Culture Of Pancreatic Islets Research Articles

THU291 Successful Reversal Of Glucose Toxicity After DKA Precipitated By SGLT2 Inhibitor

Abstract Disclosure: H. Abdelrahman: None. B. DeMoranville: None. Case: A 40 year old man with history of NAFLD and type 2 diabetes mellitus with HbA1c 12.2% on metformin 750 mg daily, presented with diabetic ketoacidosis (DKA) with blood glucose 275 mg/dL and anion gap 24 (AG 10-12). He had been started on empagliflozin 25 mg daily four days prior as outpatient. He was treated with IV insulin and hydration per DKA protocol. Labs showed C-peptide 3.0 ng/mL (1.1-4.4 ng/mL), Anti-GAD Ab 0.01 nmol/L (<0.02 mol/L), negative anti-insulin cell Ab. He was discharged on glargine 10u qHS, lispro 2u AC plus corrective scale, and metformin 750 mg BID. On outpatient follow-up, pre-meal glucose remained under 130 mg/dL, not requiring corrective insulin. He was started on semaglutide 0.5mg weekly and insulin was discontinued. At 3 months, his HbA1c improved to 5.8%. Self-monitoring glucose levels were at goal. Discussion: This case illustrates glucotoxicity due to significant hyperglycemia causing beta-cell apoptosis and resulting in insulin deficiency. The initiation of SGLT2 inhibitors (SGLT2i) when insulin deficiency and peripheral resistance are at their peak increases risk of DKA. This patient with type 2 diabetes presented with blood glucose 275 mg/dL in DKA four days after starting empagliflozin, a SGLT2i. SGLT2i increase risk of DKA due to dehydration from glucosuria, inhibiting ketone body excretion by the kidneys, causing a state of carbohydrate deficit and hypovolemia that promotes glucagon release from alpha-cells, an increased glucagon-to-insulin ratio, and thus triggers ketogenesis.1 In glucose toxicity, intra-cellular hyperglycemia-induced molecular dysfunction results in beta-cell apoptosis. Islet cells cultured in a glucotoxic environment (20 mmol/l) had suppressed insulin content, but fully reversed the intra-cellular calcium response after one week of culturing in a euglycemic environment, demonstrating recovery of the molecular steps promoting insulin release. 2 Treatment with insulin close to euglycemic goals promotes the reversal of glucose toxicity. Our patient recovered beta-cell function several weeks after the glucotoxic episode and was able to discontinue insulin therapy. With the addition of semaglutide, a GLP-1 receptor agonist, which potentiates glucose-dependent insulin release to control post-prandial hyperglycemia, he was able to maintain euglycemia and an HbA1c 5.8%. Conclusion Patients with type 2 diabetes mellitus may present in diabetic ketoacidosis due to glucose toxicity which can be reversed with short-term insulin therapy. References 1) Nasa P, et al. Euglycemic diabetic ketoacidosis: A missed diagnosis. World J Diabetes. 2021 May 15;12(5):514-523. 2) Tariq M, et al. Prolonged culture of human pancreatic islets under glucotoxic conditions changes their acute beta cell calcium and insulin secretion glucose response curves from sigmoid to bell-shaped. Diabetologia. 2022 Dec 2. Epub ahead of print. PMID: 36459178. Presentation: Thursday, June 15, 2023

Hollow Microfiber Assembly-Based Endocrine Pancreas-on-a-Chip for Sugar Substitute Evaluation.

With the increasing demand for low-sugar, low-calorie healthy diets, artificial sweeteners are widely used as substitutes for sugar in the food industry. Therefore, developing models that can better predict the effects of sugar substitutes on the human body is necessary. Here, a new type of endocrine pancreas-on-a-chip is developed based on a microfiber assembly and its stimulation of pancreatic secretion by glucose or sugar substitutes is evaluated. This new endocrine pancreas-on-a-chip is assembled using two components: (1) a cell-loaded hollow methacrylate gelatin (GelMA)/calcium alginate (CaA) composite microfiber prepared by microfluidic spinning to achieve vascular simulation and material transport, and (2) a 3D pancreatic islet culture layer, which also serves as a fiber assembly microchip. Using this established organ chip, the effects of five sweeteners (glucose, erythritol, xylitol, sodium cyclamate, and sucralose) were investigated on pancreatic islet cell viability and insulin and glucagon secretion. The constructed endocrine pancreas-on-a-chip has potential for the safety evaluation of sugar-substituted food additives, which can expand the application of organ chips in the field of food safety and provide a new platform for evaluating various food additives.

