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Defining the oxygen level that induces cell death within 3-D tissues is vital for understanding tissue hypoxia; however, obtaining accurate measurements has been technically challenging. In this study, we introduce a noninvasive, high-throughput methodology to quantify critical survival partial oxygen pressure (pO2) with high spatial resolution within spheroids by using a combination of controlled hypoxic conditions, semiautomated live/dead cell imaging, and computational oxygen modeling. The oxygen-permeable, micropyramid patterned culture plates created a precisely controlled oxygen condition around the individual spheroid. Live/dead cell imaging provided the geometric information of the live/dead boundary within spheroids. Finally, computational oxygen modeling calculated the pO2 at the live/dead boundary within spheroids. As proof of concept, we determined the critical survival pO2 in two types of spheroids: isolated primary pancreatic islets and tumor-derived pseudoislets (2.43 ± 0.08 vs. 0.84 ± 0.04 mmHg), indicating higher hypoxia tolerance in pseudoislets due to their tumorigenic origin. We also applied this method for evaluating graft survival in cell transplantations for diabetes therapy, where hypoxia is a critical barrier to successful transplantation outcomes; thus, designing oxygenation strategies is required. Based on the elucidated critical survival pO2, 100% viability could be maintained in a typically sized primary islet under the tissue pO2 above 14.5 mmHg. This work presents a valuable tool that is potentially instrumental for fundamental hypoxia research. It offers insights into physiological responses to hypoxia among different cell types and may refine translational research in cell therapies. NEW & NOTEWORTHY: Our study introduces an innovative combinatory approach for noninvasively determining the critical survival oxygen level of cells within small cell spheroids, which replicates a 3-D tissue environment, by seamlessly integrating three pivotal techniques: cell death induction under controlled oxygen conditions, semiautomated imaging that precisely identifies live/dead cells, and computational modeling of oxygen distribution. Notably, our method ensures high-throughput analysis applicable to various cell types, offering a versatile solution for researchers in diverse fields. [ABSTRACT FROM AUTHOR]

Hypothermic (cold) preservation is a limiting factor for successful cell and tissue transplantation where cell swelling (edema) usually develops, impairing cell function. University of Wisconsin (UW) solution, a standard cold preservation solution, contains effective components to suppress hypothermia-induced cell swelling. Antifreeze proteins (AFPs) found in many cold-adapted organisms can prevent cold injury of the organisms. Here, the effects of a beetle AFP from Dendroides canadensis (DAFP-1) on pancreatic β-cells preservation were first investigated. As low as 500 µg/mL, DAFP-1 significantly minimized INS-1 cell swelling and subsequent cell death during 4 °C preservation in UW solution for up to three days. However, such significant cytoprotection was not observed by an AFP from Tenebrio molitor (TmAFP), a structural homologue to DAFP-1 but lacking arginine, at the same levels. The cytoprotective effect of DAFP-1 was further validated with the primary β-cells in the isolated rat pancreatic islets in UW solution. The submilligram level supplement of DAFP-1 to UW solution significantly increased the islet mass recovery after three days of cold preservation followed by rewarming. The protective effects of DAFP-1 in UW solution were discussed at a molecular level. The results indicate the potential of DAFP-1 to enhance cell survival during extended cold preservation. [ABSTRACT FROM AUTHOR]

