Viability Of Pancreatic Islets Research Articles

Pancreatic Islet Viability Assessment Using Hyperspectral Imaging of Autofluorescence.

Islets prepared for transplantation into type 1 diabetes patients are exposed to compromising intrinsic and extrinsic factors that contribute to early graft failure, necessitating repeated islet infusions for clinical insulin independence. A lack of reliable pre-transplant measures to determine islet viability severely limits the success of islet transplantation and will limit future beta cell replacement strategies. We applied hyperspectral fluorescent microscopy to determine whether we could non-invasively detect islet damage induced by oxidative stress, hypoxia, cytokine injury, and warm ischaemia, and so predict transplant outcomes in a mouse model. In assessing islet spectral signals for NAD(P)H, flavins, collagen-I, and cytochrome-C in intact islets, we distinguished islets compromised by oxidative stress (ROS) (AUC = 1.00), hypoxia (AUC = 0.69), cytokine exposure (AUC = 0.94), and warm ischaemia (AUC = 0.94) compared to islets harvested from pristine anaesthetised heart-beating mouse donors. Significantly, with unsupervised assessment we defined an autofluorescent score for ischaemic islets that accurately predicted the restoration of glucose control in diabetic recipients following transplantation. Similar results were obtained for islet single cell suspensions, suggesting translational utility in the context of emerging beta cell replacement strategies. These data show that the pre-transplant hyperspectral imaging of islet autofluorescence has promise for predicting islet viability and transplant success.

Identification of Compounds Protecting Pancreatic Islets against Oxidative Stress using a 3D Pseudoislet Screening Platform.

Oxidative stress leads to a lower success rate of clinical islet transplantation. Here, FDA-approved compounds are screened for their potential to decrease oxidative stress and to protect or enhance pancreatic islet viability and function. Studies are performed on in vitro "pseudoislet" spheroids, which are pre-incubated with 1280 different compounds and subjected to oxidative stress. Cell viability and oxidative stress levels are determined using a high-throughput fluorescence microscopy pipeline. Initial screening on cell viability results in 59 candidates. The top ten candidates are subsequently screened for their potential to decrease induced oxidative stress, and eight compounds efficient reduction of induced oxidative stress in both alpha and beta cells by 25-50%. After further characterization, the compound sulfisoxazole is found to be the most capable of reducing oxidative stress, also at short pre-incubation times, which is validated in primary human islets, where low oxidative stress levels and islet function are maintained. This study shows an effective screening strategy with 3D cell aggregates based on cell viability and oxidative stress, which leads to the discovery of several compounds with antioxidant capacity. The top candidate, sulfisoxazole is effective after a 30 min pre-incubation, maintains baseline islet function, and may help alleviate oxidative stress in pancreatic islets.

Extracellular vesicle-mediated intercellular and interorgan crosstalk of pancreatic islet in health and diabetes.

Diabetes mellitus (DM) is a systemic metabolic disease with high mortality and morbidity. Extracellular vesicles (EVs) have emerged as a novel class of signaling molecules, biomarkers and therapeutic agents. EVs-mediated intercellular and interorgan crosstalk of pancreatic islets plays a crucial role in the regulation of insulin secretion of β-cells and insulin action in peripheral insulin target tissues, maintaining glucose homeostasis under physiological conditions, and it's also involved in pathological changes including autoimmune response, insulin resistance and β-cell failure associated with DM. In addition, EVs may serve as biomarkers and therapeutic agents that respectively reflect the status and improve function and viability of pancreatic islets. In this review, we provide an overview of EVs, discuss EVs-mediated intercellular and interorgan crosstalk of pancreatic islet under physiological and diabetic conditions, and summarize the emerging applications of EVs in the diagnosis and treatment of DM. A better understanding of EVs-mediated intercellular and interorgan communication of pancreatic islets will broaden and enrich our knowledge of physiological homeostasis maintenance as well as the development, diagnosis and treatment of DM.

