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Background & Aims Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive tumor that is almost uniformly lethal in humans. Activating mutations of KRAS are found in >90% of human PDACs and are sufficient to promote acinar-to-ductal metaplasia (ADM) during tumor initiation. The roles of miRNAs in oncogenic Kras-induced ADM are incompletely understood. Methods The Ptf1aCre/+ LSL-KrasG12D/+ and Ptf1aCre/+ LSL-KrasG12D/+ LSL-p53R172H/+ and cerulein-induced acute pancreatitis mice models were used. mir-802 was conditionally ablated in acinar cells to study the function of miR-802 in ADM. Results We show that miR-802 is a highly abundant and acinar-enriched pancreatic miRNA that is silenced during early stages of injury or oncogenic KrasG12D-induced transformation. Genetic ablation of mir-802 cooperates with KrasG12D by promoting ADM formation. miR-802 deficiency results in de-repression of the miR-802 targets Arhgef12, RhoA, and Sdc4, activation of RhoA, and induction of the downstream RhoA effectors ROCK1, LIMK1, COFILIN1, and EZRIN, thereby increasing F-actin rearrangement. mir-802 ablation also activates SOX9, resulting in augmented levels of ductal and attenuated expression of acinar identity genes. Consistently with these findings, we show that this miR-802–RhoA–F-actin network is activated in biopsies of pancreatic cancer patients and correlates with poor survival. Conclusions We show miR-802 suppresses pancreatic cancer initiation by repressing oncogenic Kras-induced ADM. The role of miR-802 in ADM fills the gap in our understanding of oncogenic Kras-induced F-actin reorganization, acinar reprogramming, and PDAC initiation. Modulation of the miR-802–RhoA–F-actin network may be a new strategy to interfere with pancreatic carcinogenesis.

Objective The miR-200–Zeb1 axis regulates the epithelial-to-mesenchymal transition (EMT), differentiation, and resistance to apoptosis. A better understanding of these processes in diabetes is highly relevant, as β-cell dedifferentiation and apoptosis contribute to the loss of functional β-cell mass and diabetes progression. Furthermore, EMT promotes the loss of β-cell identity in the in vitro expansion of human islets. Though the miR-200 family has previously been identified as a regulator of β-cell apoptosis in vivo, studies focusing on Zeb1 are lacking. The aim of this study was thus to investigate the role of Zeb1 in β-cell function and survival in vivo. Methods miR-200 and Zeb1 are involved in a double-negative feedback loop. We characterized a mouse model in which miR-200 binding sites in the Zeb1 3′UTR are mutated (Zeb1200), leading to a physiologically relevant upregulation of Zeb1 mRNA expression. The role of Zeb1 was investigated in this model via metabolic tests and analysis of isolated islets. Further insights into the distinct contributions of the miR-200 and Zeb1 branches of the feedback loop were obtained by crossing the Zeb1200 allele into a background of miR-141–200c overexpression. Results Mild Zeb1 derepression in vivo led to broad transcriptional changes in islets affecting β-cell identity, EMT, insulin secretion, cell–cell junctions, the unfolded protein response (UPR), and the response to ER stress. The aggregation and insulin secretion of dissociated islets of mice homozygous for the Zeb1200 mutation (Zeb1200M) were impaired, and Zeb1200M islets were resistant to thapsigargin-induced ER stress ex vivo. Zeb1200M mice had increased circulating proinsulin levels but no overt metabolic phenotype, reflecting the strong compensatory ability of islets to maintain glucose homeostasis. Conclusions This study signifies the importance of the miR-200–Zeb1 axis in regulating key aspects of β-cell function and survival. A better understanding of this axis is highly relevant in developing therapeutic strategies for inducing β-cell redifferentiation and maintaining β-cell identity in in vitro islet expansion., Highlights • The miR-200-Zeb1 double negative feedback loop is essential for pancreatic β-cell identity and function. • Disruption of the miR-200-Zeb1 axis leads to broad transcriptional changes affecting many aspects of β-cell biology. • Disruption of miR-200-mediated inhibition of Zeb1 protects against ER stress. • Derepression of Zeb1 leads to impaired reaggregation of dissociated islets and perturbed insulin secretion.