Long-term cultures of human pancreatic islets in self-assembling peptides hydrogels.

Human pancreatic islets transplantation is an experimental therapeutic treatment for Type I Diabetes. Limited islets lifespan in culture remains the main drawback, due to the absence of native extracellular matrix as mechanical support after their enzymatic and mechanical isolation procedure. Extending the limited islets lifespan by creating a long-term in vitro culture remains a challenge. In this study, three biomimetic self-assembling peptides were proposed as potential candidates to recreate in vitro a pancreatic extracellular matrix, with the aim to mechanically and biologically support human pancreatic islets, by creating a three-dimensional culture system. The embedded human islets were analyzed for morphology and functionality in long-term cultures (14-and 28-days), by evaluating β-cells content, endocrine component, and extracellular matrix constituents. The three-dimensional support provided by HYDROSAP scaffold, and cultured into MIAMI medium, displayed a preserved islets functionality, a maintained rounded islets morphology and an invariable islets diameter up to 4 weeks, with results analogues to freshly-isolated islets. In vivo efficacy studies of the in vitro 3D cell culture system are ongoing; however, preliminary data suggest that human pancreatic islets pre-cultured for 2weeks in HYDROSAP hydrogels and transplanted under subrenal capsule may restore normoglycemia in diabetic mice. Therefore, engineered self-assembling peptide scaffolds may provide a useful platform for long-term maintenance and preservation of functional human pancreatic islets in vitro.

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 400 µm-thick, oxygen-permeable polydimethylsiloxane sheet topped with micropyramid pattern of 400 µm-base and 200 µm-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.

Prolonged culture of human pancreatic islets under glucotoxic conditions changes their acute beta cell calcium and insulin secretion glucose response curves from sigmoid to bell-shaped.

The rapid remission of type 2 diabetes by a diet very low in energy correlates with a marked improvement in glucose-stimulated insulin secretion (GSIS), emphasising the role of beta cell dysfunction in the early stages of the disease. In search of novel mechanisms of beta cell dysfunction after long-term exposure to mild to severe glucotoxic conditions, we extensively characterised the alterations in insulin secretion and upstream coupling events in human islets cultured for 1-3 weeks at ~5, 8, 10 or 20 mmol/l glucose and subsequently stimulated by an acute stepwise increase in glucose concentration. Human islets from 49 non-diabetic donors (ND-islets) and six type 2 diabetic donors (T2D-islets) were obtained from five isolation centres. After shipment, the islets were precultured for 3-7 days in RPMI medium containing ~5 mmol/l glucose and 10% (vol/vol) heat-inactivated FBS with selective islet picking at each medium renewal. Islets were then cultured for 1-3 weeks in RPMI containing ~5, 8, 10 or 20 mmol/l glucose before measurement of insulin secretion during culture, islet insulin and DNA content, beta cell apoptosis and cytosolic and mitochondrial glutathione redox state, and assessment of dynamic insulin secretion and upstream coupling events during acute stepwise stimulation with glucose [NAD(P)H autofluorescence, ATP/(ATP+ADP) ratio, electrical activity, cytosolic Ca2+ concentration ([Ca2+]c)]. Culture of ND-islets for 1-3 weeks at 8, 10 or 20 vs 5 mmol/l glucose did not significantly increase beta cell apoptosis or oxidative stress but decreased insulin content in a concentration-dependent manner and increased beta cell sensitivity to subsequent acute stimulation with glucose. Islet glucose responsiveness was higher after culture at 8 or 10 vs 5 mmol/l glucose and markedly reduced after culture at 20 vs 5 mmol/l glucose. In addition, the [Ca2+]c and insulin secretion responses to acute stepwise stimulation with glucose were no longer sigmoid but bell-shaped, with maximal stimulation at 5 or 10 mmol/l glucose and rapid sustained inhibition above that concentration. Such paradoxical inhibition was, however, no longer observed when islets were acutely depolarised by 30 mmol/l extracellular K+. The glucotoxic alterations of beta cell function were fully reversible after culture at 5 mmol/l glucose and were mimicked by pharmacological activation of glucokinase during culture at 5 mmol/l glucose. Similar results to those seen in ND-islets were obtained in T2D-islets, except that their rate of insulin secretion during culture at 8 and 20 mmol/l glucose was lower, their cytosolic glutathione oxidation increased after culture at 8 and 20 mmol/l glucose, and the alterations in GSIS and upstream coupling events were greater after culture at 8 mmol/l glucose. Prolonged culture of human islets under moderate to severe glucotoxic conditions markedly increased their glucose sensitivity and revealed a bell-shaped acute glucose response curve for changes in [Ca2+]c and insulin secretion, with maximal stimulation at 5 or 10 mmol/l glucose and rapid inhibition above that concentration. This novel glucotoxic alteration may contribute to beta cell dysfunction in type 2 diabetes independently from a detectable increase in beta cell apoptosis.