Automated pancreatic islet viability assessment for transplantation using bright-field deep morphological signature

Islets transplanted for type-1 diabetes have their viability reduced by warm ischemia, dimethyloxalylglycine (DMOG; hypoxia model), oxidative stress and cytokine injury. This results in frequent transplant failures and the major burden of patients having to undergo multiple rounds of treatment for insulin independence. Presently there is no reliable measure to assess islet preparation viability prior to clinical transplantation. We investigated deep morphological signatures (DMS) for detecting the exposure of islets to viability compromising insults from brightfield images. Accuracies ranged from 98 % to 68 % for; ROS damage, pro-inflammatory cytokines, warm ischemia and DMOG. When islets were disaggregated to single cells to enable higher throughput data collection, good accuracy was still obtained (83–71 %). Encapsulation of islets reduced accuracy for cytokine exposure, but it was still high (78 %). Unsupervised modelling of the DMS for islet preparations transplanted into a syngeneic mouse model was able to predict whether or not they would restore glucose control with 100 % accuracy. Our strategy for constructing DMS' is effective for the assessment of islet pre-transplant viability. If translated into the clinic, standard equipment could be used to prospectively identify non-functional islet preparations unable to contribute to the restoration of glucose control and reduce the burden of unsuccessful treatments.

Comparison of two techniques for assessing pancreatic islet viability: flow cytometry and fluorescein diacetate/propidium iodide staining

Introduction: The success of islet transplantation for patients with unstable type 1 diabetes mellitus is dependent, in part, on the number of isolated islets as well as their quality, which is assessed by functional and viability tests. The currently employed test to evaluate islet viability, used by the Collaborative Islet Transplant Registry to release clinical islet product for transplantation, is the FDA/PI staining. However, the efficacy of this method is dependent on the researcher experience. In this context, a quantitative method may be useful. Therefore, the aim of this study was to compare islet viability assessed by flow cytometry and FDA/PI assay. Methods: Viability was evaluated in islets isolated from 10 male Wistar rats. Upon FDA and PI staining, 50 islets from each animal were analyzed under fluorescence microscope by two well-trained researchers. For flow cytometry, islets were dispersed, and then 100,000 single cells were incubated with 7AAD fluorophore (dyes necrotic and late apoptotic cells) and Annexin V-APC antibody (marker of early apoptotic cells). Results: A moderate correlation was found between the two techniques (r = 0.6; P= 0.047). The mean islet viability measured by flow cytometry was higher than the viability estimated using FDA/PI staining (95.5 ± 1.4% vs . 89.5 ± 5.0%; P= 0.002). Conclusions: Although flow cytometry is more expensive and time-consuming than FDA/PI staining, it is a quantitative technique, not dependent of the researcher experience. Thus, flow cytometry appears to be the technique of choice when aiming a more precise determination of islet viability.

P.127: The Influence of Temperature Conditions and Reconstruction of the Extracellular Matrix Environment on the Viability of Pancreatic Islets

Introduction: Pancreatic islet transplantation is one of the methods of treating patients with T1D. However, for the procedure to be successful, the high quality of the implanted islets is of key importance for a successful islet transplant. Following islet isolation, it has been shown that mainly β-cells undergo apoptosis due to vascular loss and hypoxia. The cellular stresses to which islets are subjected during isolation (for example disturbance of the cell-matrix relationship). Appropriate storage conditions for insulated islands are of key importance for their functionality. The most common method of islet storage is the introduction into a suspension culture. However, because these are micro organs, the storage of pancreatic islets is a more accurate term. Therefore, this study aimed to demonstrate whether for pancreatic islets it is better to cultivate them in standard conditions (37oC; 5% CO2), or maybe stored them at 4oC. Material: The research material was porcine pancreatic islets. Islets were isolated from the native organ using collagenase and a Ricordi chamber. After isolation, the material was divided into 4 research groups: 1. control (islands suspended in CMRL 1066 medium) - traditional breeding conditions (temperature 37oC; 5% CO2); 2. CMRL 1066 suspended islets stored at 4oC; 3. Islets in CMRL 1066 medium with the addition of dECM (decellularized extracellular matrix) - in traditional culture conditions; 4. Islets in CMRL 1066 supplemented with dECM stored at 4oC. Each variant was tested in 3 replicates and each trial was 3000iEq. Additionally, each tested variant was tested on pancreatic islets that were cleaned or not immediately after isolation. Results: The research showed that the storage temperature, reduced to 4oC, and the addition of ECM significantly influenced the functionality of the islets. This variant tested (uncleaned islands) showed functionality even 5 days after isolation. On the other hand, islets incubated at 37°C without the addition of dECM showed no functionality after 2 days of the experiment. The remaining variants showed a gradual loss of functionality. However, the 4oC variant was better in each case. Conclusion: Pancreatic islets, as micro organs with their vascularization, need appropriate storage conditions after isolation to ensure their proper functioning. Based on these studies, it was shown that the islands should be stored and not cultivated as single-cell lines. In addition, the addition of dECM allows for the recreation of the matrix-cell environment, which is appropriate storage conditions - corresponding to the native environment. This study was financially supported by The National Centre for Research and Development STRATEGMED3/305813/2/NCBR/2017.