Umbilical cord mesenchymal stem cell-derived extracellular vesicles (UC-MSC-EVs) have become an emerging strategy for treating various autoimmune and metabolic disorders, particularly diabetes. Delivery of UC-MSC-EVs is essential to ensure optimal efficacy of UC-MSC-EVs. To develop safe and superior EVs-based delivery strategies, we explored nuclear techniques including positron emission tomography (PET) to evaluate the delivery of UC-MSC-EVs in vivo. In this study, human UC-MSC-EVs were first successfully tagged with I-124 to permit PET determination. Intravenous (I.V.) and intra-arterial (I.A.) administration routes of [124I]I-UC-MSC-EVs were compared and evaluated by in vivo PET-CT imaging and ex vivo biodistribution in a non-diabetic Lewis (LEW) rat model. For I.A. administration, [124I]I-UC-MSC-EVs were directly infused into the pancreatic parenchyma via the celiac artery. PET imaging revealed that the predominant uptake occurred in the liver for both injection routes, and further imaging characterized clearance patterns of [124I]I-UC-MSC-EVs. For biodistribution, the uptake (%ID/gram) in the spleen was significantly higher for I.V. administration compared to I.A. administration (1.95 ± 0.03 and 0.43 ± 0.07, respectively). Importantly, the pancreas displayed similar uptake levels between the two modalities (0.20 ± 0.06 for I.V. and 0.24 ± 0.03 for I.A.). Therefore, our initial data revealed that both routes had similar delivery efficiency for [124I]I-UC-MSC-EVs except in the spleen and liver, considering that higher spleen uptake could enhance immunomodulatory application of UC-MSC-EVs. These findings could guide the development of safe and efficacious delivery strategies for UC-MSC-EVs in diabetes therapies, in which a minimally invasive I.V. approach would serve as a better delivery strategy. Further confirmation studies are ongoing. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background/Aims Pancreatic islet transplantation is an effective treatment for Type 1 diabetic patients to eliminate insulin injections; however, a shortage of donor organs hinders the widespread use. Although long-term islet storage, such as cryopreservation, is considered one of the key solutions, transplantation of cryopreserved islets is still not practical due to the extensive loss during the cryopreservation-rewarming process. We have previously reported that culturing islets in a hyperoxic environment is an effective treatment to prevent islet death from the hypoxic injury during culture. In this study, we explored the effectiveness of thawing and rewarming cryopreserved islets in a hyperoxic environment. Methods Following cryopreservation of isolated human islets, the thawing solution and culture media were prepared with or without pre-equilibration to 50% oxygen. Thawing/rewarming and the pursuant two-day culture were performed with or without oxygenation. Short-term recovery rate, defined as the volume change during cryopreservation and thawing/rewarming, was assessed. Ischemia-associated and inflammation-associated gene expressions were examined using qPCR after the initial rewarming period. Long-term recovery rate, defined as the volume change during the two-day culture after the thawing/rewarming, was also examined. Islet metabolism and function were assessed by basal oxygen consumption rate and glucose stimulated insulin secretion after long-term recovery. Results Oxygenated thawing/rewarming did not alter the short-term recovery rate. Inflammation-associated gene expressions were elevated by the conventional thawing/rewarming method and suppressed by the oxygenated thawing/rewarming, whereas ischemia-associated gene expressions did not change between the thawing/rewarming methods. Long-term recovery rate experiments revealed that only the combination therapy of oxygenated thawing/rewarming and oxygenated culture alleviated islet volume loss. These islets showed higher metabolism and better function among the conditions examined. Conclusion Oxygenated thawing/rewarming alleviated islet volume loss, with the help of oxygenated culture. [ABSTRACT FROM AUTHOR]

Pancreatic islet transplantation has been recognized as an effective treatment for Type 1 diabetes; however, there is still plenty of room to improve transplantation efficiency. Because islets are metabolically active they require high oxygen to survive; thus hypoxia after transplant is one of the major causes of graft failure. Knowing the optimal oxygen tension for isolated islets would allow a transplant team to provide the best oxygen environment during pre- and post-transplant periods. To address this issue and begin to establish empirically determined guidelines for islet maintenance, we exposed in vitro cultured islets to different partial oxygen pressures (pO 2 ) and assessed changes in islet volume, viability, metabolism, and function. Human islets were cultured for 7 days in different pO 2 media corresponding to hypoxia (90 mmHg), normoxia (160 mmHg), and hyerpoxia (270 or 350 mmHg). Compared to normoxia and hypoxia, hyperoxia alleviated the loss of islet volume, maintaining higher islet viability and metabolism as measured by oxygen consumption and glucose-stimulated insulin secretion responses. We predict that maintaining pre- and post-transplanted islets in a hyperoxic environment will alleviate islet volume loss and maintain islet quality thereby improving transplant outcomes. [ABSTRACT FROM AUTHOR]

GNAS, a gene encoding G protein stimulating α subunit, is frequently mutated in intraductal papillary mucinous neoplasms (IPMNs), which are indolent and slow-growing pancreatic tumors that secrete abundant mucin. The GNAS mutation is not observed in conventional ductal adenocarcinomas of the pancreas. To determine the functional significance of the GNAS mutation in pancreatic ductal lineage cells, we examined in vitro phenotypes of cells of pancreatic ductal lineage, HPDE, PK-8, PCI-35, and MIA PaCa-2, with exogenous expression of either wild-type or mutated (R201H) GNAS. We found that exogenous GNAS upregulated intracellular cyclic adenine monophosphate (cAMP), particularly in mutated GNAS transfectants, and upregulated expression of MUC2 and MUC5AC in HPDE and PK-8 cells. By contrast, exogenous GNAS inhibited expression of mucin genes in PCI-35 and MIA PaCa-2 cells, despite upregulation of cAMP. We examined global gene expression profiles of some of the cells transfected with exogenous mutated GNAS (PK-8, PCI-35, and MIA PaCa-2), and found that PK-8 cells exhibited drastic alterations of the gene expression profile, which contrasted with modest alterations in PCI-35 and MIA PaCa-2 cells. To identify a cause of these different effects of exogenous mutated GNAS on phenotypes of the cells, we examined effects of interactions of the signaling pathways of G protein-coupled receptor (GPCR), mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K) on expression of mucin genes. The MAPK and PI3K pathways significantly influenced the expression of mucin genes. Exogenous GNAS did not promote cell growth but suppressed it in some of the cells. In conclusion, mutated GNAS found in IPMNs may extensively alter gene expression profiles, including expression of mucin genes, through the interaction with MAPK and PI3K pathways in pancreatic ductal cells; these changes may determine the characteristic phenotype of IPMN. PK-8 cells expressing exogenous mutated GNAS may be an ideal in vitro model of IPMN. [ABSTRACT FROM AUTHOR]