OR22-4 Homing of Antigen-specific Engineered Regulatory T Cells To Human Pancreatic Islets

Abstract The therapeutic application of regulatory T cells (Tregs) for the treatment of autoimmune disorders, although efficacious, has been limited by the scarcity of antigen-specific Tregs. If antigen-specific Tregs, capable of reaching the desired autoimmune target, could be produced on demand, antigen-specific immune suppression of autoimmune diseases would be achievable. One approach to endow T cells with a desired antigen-specificity uses chimeric T cell antigen-receptors (CAR) with antibody-type specificity. Adoptive cell transfer therapies with CAR-re-directed cytotoxic T cells, have shown impressive efficacy in the treatment of hematologic malignancies. Accordingly, employing such technology to re-direct Tregs to sites of autoimmune attack may be a useful therapeutic approach to alleviate a broad spectrum of diseases in which uncontrolled auto and alloimmune responses play a major role. We recently developed pancreatic beta-cell, antigen-specific, CAR Tregs (1) and explored their therapeutic potential against T1D in our humanized mouse model (2). Ours is the first successful, antigen-specific CAR-Treg treatment of T1D in a humanized mouse model that closely resembles the human disease. Based on our mice data, we believe treatment with pancreatic beta-cell, antigen-specific CAR-Tregs will allow for recovery and reconstitution of beta cells in human T1D patients as well. The purpose of this study was to determine if antigen-specific human CAR Tregs could also identify target and home to human pancreatic islets in culture as they do in mice. The study involved drawing 10 cc of blood 1-2 weeks prior to pancreas surgery; followed by collection of a small piece of pancreas (5 cc wedge) once the pancreas was removed for a clinical indication (cancer, pancreatitis). We first isolated Tregs from peripheral blood of these human donors and expanded them in vitro. Treg cells were genetically modified to express either a beta-cell antigen-specific (GAD65) CAR or an irrelevant (EPCAM) CAR construct together with GFP marker. CAR Tregs were selectively expanded in the presence of rhGAD65 antigen. Once pancreas tissue became available, it was processed for islet separation using the collagenase method. Pancreatic islets were then co-cultured with syngeneic CAR Tregs for 7 days. IncuCyte S3 Live-cell System (Sartorius) is a real-time system that automatically acquires and analyzes HD, phase and fluorescent images of cell cultures, around the clock, for 7 days, while cells remain undisturbed. Live immunofluorescence microscopy demonstrated the distinct homing of GAD65 CAR Tregs to islets as compared to control EPCAM CAR Tregs as early as 24 hours of co-culture. Importantly, proliferation of GAD65 CAR Tregs was clearly demonstrated within 72 hours. GAD65 CAR Treg cytokine profile from co-culture supernatant was characteristic of activated Tregs. 1 PATENT #WO2020097546 2 PATENT #US20200037586 Presentation: Monday, June 13, 2022 11:45 a.m. - 12:00 p.m.