Bioluminescence Imaging In Vivo Confirms the Viability of Pancreatic Islets Transplanted into the Greater Omentum.

The liver is the most widely used site for pancreatic islet transplantation. However, several site-specific limitations impair functional success, with instant blood-mediated inflammatory reaction being the most important. The aim of this study was to develop a preclinical model for placement of the islet graft into a highly vascularized omental flap using a fibrin gel. For this purpose, we tested islet viability by bioluminescence imaging (BLI). Pancreatic islets were isolated from luciferase-positive and luciferase-negative rats, mixed at a 1:1 ratio, placed into a plasma-thrombin bioscaffold, and transplanted in standard (10 pancreatic islets/g wt; n = 10) and marginal (4 pancreatic islets/g wt; n = 7) numbers into the omentums of syngeneic diabetic animals. For the control, 4 pancreatic islets/g were transplanted into the liver using the standard procedure (n = 7). Graft viability was tested by bioluminescence at days 14, 30, 60, and 90 post transplant. Glucose levels, intravenous glucose tolerance, and serum C-peptide were assessed regularly. Nonfasting glucose levels < 10 mmol/l were restored in all animals. While islet viability in the omentum was clearly detected by stable luminescence signals throughout the whole study period, no signals were detected from islets transplanted into the liver. The bioluminescence signals were highly correlated with stimulated C-peptide levels detected at 80 days post transplant. Glucose tolerance did not differ among the 3 groups. We successfully tested a preclinical model of islet transplantation into the greater omentum using a biocompatible scaffold made from autologous plasma and human thrombin. Both standard and marginal pancreatic islet numbers in a gel-form bioscaffold placed in the omentum restored glucose homeostasis in recipients with diabetes. Bioluminescence was shown promising as a direct proof of islet viability.

Collagen and Endothelial Cell Coculture Improves β-Cell Functionality and Rescues Pancreatic Extracellular Matrix.

The use of biomaterials and biomaterial functionalization is a promising approach to support pancreatic islet viability posttransplantation in an effort to reduce insulin dependence for patients afflicted with diabetes mellitus type 1. Extracellular matrix (ECM) proteins are known to impact numerous reparative functions in the body. Assessing how endogenously expressed pancreatic ECM proteins are affected by posttransplant-like hypoxic conditions may provide significant insights toward the development of tissue-engineered therapeutic strategies to positively influence β-cell survival, proliferation, and functionality. Here, we investigated the expression of three relevant groups of pancreatic ECM proteins in human native tissue, including basement membrane (BM) proteins (collagen type 4 [COL4], laminins [LAM]), proteoglycans (decorin [DCN], nidogen-1 [NID1]), and fibril-forming proteins (fibronectin [FN], collagen type 1 [COL1]). In an in vitro hypoxia model, we identified that ECM proteins were differently affected by hypoxic conditions, contributing to an overall loss of β-cell functionality. The use of a COL1 hydrogel as carrier material demonstrated a protective effect on β-cells mitigating the effect of hypoxia on proteoglycans as well as fibril-forming protein expression, supporting β-cell functionality in hypoxia. We further showed that providing endothelial cells (ECs) into the COL1 hydrogel improves β-cell response as well as the expression of relevant BM proteins. Our data show that β-cells benefit from a microenvironment composed of structure-providing COL1 with the incorporation of ECs to withstand the harsh conditions of hypoxia. Such hydrogels support β-cell survival and can serve as an initial source of ECM proteins to allow cell engraftment while preserving cell functionality posttransplantation. Impact statement Expression analysis identifies hypoxia-induced pathological changes in extracellular matrix (ECM) homeostasis as potential targets to support β-cell transplants by encapsulation in biomaterials for the treatment of diabetes mellitus. A collagen-1 hydrogel is shown to attenuate the effect of hypoxia on β-cells and their ECM expression. The functionalization of the hydrogel with endothelial cells increases the β-cell response to glucose and rescues essential basement membrane proteins.