Background A robust scalable method for producing enucleated red blood cells (RBCs) is not only a process to produce packed RBC units for transfusion but a potential platform to produce modified RBCs with applications in advanced cellular therapy. Current strategies for producing RBCs have shortcomings in the limited self-renewal capacity of progenitor cells, or difficulties in effectively enucleating erythroid cell lines. We explored a new method to produce RBCs by inducibly expressing c-Myc in primary erythroid progenitor cells and evaluated the proliferative and maturation potential of these modified cells. Results Primary erythroid progenitor cells were genetically modified with an inducible gene transfer vector expressing a single transcription factor, c-Myc, and all the gene elements required to achieve dox-inducible expression. Genetically modified cells had enhanced proliferative potential compared to control cells, resulting in exponential growth for at least 6 weeks. Inducibly proliferating erythroid (IPE) cells were isolated with surface receptors similar to colony forming unit-erythroid (CFU-Es), and after removal of ectopic c-Myc expression cells hemoglobinized, decreased in cell size to that of native RBCs, and enucleated achieving cultures with 17% enucleated cells. Experiments with IPE cells at various levels of ectopic c-Myc expression provided insight into differentiation dynamics of the modified cells, and an optimized two-stage differentiation strategy was shown to promote greater expansion and maturation. Conclusions Genetic engineering of adult erythroid progenitor cells with an inducible c-Myc vector established an erythroid progenitor cell line that could produce RBCs, demonstrating the potential of this approach to produce large quantities of RBCs and modified RBC products. Electronic supplementary material The online version of this article (10.1186/s12896-019-0515-9) contains supplementary material, which is available to authorized users.

On-chip imaging of intact three-dimensional tissues within microfluidic devices is fundamentally hindered by intratissue optical scattering, which impedes their use as tissue models for high-throughput screening assays. Here, we engineered a microfluidic system that preserves and converts tissues into optically transparent structures in less than 1 d, which is 20× faster than current passive clearing approaches. Accelerated clearing was achieved because the microfluidic system enhanced the exchange of interstitial fluids by 567-fold, which increased the rate of removal of optically scattering lipid molecules from the cross-linked tissue. Our enhanced clearing process allowed us to fluorescently image and map the segregation and compartmentalization of different cells during the formation of tumor spheroids, and to track the degradation of vasculature over time within extracted murine pancreatic islets in static culture, which may have implications on the efficacy of beta-cell transplantation treatments for type 1 diabetes. We further developed an image analysis algorithm that automates the analysis of the vasculature connectivity, volume, and cellular spatial distribution of the intact tissue. Our technique allows whole tissue analysis in microfluidic systems, and has implications in the development of organ-on-a-chip systems, high-throughput drug screening devices, and in regenerative medicine.

Fibroblast growth factor receptor-1 (FGFR1) activity at the plasma membrane is tightly controlled by the availability of co-receptors and competing receptor isoforms. We have previously shown that FGFR1 activity in pancreatic beta-cells modulates a wide range of processes, including lipid metabolism, insulin processing, and cell survival. More recently, we have revealed that co-expression of FGFR5, a receptor isoform that lacks a tyrosine-kinase domain, influences FGFR1 responses. We therefore hypothesized that FGFR5 is a co-receptor to FGFR1 that modulates responses to ligands by forming a receptor heterocomplex with FGFR1. We first show here increased FGFR5 expression in the pancreatic islets of nonobese diabetic (NOD) mice and also in mouse and human islets treated with proinflammatory cytokines. Using siRNA knockdown, we further report that FGFR5 and FGFR1 expression improves beta-cell survival. Co-immunoprecipitation and quantitative live-cell imaging to measure the molecular interaction between FGFR5 and FGFR1 revealed that FGFR5 forms a mixture of ligand-independent homodimers (∼25%) and homotrimers (∼75%) at the plasma membrane. Interestingly, co-expressed FGFR5 and FGFR1 formed heterocomplexes with a 2:1 ratio and subsequently responded to FGF2 by forming FGFR5/FGFR1 signaling complexes with a 4:2 ratio. Taken together, our findings identify FGFR5 as a co-receptor that is up-regulated by inflammation and promotes FGFR1-induced survival, insights that reveal a potential target for intervention during beta-cell pathogenesis.