Characterization of the Secretome, Transcriptome, and Proteome of Human β Cell Line EndoC-βH1

Early diabetes research is hampered by limited availability, variable quality, and instability of human pancreatic islets in culture. Little is known about the human β cell secretome, and recent studies question translatability of rodent β cell secretory profiles. Here, we verify representativeness of EndoC-βH1, one of the most widely used human β cell lines, as a translational human β cell model based on omics and characterize the EndoC-βH1 secretome. We profiled EndoC-βH1 cells using RNA-seq, data-independent acquisition, and tandem mass tag proteomics of cell lysate. Omics profiles of EndoC-βH1 cells were compared to human β cells and insulinomas. Secretome composition was assessed by data-independent acquisition proteomics. Agreement between EndoC-βH1 cells and primary adult human β cells was ∼90% for global omics profiles as well as for β cell markers, transcription factors, and enzymes. Discrepancies in expression were due to elevated proliferation rate of EndoC-βH1 cells compared to adult β cells. Consistently, similarity was slightly higher with benign nonmetastatic insulinomas. EndoC-βH1 secreted 783 proteins in untreated baseline state and 3135 proteins when stressed with nontargeting control siRNA, including known β cell hormones INS, IAPP, and IGF2. Further, EndoC-βH1 secreted proteins known to generate bioactive peptides such as granins and enzymes required for production of bioactive peptides. EndoC-βH1 secretome contained an unexpectedly high proportion of predicted extracellular vesicle proteins. We believe that secretion of extracellular vesicles and bioactive peptides warrant further investigation with specialized proteomics workflows in future studies.

Isolation, Purification, and Culture of Mouse Pancreatic Islets of Langerhans.

Pancreatic islets constitute an important tool for research and clinical applications in the field of diabetes. They are used for transplantation, unraveling new mechanisms in insulin secretion, studying pathophysiological pathways in diseased cells, and pharmacological research aimed at developing improved therapeutic strategies. Therefore, fine-tuning islet isolation protocols remains an important objective for reliable investigations. Here we describe a relatively simple mouse islet isolation protocol that relies on enzymatic digestion using low-activity collagenase and several sedimentation and Percoll gradient steps.

Heat shock protein B1 is required for the prolactin-induced cytoprotective effects on pancreatic islets.

The success of islet transplantation has improved lately. Unfortunately, it is still compromised by cell loss. We have shown that prolactin (PRL) inhibits beta-cell apoptosis and up-regulates the antiapoptotic Heat Shock Protein B1 (HSPB1) in human islets. Since its function in pancreatic islets has not been studied, we explored the role of HSPB1 in PRL-induced beta-cell survival. The significant PRL-induced cytoprotection in control cells was abrogated in HSPB1 silenced cells, overexpression of HSPB1 recovered survival. PRL-mediated inhibition of cytokine-induced caspase activities and cytokine-induced decrease of BCL-2/BAX ratio was significantly reverted in knocked-down cells. Kinetics of HSPB1 and HSF1 expression were studied in primary cultures of murine and human pancreatic islets. These findings are highly relevant for the improvement of clinical islet transplantation success rate since our results demonstrated a key role for HSPB1 pointing it as a promising target for beta-cell cytoprotection through the up-regulation of an endogenous protective pathway.

Biomimetic Surfaces Supporting Dissociated Pancreatic Islet Cultures

This study describes a method to screen biomimetic surfaces based on intracellular insulin content of either fully or partly dissociated primary endocrine islet tissue. It is challenging to maintain endocrine pancreatic islets and more so, dissociated ones. Physiological activity of isolated islet cells in vitro declines due to loss of cell-to-cell and cell-to-extracellular matrix interactions. An in vitro model was developed to evaluate specific extracellular binding components potentially affecting islet biology, with the intention to identify in vivo-like peptides promoting survival and function. Synthetic peptides were bound to low-fouling carboxy-methyl-dextran surfaces, effectively presenting defined surfaces while minimizing non-specific interactions. These biomimetic surfaces were screened based on intracellular insulin content of applied mouse primary islet tissue by analysis with an anti-insulin cell-ELISA. Three active biomimetic surfaces were identified, two laminin- (IKLLI and PDSGR) and one cadherin (HAVDI)-derived, which supported adhesion and survival of insulin-containing cultures for 5days, respectively suggesting a benefit from both cell-extracellular matrix and cell–cell interactions. Cells from dissociated islets show progression over 10days on the HAVDI-biomimetic for the insulin immunoreactivity and cell density. The three surfaces did not act additively or synergistically. A favorable reaction to glucose-stimulated insulin secretion on the cadherin-biomimetic indicated the cultures were physiologically functional. This supportive role of biomimetic peptides represents initial progress in defining minimal extracellular binding requirements influencing islet cell physiology. This will influence further optimization of growth surfaces and promote the basic understanding of islet biology. Low-fouling biomimetics are predicted to be applicable to additional diverse culture systems.