Development of a Coaxial 3D Printing Platform for Biofabrication of Implantable Islet-Containing Constructs.

Over the last two decades, pancreatic islet transplantations have become a promising treatment for Type I diabetes. However, although providing a consistent and sustained exogenous insulin supply, there are a number of limitations hindering the widespread application of this approach. These include the lack of sufficient vasculature and allogeneic immune attacks after transplantation, which both contribute to poor cell survival rates. Here, these issues are addressed using a biofabrication approach. An alginate/gelatin-based bioink formulation is optimized for islet and islet-related cell encapsulation and 3D printing. In addition, a custom-designed coaxial printer is developed for 3D printing of multicellular islet-containing constructs. In this work, the ability to fabricate 3D constructs with precise control over the distribution of multiple cell types is demonstrated. In addition, it is shown that the viability of pancreatic islets is well maintained after the 3D printing process. Taken together, these results represent the first step toward an improved vehicle for islet transplantation and a potential novel strategy to treat Type I diabetes.

Influence of rat bone marrow mesenchymal stem cells to rat Langerhans islets viability during co-cultivation with microst ructured collagen‑containing hydrogel

Aim. To research the rat bone marrow mesenchymal stem cells (MSC BM) effect to the viability of isolated rat OL during cultivation with microstructured collagen-containing hydrogel (BMCH).Materials and methods. Rat islets and MSC BM were isolated according to classical technique with some modifications. The experiments on the co- cultivation were carried out under standard conditions. Islet’s viability was determined by immunofluorescence staining.Results. TheOL cultivation with BMCH and MSCBM resulted in destructive changes of isletson the third day, and complete destruction of OL was observed on the seventh day. At the same time, OL cultured with BMCH remained viable for 14 days of incubation.Conclusion. Cultivation of OL with BMCH and MSC BM did not contribute to the long-term survival of the islets. Thus, there was no positive effect of MSC BM on the viability of pancreatic islets.

Comparison of two techniques for evaluation of pancreatic islet viability: flow cytometry and FDA/PI staining

Background Type 1 diabetes (T1D) accounts for approximately 10% of all diabetes cases, and it is caused by autoimmune destruction of pancreatic beta-cells, which leads to insulin deficiency and fates individuals to require insulin treatment to survive. Although important advances in the treatment of T1D have been achieved in recent yrs., this disease is associated with chronic complications that lead to high morbidity and mortality rates in young adults of productive age. In patients with unstable T1D, pancreatic islet transplantation is a therapeutic option to restore insulin secretion and improve glycemic control. However, the success of islet transplantation is dependent, in part, on the number of isolated islets as well as factors associated with their quality, which is assessed by functional and viability tests. In this context, the method currently used for islet viability evaluation [fluorescein diacetate (FDA)/propidium iodide (PI) staining] is not accurate enough, and new methods have been researched, such as flow cytometry.

Mesenchymal stem cells and ligand incorporation in biomimetic poly(ethylene glycol) hydrogels significantly improve insulin secretion from pancreatic islets.