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. • Pancreata from older donors yield fewer islets. • Isolated islets from older donors are morphologically more fragmented. • Insulin secreting function of islets deteriorates as donor age increases. • Metabolic increment by glucose stimulation declines as donor age increases. • Metabolic response to high glucose is slower in islets from older donors. [ABSTRACT FROM AUTHOR]

Objective: Islet transplantation is an effective treatment for type 1 diabetes. However, transplant success depends on quick islet assessment because islets deteriorate 2-3 days after isolation. A new tool, single-cell Western blot (scWestern), offers results within one day. In this study, we aimed to test the suitability of scWestern to detect protein markers for beta (insulin), alpha (glucagon), and delta (somatostatin) cells, the three major endocrine cell types in islets., Methods: We characterized the antibody specificity, signal intensity, and cell identification on the scWestern platform, and then compared the islet cell composition analysis between scWestern and immunohistochemistry performed by the Integrated Islet Distribution Program (IIDP)., Results: Islet cell composition is comparable for alpha and beta cells, but not delta cells. Protein expression levels of insulin, glucagon, and somatostatin in individual islet cells varied greatly, highlighting cell type heterogeneity. Surprisingly, scWestern revealed double-hormonal cells (~1%), co-expressing insulin and somatostatin or insulin and glucagon, in non-diabetic and non-obese adult human islets, which was confirmed by confocal immunofluorescence microscopy., Conclusions: These results demonstrate that each alpha, beta, and delta cells express varying levels of peptide hormones, and a small subpopulation co-expresses double hormones in normal human islets. The scWestern platform will enable timely assessment of beta cell mass in isolated islets before clinical transplantation., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

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/cm 2 ). 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., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: This study was performed as a collaborative study between Tokai Hit and Arthur Riggs Diabetes & Metabolism Research Institute of City of Hope. KI, NK, and TT are the employees at Tokai Hit. The remaining authors declare no competing interests.

Fireflies produce light through luciferase-catalyzed reactions involving luciferin, oxygen, and adenosine triphosphate, distinct from other luminescent organisms. This unique feature has revolutionized molecular biology and physiology, serving as a valuable tool for cellular research. Luciferase-based bioluminescent imaging enabled the creation of transgenic animals, such as Firefly Rats. Firefly Rats, created in 2006, ubiquitously express luciferase and have become a critical asset in scientific investigations. These rats have significantly contributed to transplantation and tissue engineering studies. Their low immunogenicity reduces graft rejection risk, making them ideal for long-term tracking of organ/tissue/cellular engraftments. Importantly, in the islet transplantation setting, the ubiquitous luciferase expression in these rats does not alter islet morphology or function, ensuring accurate assessments of engrafted islets. Firefly Rats have illuminated the path of transplantation research worldwide for over a decade and continue accelerating scientific advancements in many fields., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

"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.

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 400 µ m-thick, oxygen-permeable polydimethylsiloxane sheet topped with micropyramid pattern of 400 µ m-base and 200 µ m-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., (© 2022 IOP Publishing Ltd.)

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., Competing Interests: This study was performed as a collaborative study between AGC Techno Glass and Arthur Riggs Diabetes & Metabolism Research Institute of City of Hope. TM, AW, and KT are the employees at AGC Techno Glass. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kato, Miwa, Quijano, Medrano, Ortiz, Desantis, Omori, Wada, Tatsukoshi, Kandeel, Mullen, Ku and Komatsu.)

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.

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.