The Optimization of Short-Term Hepatocyte Preservation Before Transplantation.

BackgroundNo optimal methods for short-term hepatocyte preservation have been established. We have recently developed a prominent oxygen-permeable bag (Tohoku Device [TD]) for pancreatic islet culture and transplantation. In this study, we investigated whether TD is also effective for hepatocyte preservation and tried to optimize other conditions.MethodsHepatocytes were preserved in the following conditions, and their outcomes were observed. First, the effectiveness of TD was investigated. Second, hepatocyte medium (HM) and organ preservation solutions with or without fetal bovine serum (FBS) were compared. Third, as supplementations, FBS and human serum albumin (HSA) were compared. Fourth, low, room and high temperature were compared. And finally, hepatocytes preserved in various conditions were transplanted into the subrenal capsule space of nonalbumin rats and engrafted areas were assessed.ResultsThe survival rate of hepatocytes preserved in TD tended to be higher and their viability and function were maintained significantly greater than those of non-TD group. Irrespective of FBS supplementation, the survival rate of HM group was significantly higher than those of organ preservation solution group while viabilities and plating efficiency were similar among them. Although survival rates of groups without FBS were extremely low, results of HSA supplemented group were not inferior to FBS supplemented group. Hepatocytes preserved at high temperature had the worst results. The engrafted area of TD group tended to be higher than those of other groups.ConclusionsTD is effective for short-term hepatocyte preservation. HSA is a useful substitute for FBS, and preserving in HM at low temperature is recommended.

Real architecture For 3D Tissue (RAFT\u2122) culture system improves viability and maintains insulin and glucagon production of mouse pancreatic islet cells

There is an unmet medical need for the improvement of pancreatic islet maintenance in culture. Due to restricted donor availability it is essential to ameliorate islet viability and graft engraftment. The aim of this study was to compare the standard tissue culture techniques with the advanced Real Architecture For 3D Tissue (RAFT™) culture system in terms of viability and hormone production. Here, we first report that islets embedded in RAFT™ collagen type I advanced tissue culture system maintain their tissue integrity better than in monolayer and suspension cultures. The Calcein violet assay and Annexin V/propidium-iodide staining show higher cell viability in the RAFT™ culture system. Quantitative real-time PCR data showed that RAFT™ increases insulin expression after 18 days in culture compared to traditional methods. Enhanced insulin and glucagon production was further verified by immunofluorescent staining in a time-course manner. These results indicate that RAFT™ tissue culture platform can be a promising tool to maintain pancreatic islet spheroid integrity and culture islets for downstream high throughput pharmacological studies ex vivo.

IGF-2 coated porous collagen microwells for the culture of pancreatic islets.

Islet transplantation, the only curative therapy for type I diabetes, requires isolation of the graft in highly specialized facilities for its later dispatch to remote transplantation centres. During transport and culture, many valuable cells are lost due to several factors such as mechanical stress, islet aggregation and dissociation. Here, we evaluate a porous microwell array sheet made of natural collagen type I extracellular matrix (ECM) protein as a novel islet culture substrate. This culture platform can be coated with IGF-2, a growth factor favorable for islet survival, and allows segregation of the islets within the porous microwell sheet, preventing aggregation. This design shows promising results for improving human pancreatic islets viability and function during culture and could form a novel paradigm for the transport of islets between isolation and transplantation centres.