The main goal of this study was to investigate pancreatic islet function with mesenchymal stem cells (MSCs) in a ligand-functionalized poly(ethylene glycol) (PEG) hydrogel for the treatment of type 1 diabetes (T1D). Rat bone marrow-derived MSCs (rBM-MSCs) were encapsulated within synthetic PEG hydrogel, and cell viability and apoptosis within this 3D environment was examined in detail. ATP content and caspase-3 activity of encapsulated MSCs showed that fibronectin-derived RGDS, laminin-derived IKVAV and/or insulinotropic glucagon-like peptide (GLP-1) were required to maintain MSC survival. Incorporation of these peptides into the hydrogel environment also improved pancreatic islet viability, where combinations of peptides had altered effects on islet survival. GLP-1 alone was the leading stimulator for insulin secretion. Cell adhesion peptides RGDS and IKVAV improved insulin secretion only when they were used in combination, but could not surpass the effect of GLP-1. Further, when pancreatic islets were co-encapsulated with MSCs within synthetic PEG hydrogel, a two-fold increase in the stimulation index was measured. Synergistic effects of MSCs and peptides were observed, with a seven-fold increase in the stimulation index. The results are promising and suggest that simultaneous incorporation of MSCs and ECM-derived peptides and/or GLP-1 can improve pancreatic islet function in response to altered glucose levels in the physiological environment. Copyright © 2014 John Wiley & Sons, Ltd.

Ileal Interposition and Viability of Pancreatic Islets Transplanted into Intramuscular Site of Diabetic Rats

ABSTRACTBackground: Assuming that ileal stimulation by food may increase incretin secretion, we aimed to investigate whether ileal interposition obtains adequate pancreatic islet viability and function after intramuscular islet transplantation in diabetic rats. Methods: We investigated four groups of eight Wistar rats: ileal interposition + islet transplantation, islet transplantation, ileal interposition, and diabetic control. All rats were subjected to streptozotocin-induced diabetes. We used the C-peptide/glucose ratio and islet image to investigate beta cell mass, and plasma glucagon like peptide-1 (GLP-1) measure. Results: Ileal interposition was effective in preserving function and increasing islet mass in animals with islets transplanted into alginate microcapsules. The plasma GLP-1 level in the diabetic control rats was a basal concentration (4.1 ± 1.2 pM). GLP-1 level after ileal interposition + islet transplantation (12.3 ± 3.3 pM) was significantly higher (p < .05) than in the islet transplantation group (8.2 ± 2.4 pM) and ileal interposition group rats (7.6 ± 1.8 pM). Conclusions: Ileal interposition positively influenced beta cell viability after intramuscular transplantation of pancreatic islets in diabetic rats.

Bioorthogonal layer-by-layer encapsulation of pancreatic islets via hyperbranched polymers.

Encapsulation of viable tissues via layer-by-layer polymer assembly provides a versatile platform for cell surface engineering, with nanoscale control over the capsule properties. Herein, we report the development of a hyperbranched polymer-based, ultrathin capsule architecture expressing bioorthogonal functionality and tailored physiochemical properties. Random carbodiimide-based condensation of 3,5-dicarboxyphenyl glycineamide on alginate yielded a highly branched polysaccharide with multiple, spatially restricted, and readily functionalizable terminal carboxylate moieties. Poly(ethylene glycol) (PEG) was utilized to link azido end groups to the structured alginate. Together with a phosphine-functionalized poly(amidoamine) dendrimer, nanoscale layer-by-layer coatings, covalently stabilized via Staudinger ligation, were assembled onto solid surfaces and pancreatic islets. The effects of electrostatic and/or bioorthogonal covalent interlayer interactions on the resulting coating efficiency and stability, as well as pancreatic islet viability and function, were studied. These hyperbranched polymers provide a flexible platform for the formation of covalently stabilized, ultrathin coatings on viable cells and tissues. In addition, the hyperbranched nature of the polymers presents a highly functionalized surface capable of bioorthogonal conjugation of additional bioactive or labeling motifs.