Islet transplantation is an effective therapy that allows the achievement of insulin independence in patients with type 1 diabetes (T1D). To ensure successful transplantation, islet viability and function are of great importance. Viability assessments most often use fluorescein diacetate (FDA)/propidium iodide (PI) staining. However, results using this method often do not correlate well with graft function. Because FDA nonspecifically penetrates all cells present in the islet preparation, including islets and contaminating acinar cells, its use often complicates viability assessments of the overall cell population. Furthermore, the manual method for determining viability percentages is highly subjective. Shortcomings of the conventional islet viability assay can be potentially improved by staining cells with Newport Green (NG). NG, is a zinc-specific fluorescent dye that specifically reacts with zinc-rich β cells. Two kinds of NG dyes, NG-DCF and NG-PDX, are currently available. We examined the zinc specificity of these NG dyes and compared NG staining with traditional FDA staining to explore the potential of NG dyes to improve islet viability assessment. Of the two NGs tested, NG-DCF showed the higher specificity toward a β-cell line as well as human islets. NG-DCF accurately identified the islet area, even in low-purity islets, while neither FDA nor NG-PDX did. Although NG-DCF staining required a longer incubation time, the addition of poloxamer F127 and incubation at 37°C allowed viability assessment to take place within 30 min. Unlike FDA/PI staining, NG-DCF/PI staining allowed for islet-specific assessment. We also introduced a semiautomated measurement to determine NG-DCF/PI staining results, which enabled us to obtain objective and reproducible results. NG-DCF/PI staining is easy and reliable, and this method permits highly objective islet-specific viability assessments.

Long-term pancreatic cold ischemia contributes to decreased islet number and viability after isolation and culture, leading to poor islet transplantation outcome in patients with type 1 diabetes. In this study, we examined mechanisms of pancreatic cold preservation and rewarming-induced injury by interrogating the proapoptotic gene BBC3/Bbc3, also known as Puma (p53 upregulated modulator of apoptosis), using three experimental models: 1) bioluminescence imaging of isolated luciferase-transgenic ("Firefly") Lewis rat islets, 2) cold preservation of en bloc-harvested pancreata from Bbc3-knockout (KO) mice, and 3) cold preservation and rewarming of human pancreata and isolated islets. Cold preservation-mediated islet injury occurred during rewarming in "Firefly" islets. Silencing Bbc3 by transfecting Bbc3 siRNA into islets in vitro prior to cold preservation improved postpreservation mitochondrial viability. Cold preservation resulted in decreased postisolation islet yield in both wild-type and Bbc3 KO pancreata. However, after culture, the islet viability was significantly higher in Bbc3-KO islets, suggesting that different mechanisms are involved in islet damage/loss during isolation and culture. Furthermore, Bbc3-KO islets from cold-preserved pancreata showed reduced HMGB1 (high-mobility group box 1 protein) expression and decreased levels of 4-hydroxynonenal (4-HNE) protein adducts, which was indicative of reduced oxidative stress. During human islet isolation, BBC3 protein was upregulated in digested tissue from cold-preserved pancreata. Hypoxia in cold preservation increased BBC3 mRNA and protein in isolated human islets after rewarming in culture and reduced islet viability. These results demonstrated the involvement of BBC3/Bbc3 in cold preservation/rewarming-mediated islet injury, possibly through modulating HMGB1- and oxidative stress-mediated injury to islets., (Copyright © 2016 the American Physiological Society.)

GNAS, a gene encoding G protein stimulating α subunit, is frequently mutated in intraductal papillary mucinous neoplasms (IPMNs), which are indolent and slow-growing pancreatic tumors that secrete abundant mucin. The GNAS mutation is not observed in conventional ductal adenocarcinomas of the pancreas. To determine the functional significance of the GNAS mutation in pancreatic ductal lineage cells, we examined in vitro phenotypes of cells of pancreatic ductal lineage, HPDE, PK-8, PCI-35, and MIA PaCa-2, with exogenous expression of either wild-type or mutated (R201H) GNAS. We found that exogenous GNAS upregulated intracellular cyclic adenine monophosphate (cAMP), particularly in mutated GNAS transfectants, and upregulated expression of MUC2 and MUC5AC in HPDE and PK-8 cells. By contrast, exogenous GNAS inhibited expression of mucin genes in PCI-35 and MIA PaCa-2 cells, despite upregulation of cAMP. We examined global gene expression profiles of some of the cells transfected with exogenous mutated GNAS (PK-8, PCI-35, and MIA PaCa-2), and found that PK-8 cells exhibited drastic alterations of the gene expression profile, which contrasted with modest alterations in PCI-35 and MIA PaCa-2 cells. To identify a cause of these different effects of exogenous mutated GNAS on phenotypes of the cells, we examined effects of interactions of the signaling pathways of G protein-coupled receptor (GPCR), mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K) on expression of mucin genes. The MAPK and PI3K pathways significantly influenced the expression of mucin genes. Exogenous GNAS did not promote cell growth but suppressed it in some of the cells. In conclusion, mutated GNAS found in IPMNs may extensively alter gene expression profiles, including expression of mucin genes, through the interaction with MAPK and PI3K pathways in pancreatic ductal cells; these changes may determine the characteristic phenotype of IPMN. PK-8 cells expressing exogenous mutated GNAS may be an ideal in vitro model of IPMN.