Oxygen considerations for the preservation of pancreata and culture of pancreatic islets

Oxygen considerations for the preservation of pancreata and culture of pancreatic islets

The Effects of Cord Blood Serum on Survival of Rat Pancreatic Islets during In vitro Culturing

Background: Transplantation of islets of Langerhans can improve glycometabolic control in type 1 diabetic patients; however, recipients usually require islets from 1-3 donor pancreas. Current isolation techniques generally recover <50% of the islets from a given pancreas. This study aimed to determine the effect of cord blood serum (CBS) on isolated pancreatic islet cells in vitro. Methods: Rat pancreatic islets were isolated via collagenase digestion, and then cultured with and without the presence of 50 μl, 100 μl, and 150 μl of CBS. Next, islet cell viability was determined using a fluorescein diacetate and propidium iodide mixture via fluorescence microscopy. Additionally, islet function was evaluated based on insulin secretion. Results: Treatment of primary cultures of rat pancreatic islets with CBS resulted in augmented β-cell viability. It was observed that the viability rate increased as the concentration of CBS increased and that viability was higher in the 150 μl of CBS group than in the control group. CBS was observed to preserve β-cell glucose responsiveness and insulin secretion under basal (3.3 mmoll-1) and stimulated (16.7 mmoll-1) glucose conditions. Moreover, a significant increase in insulin secretion was observed following treatment with 50 μl of CBS. Conclusions: The present findings show that cord blood serum treatment of freshly isolated rat islets had a positive effect on islet survival by increasing their viability. We think these findings suggest that CBS might be a suitable factor for optimizing and stimulating recovery, and subsequent function of islets during in vitro culturing, indicating that CBS might play an important role in the biophysiological function of pancreatic islets.

In Vitro Assessment of Human Islet Vulnerability to Instant Blood-Mediated Inflammatory Reaction (IBMIR) and Its Use to Demonstrate a Beneficial Effect of Tissue Culture.

Culture of human pancreatic islets is now routinely carried out prior to clinical islet allotransplantation, using conditions that have been developed empirically. One of the major causes of early islet destruction after transplantation is the process termed instant blood-mediated inflammatory reaction (IBMIR). The aim of this study was to develop in vitro methods to investigate IBMIR and apply them to the culture conditions used routinely in our human islet isolation laboratory. Freshly isolated or precultured (24 h, 48 h) human islets were incubated in either ABO-compatible allogeneic human blood or Hank's buffered salt solution (HBSS) for 1 h at 37°C. Tissue factor (TF) expression and leukocyte migration were assessed by light microscopy. TF was also quantified by ELISA. To assess β-cell function, glucose-stimulated insulin secretion (GSIS) assay was carried out. The extent of islet β-cell damage was quantified using a proinsulin assay. Islets cultured for 24 h had higher GSIS when compared to freshly isolated or 48-h precultured islets. Freshly isolated islets had significantly higher TF content than 24-h and 48-h precultured islets. Incubation of freshly isolated human islets in allogeneic human blood released 6.5-fold higher level of proinsulin in comparison to freshly isolated human islets in HBSS. The high level of proinsulin released was significantly attenuated when precultured islets (24 h or 48 h) were exposed to fresh blood. Histological examination of fresh islets in blood clot showed that some islets were fragmented, showing signs of extraislet insulin leakage and extensive neutrophil infiltration and necrosis. These features were markedly reduced when the islets were cultured for 24 h. These results suggest that our standard 24-h islet culture is markedly beneficial in attenuating IBMIR, as evidenced by increased GSIS, lower content of TF, decrease islet fragmentation, and proinsulin release.

Beta Cells Secrete Significant and Regulated Levels of Insulin for Long Periods when Seeded onto Acellular Micro-Scaffolds.

The aim of this work is to obtain significant and regulated insulin secretion from human beta cells ex vivo. Long-term culture of human pancreatic islets and attempts at expanding human islet cells normally result in loss of beta-cell phenotype. We propose that to obtain proper ex vivo beta cell function, there is a need to develop three-dimensional structures that mimic the natural islet tissue microenvironment. We here describe the preparation of endocrine micro-pancreata (EMPs) that are made up of acellular organ-derived micro-scaffolds seeded with human intact or enzymatically dissociated islets. We show that EMPs constructed by seeding whole islets, freshly enzymatically-dissociated islets or even dissociated islets grown first in standard monolayer cultures express high levels of key beta-cell specific genes and secrete quantities of insulin per cell similar to freshly isolated human islets in a glucose-regulated manner for more than 3 months in vitro.

Evaluation of low doses BPA-induced perturbation of glycemia by toxicogenomics points to a primary role of pancreatic islets and to the mechanism of toxicity.