A Physiological Pattern of Oxygenation Using Perfluorocarbon-Based Culture Devices Maximizes Pancreatic Islet Viability and Enhances β-Cell Function

Conventional culture vessels are not designed for physiological oxygen (O2) delivery. Both hyperoxia and hypoxia-commonly observed when culturing cells in regular plasticware-have been linked to reduced cellular function and death. Pancreatic islets, used for the clinical treatment of diabetes, are especially sensitive to sub- and supraphysiological O2 concentrations. A result of current culture standards is that a high percentage of islet preparations are never transplanted because of cell death and loss of function in the 24-48 h postisolation. Here, we describe a new culture system designed to provide quasiphysiological oxygenation to islets in culture. The use of dishes where islets rest atop a perfluorocarbon (PFC)-based membrane, coupled with a careful adjustment of environmental O2 concentration to target the islet physiological pO2 range, resulted in dramatic gains in viability and function. These observations underline the importance of approximating culture conditions as closely as possible to those of the native microenvironment, and fill a widely acknowledged gap in our ability to preserve islet functionality in vitro. As stem cell-derived insulin-producing cells are likely to suffer from the same limitations as those observed in real islets, our findings are especially timely in the context of current efforts to define renewable sources for transplantation.

Differentiation of Mesenchymal Stem Cells towards an insulin-releasing phenotype after co-culture with Pancreatic Islets

Transplantation of pancreatic islets has become a promising clinical option to treat patients with type 1 diabetes, alternative to the standard therapy with insulin injections. Islet transplantation is a minimally invasive therapeutic approach, and it allows a better metabolic control and a long-term insulin independence in more than 80% of patients (Ryan et al., 2002). However this therapeutic treatment has some side effects, such as the poor yield of pancreatic islet explants and even more the immune graft rejection, which have as a consequence the very limited lifespan of transplanted pancreatic islets. To avoid these side effects several strategies have been proposed and, besides the treatment with immunosuppressive drugs, promising results have been obtained with the use of Mesenchymal Stem cells (MSCs), already known in literature to be able to support the survival of many cell types (Scuteri et al., 2006). Several in vivo studies have demonstrated that the concurrent transplantation of pancreatic islets with MSCs reduces the number of islets required to achieve glycemic control in diabetic rats, but the mechanisms of these encouraging results are still unknown (Figliuzzi et al., 2009). For these reasons in this in vitro study we characterized the effect of co-culture of rat MSC on survival and functioning of rat pancreatic islets, by evaluating for 4 weeks: i) MSC adhesion to pancreatic islets; ii) viability of pancreatic islets co-cultured with MSCs; iii) the expression of insulin after co-culture; iv) the ability of co-cultured pancreatic islets to correctly adjust insulin release after variation of glucose concentration. Our results demonstrated that MSCs are able to adhere to pancreatic islets, but to increase only partly the pancreatic islet survival, which retain the ability to express and correctly release insulin after glucose variation in medium culture. Noteworthy that the insulin level in the medium of co-cultured pancreatic islets is always higher with respect to medium of pancreatic islets alone. The immunofluorescence analysis reveals that also MSCs (and not only pancreatic islets) are able to express insulin, but only in co-culture. These results, which justify the in vivo observation reported above, suggest that MSCs undergo to differentiation into a insulin-releasing phenotype after co-culture with pancreatic islets. We are now evaluating the molecular mechanisms which drive this effect, by analyzing the role of soluble factors and of proteins able to induce insulin expression. This study was granted by MIUR – FIRB Futuro in Ricerca 2008 RBFR08VSVI_001.

MTOR links incretin signaling to HIF induction in pancreatic beta cells

Under feeding conditions, the incretin hormone GLP-1 promotes pancreatic islet viability by triggering the cAMP pathway in beta cells. Increases in PKA activity stimulate the phosphorylation of CREB, which in turn enhances beta cell survival by upregulating IRS2 expression. Although sustained GLP-1 action appears important for its salutary effects on islet function, the transient nature of CREB activation has pointed to the involvement of additional nuclear factors in this process. Following the acute induction of CREB-regulated genes, cAMP triggers a second delayed phase of gene expression that proceeds via the HIF transcription factor. Increases in cAMP promote the accumulation of HIF1α in beta cells by activating the mTOR pathway. As exposure to rapamycin disrupts GLP-1 effects on beta cell viability, these results demonstrate how a pathway associated with tumor growth also mediates salutary effects of an incretin hormone on pancreatic islet function.

