mmHg within grafts; in contrast, a control device that did not transport oxygen showed <6.5 mmHg. In vitro experiments demonstrated similar results. To test the effectiveness of the oxygen transporter in vivo, we transplanted pancreatic islets, which are susceptible to hypoxia, subcutaneously into diabetic rats. Islets transplanted using the oxygen transporter showed improved graft viability and cellular function over the control device. These results indicate that our oxygen transporter, which is safe and easily fabricated, effectively supplies oxygen locally. Such a device would be suitable for multiple clinical applications, including cell transplantations that require changing a hypoxic microenvironment into an oxygen-rich site.

Surfactants Improve Live Cell Imaging of Human Pancreatic Islets.

OBJECTIVES: Newport Green is a zinc-specific fluorescent dye developed to monitor cellular zinc transport. In pancreatic islets with zinc-rich ß-cells, Newport Green is expected to be useful as an islet-specific indicator for live imaging. However, the low penetration of Newport Green into islets hinders clear detection. The aim of this study was to develop a practical method of live islet imaging by using surfactants to enhance the penetration efficiency. METHODS: Surfactants (F127, Tween 20, and Triton X-100) were co-incubated with Newport Green for fluorescent imaging of live isolated human islet and nonislet tissues. Toxicity, enhancement of Newport Green fluorescence, and effects on specificity to islets were examined. RESULTS: Newport Green fluorescent intensity was increased after co-incubation with all surfactants tested (0.2-3.2 mM); however, surfactants were toxic to islets at high concentrations. Within the nontoxic range, high specificity to islets was observed when co-incubated with Tween 20 at 0.2-0.4 mM, compared with F127 and Triton X-100. This optimized range successfully distinguished islets from nonislet tissues using statistically calculated cutoff value of Newport Green fluorescent intensity. CONCLUSIONS: Surfactants, particularly Tween 20 in the optimized range, effectively and selectively enhanced Newport Green fluorescence in live islets without increasing islet toxicity.

Impact of Oxygen on Pancreatic Islet Survival.

Pancreatic islet transplantation is a promising treatment option for individuals with type 1 diabetes; however, maintaining islet function after transplantation remains a large challenge. Multiple factors, including hypoxia associated events, trigger pretransplant and posttransplant loss of islet function. In fact, islets are easily damaged in hypoxic conditions before transplantation including the preparation steps of pancreas procurement, islet isolation, and culture. Furthermore, after transplantation, islets are also exposed to the hypoxic environment of the transplant site until they are vascularized and engrafted. Because islets are exposed to such drastic environmental changes, protective measures are important to maintain islet viability and function. Many studies have demonstrated that the prevention of hypoxia contributes to maintaining islet quality. In this review, we summarize the latest oxygen-related islet physiology, including computational simulation. Furthermore, we review recent advances in oxygen-associated treatment options used as part of the transplant process, including up-to-date oxygen generating biomaterials as well as a classical oxygen inhalation therapy.

Gene expression signature predicts human islet integrity and transplant functionality in diabetic mice.

There is growing evidence that transplantation of cadaveric human islets is an effective therapy for type 1 diabetes. However, gauging the suitability of islet samples for clinical use remains a challenge. We hypothesized that islet quality is reflected in the expression of specific genes. Therefore, gene expression in 59 human islet preparations was analyzed and correlated with diabetes reversal after transplantation in diabetic mice. Analysis yielded 262 differentially expressed probesets, which together predict islet quality with 83% accuracy. Pathway analysis revealed that failing islet preparations activated inflammatory pathways, while functional islets showed increased regeneration pathway gene expression. Gene expression associated with apoptosis and oxygen consumption showed little overlap with each other or with the 262 probeset classifier, indicating that the three tests are measuring different aspects of islet cell biology. A subset of 36 probesets surpassed the predictive accuracy of the entire set for reversal of diabetes, and was further reduced by logistic regression to sets of 14 and 5 without losing accuracy. These genes were further validated with an independent cohort of 16 samples. We believe this limited number of gene classifiers in combination with other tests may provide complementary verification of islet quality prior to their clinical use.

Oxygen environment and islet size are the primary limiting factors of isolated pancreatic islet survival.

BACKGROUND: Type 1 diabetes is an autoimmune disease that destroys insulin-producing beta cells in the pancreas. Pancreatic islet transplantation could be an effective treatment option for type 1 diabetes once several issues are resolved, including donor shortage, prevention of islet necrosis and loss in pre- and post-transplantation, and optimization of immunosuppression. This study seeks to determine the cause of necrotic loss of isolated islets to improve transplant efficiency. METHODOLOGY: The oxygen tension inside isolated human islets of different sizes was simulated under varying oxygen environments using a computational in silico model. In vitro human islet viability was also assessed after culturing in different oxygen conditions. Correlation between simulation data and experimentally measured islet viability was examined. Using these in vitro viability data of human islets, the effect of islet diameter and oxygen tension of the culture environment on islet viability was also analyzed using a logistic regression model. PRINCIPAL FINDINGS: Computational simulation clearly revealed the oxygen gradient inside the islet structure. We found that oxygen tension in the islet core was greatly lower (hypoxic) than that on the islet surface due to the oxygen consumption by the cells. The hypoxic core was expanded in the larger islets or in lower oxygen cultures. These findings were consistent with results from in vitro islet viability assays that measured central necrosis in the islet core, indicating that hypoxia is one of the major causes of central necrosis. The logistic regression analysis revealed a negative effect of large islet and low oxygen culture on islet survival. CONCLUSIONS/SIGNIFICANCE: Hypoxic core conditions, induced by the oxygen gradient inside islets, contribute to the development of central necrosis of human isolated islets. Supplying sufficient oxygen during culture could be an effective and reasonable method to maintain isolated islets viable.