Epidemiologic and experimental studies have associated changes of blood glucose homeostasis to Bisphenol A (BPA) exposure. We took a toxicogenomic approach to investigate the mechanisms of low-dose (1 × 10−9 M) BPA toxicity in ex vivo cultures of primary murine pancreatic islets and hepatocytes. Twenty-nine inhibited genes were identified in islets and none in exposed hepatocytes. Although their expression was slightly altered, their impaired cellular level, as a whole, resulted in specific phenotypic changes. Damage of mitochondrial function and metabolism, as predicted by bioinformatics analyses, was observed: BPA exposure led to a time-dependent decrease in mitochondrial membrane potential, to an increase of ROS cellular levels and, finally, to an induction of apoptosis, attributable to the bigger Bax/Bcl-2 ratio owing to activation of NF-κB pathway. Our data suggest a multifactorial mechanism for BPA toxicity in pancreatic islets with emphasis to mitochondria dysfunction and NF-κB activation. Finally, we assessed in vitro the viability of BPA-treated islets in stressing condition, as exposure to high glucose, evidencing a reduced ability of the exposed islets to respond to further damages. The result was confirmed in vivo evaluating the reduction of glycemia in hyperglycemic mice transplanted with control and BPA-treated pancreatic islets. The reported findings identify the pancreatic islet as the main target of BPA toxicity in impairing the glycemia. They suggest that the BPA exposure can weaken the response of the pancreatic islets to damages. The last observation could represent a broader concept whose consideration should lead to the development of experimental plans better reproducing the multiple exposure conditions.

Dielectric spectroscopy for monitoring human pancreatic islet differentiation within cell-seeded scaffolds in a perfusion bioreactor system.

The long-term in vitro culture and differentiation of human pancreatic islets is still hindered by the inability to emulate a suitable microenvironment mimicking physiological extracellular matrix (ECM) support and nutrient/oxygen perfusion. This is further amplified by the current lack of a non-invasive and rapid monitoring system to readily evaluate cellular processes. In this study, we realized a viable method for non-invasively monitoring isolated human pancreatic islets in vitro. Islets are induced to dedifferentiate into proliferative duct-like structures (DLS) in preparation for potential and subsequent re-differentiation into functional islet-like structures (ILS) in a process reminiscent of islet regeneration strategies. This long-term in vitro process is conducted within a three-dimensional microenvironment involving islets embedded in an optimized ECM gel supported by microfabricated three-dimensional scaffolds. The islet-scaffold is then housed and continuously perfused within chambers of a bioreactor platform. The process in its entirety is monitored through dielectric spectroscopy measurements, yielding an accurate representation of cellular morphology, functionality, and volume fraction. This non-invasive and real-time monitoring tool can be further manipulated to elucidate important information about the optimized cellular microenvironment required for maintaining long-term culture and achieve efficient differentiation for islet regeneration.

Long-term culture and functionality of pancreatic islets monitored using microelectrode arrays

Extracellular recording of the glucose-induced electrical activity of mouse islets of Langerhans on microelectrode arrays (MEAs) is an innovative and powerful tool to address beta-cell (patho-)physiology. In a dual approach we tested whether this technique can detect concentration-dependent drug effects as well as characterize alterations in beta-cell activity during prolonged culture. First we established conditions that allow long-term investigation of beta-cell function by recording electrical activity. The results provide the first measurements of beta-cell membrane potential oscillations of individual murine islets during long-term culture. Oscillations were recorded for up to 34 days after islet isolation. Importantly, the glucose dependence of electrical activity did not change over a period of one month. Thus we can follow electrophysiological changes of individual islets induced by alterations in the beta-cell environment over weeks. Second, we used the MEA technique to assay beta-cell damage induced by oxidative stress and to evaluate appropriate protection mechanisms. Oxidative stress plays a key role in the development of type 2 diabetes mellitus (T2DM). Examination of the acute effects of H2O2 on electrical activity showed that the oxidant reduced the electrical activity in a concentration-dependent manner. The superoxide dismutase mimetic, tempol, protected against the detrimental effects of H2O2. In conclusion, we demonstrated that MEA recordings can be used to address disease-related mechanisms and protective interventions in beta-cells. In the future, this fundamental work should enable the monitoring of the electrical activity of islets of Langerhans under controlled ex vivo conditions including long-term exposure to oxidative stress, glucolipotoxicity, and other diabetes-inducing agents.