Standardization of islet isolation outcome – A new automatic system to determine pancreatic islet viability

Pancreatic islet transplantation is emerging as a therapeutic approach for patients affected by diabetes. This approach consists of a minimally invasive procedure replacing insulin-producing cells (pancreatic islets). The technique has been proven successful, but limitations have been identified. One of the major challenges of the procedure is the counting of the isolated pancreatic islets, which is currently jeopardized by subjectivity and inaccuracy. Determination of the accurate islet number is a crucial factor in determining the correlation between the isolation product and clinical outcome. In the proposed study, we have developed software capable of objectively evaluating islet numbers and other viability variables by image analysis. This software is based on image processing and feature extraction algorithms for recognition of the area of interest. This is the first step toward standardization of the isolation outcome and potential clinical success predictability.

Overexpression of Vascular Endothelial Growth Factor In Vitro Using Deferoxamine: A New Drug to Increase Islet Vascularization During Transplantation

During pancreatic islet transplantation, delayed and insufficient revascularization can deprive islets of oxygen and nutrients, resulting in cell death and early graft failure. Deferoxamine (DFO), an iron chelator, increases vascular endothelial growth factor (VEGF) expression in cells. The aim of this work was to study the effect of DFO on β-cell and pancreatic islet viability as well as VEGF expression. β-cell lines from rat insulinoma (Rin m5f) and primary cultures of pancreatic islets from Wistar rats were incubated with DFO (10, 100, and 1000 μmol/L). The viability was evaluated using fluorescein diacetate/propidium iodide for dying pancreatic islets and using cell titers for Rin m5f. Expression of VEGF messenger RNA (mRNA) was quantified using reverse transcriptase polymerase chain reaction (RT-PCR). Finally, VEGF secretion was determined using enzyme-linked immunosorbent assays at 1 to 3 days after treatment. The addition of 10 μmol/L of DFO preserved Rin m5F viability at 24 hours after treatment (10 μmol/L; 101.33% ± 5.66%; n = 7). However, 100 and 1000 μmol/L of DFO induced cell death (68.92% ± 5.83% and 65.89% ± 5.83%, respectively; n = 4). In the same way, viability of pancreatic islets in the presence of DFO was preserved. RT-PCR analysis showed stimulation of VEGF mRNA in the presence of 10 μmol/L of DFO in islets at 3 days after culture. Finally, 10 μmol/L of DFO stimulated secretion of VEGF 7.95 ± 0.84 versus 1.80 ± 1.10 pg/μg total protein with 10 μmol/L of DFO in rat islets at 3 days after culture, n = 3; P < .001). The use of DFO to stimulate VEGF expression and increase islet vascularization may be a realistic approach to improve islet viability during transplantation.

The Use of Iodixanol for the Purification of Rat Pancreatic Islets

Transplantation of pancreatic islets is a promising therapeutic treatment for type 1 diabetes mellitus. For clinical and experimental transplantation, a large number of pure pancreatic islets are required for transplantation. Thus, the improvement of islet isolation and purification techniques are crucial. In this context, iodixanol-based solution, successfully used for the purification of porcine islets, seems to be a possible alternative to Ficoll for purification of islets. The aim of this study was to test the efficacy of iodixanol compared with Ficoll density gradients for the purification of rat pancreatic islets. Twelve Wistar rats were used for isolation and purification of pancreatic islets. Pancreata were digested with Liberase R1 and islets purified by two gradients: Ficoll or iodixanol gradient. The number and the purity of the pancreatic islets were assessed. To analyze the response of isolated pancreatic islet to glucose challenge, in vitro experiments were performed by measuring the insulin concentration in the Supernatant. The results demonstrated that the iodixanol gradient provided a higher purity of pancreatic islets compared to the Ficoll gradient. In addition, the rat islet yield by iodixanol gradient was significantly higher compared to a Ficoll gradient (751 ± 16 versus 464 ± 19 pancreatic islets, respectively; P < .001). The viability of pancreatic islets isolated by an iodixanol gradient was confirmed by high glucose challenge, with more than twofold higher increase in insulin secretion. The present study demonstrated that iodixanol density gradient overcomes Ficoll density gradient, providing a greater number of pure and functional rat pancreatic islets.