Isolating subpopulations of heterogeneous cancer cells is an important capability for the meaningful characterization of circulating tumor cells at different stages of tumor progression and during the epithelial-to-mesenchymal transition. Here, we present a microfluidic device that can separate phenotypically distinct subpopulations of cancer cells. Magnetic nanoparticles coated with antibodies against the epithelial cell adhesion molecule (EpCAM) are used to separate breast cancer cells in the microfluidic platform. Cells are sorted into different zones on the basis of the levels of EpCAM expression, which enables the detection of cells that are losing epithelial character and becoming more mesenchymal. The phenotypic properties of the isolated cells with low and high EpCAM are then assessed using matrix-coated surfaces for collagen uptake analysis, and an NAD(P)H assay that assesses metabolic activity. We show that low-EpCAM expressing cells have higher collagen uptake and higher folate-induced NAD(P)H responses compared to those of high-EpCAM expressing cells. In addition, we tested SKBR3 cancer cells undergoing chemically induced hypoxia. The induced cells have reduced expression of EpCAM, and we find that these cells have higher collagen uptake and NAD(P)H metabolism relative to noninduced cells. This work demonstrates that nanoparticle-mediated binning facilitates the isolation of functionally distinct cell subpopulations and allows surface marker expression to be associated with invasiveness, including collagen uptake and metabolic activity.

Tissues are challenging to genetically manipulate due to limited penetration of viral particles resulting in low transduction efficiency. We are particularly interested in expressing genetically-encoded sensors in ex vivo pancreatic islets to measure glucose-stimulated metabolism, however poor viral penetration biases these measurements to only a subset of cells at the periphery. To increase mass transfer of viral particles, we designed a microfluidic device that holds islets in parallel hydrodynamic traps connected by an expanding by-pass channel. We modeled viral particle flow into the tissue using fluorescently-labelled gold nanoparticles of varying sizes and showed a penetration threshold of only ∼5 nm. To increase this threshold, we used EDTA to transiently reduce cell–cell adhesion and expand intercellular space. Ultimately, a combination of media flow and ETDA treatment significantly increased adenoviral transduction to the core of the islet. As proof-of-principle, we used this protocol to transduce an ER-targeted redox sensitive sensor (eroGFP), and revealed significantly greater ER redox capacity at core islet cells. Overall, these data demonstrate a robust method to enhance transduction efficiency of islets, and potentially other tissues, by using a combination of microfluidic flow and transient tissue expansion.

Fibroblast growth factor receptors (FGFRs) are transmembrane tyrosine kinase receptors that modulate many cellular processes including adhesion and survival. FGFR5 is a non-canonical family member that lacks a kinase domain and is presumed to be a negative regulator of signaling. However, we previously showed that overexpression of FGFR5 results in activation of downstream signaling targets, thereby challenging our understanding of this receptor. In particular, it was unclear whether FGFR5 and FGFR1 interact on the plasma membrane to form a signaling complex. We previously used quantitative fluorescence microscopy, including number and brightness analysis (N&B) and homo-forster resonance energy transfer (homoFRET), to study the dynamics and the stoichiometry of the FGFR1 signaling complex. We therefore created a library of fluorescent protein tagged FGFR1 and FGFR5 constructs to conduct similar studies of receptor membrane aggregation. FGFR5 has a unique cytoplasmic C-terminus that includes a histidine-rich zinc binding region as well as a putative SH2-binding motif. HomoFRET and anisotropy imaging revealed that FGFR5 forms homo-dimers while a C-terminal deficient isoform (FGFR5ΔC) does not, suggesting that the C-terminus drives the interaction. We validated these findings using N&B analysis of Venus-tagged FGFR5ΔC observing no change in relative brightness in the presence or absence of non-Venus tagged FGFR5ΔC (i.e. “dark receptor”). Finally, we measured changes in anisotropy of Cerulean-tagged FGFR5 when co-expressed with an excess dark FGFR1 (i.e. mCherry-tagged FGFR1). We observed an increase in FGFR5 anisotropy indicative of dimer to monomer transition, suggesting that FGFR5 also forms heterodimers with FGFR1. Currently, we are investigating the effect of FGFs and scaffolding proteins on modulating the FGFR5-FGFR1 heterocomplex. These studies will allow us to better understand the regulatory mechanisms that govern FGFR-signaling and the downstream cellular processes.

The Jagn1 protein was indentified in a SILAC proteomic screen of proteins that are increased in insulinoma cells expressing a folding-deficient proinsulin. Jagn1 mRNA was detected in primary rodent islets and in insulinoma cell lines and the levels were increased in response to ER stress. The function of Jagn1 was assessed in insulinoma cells by both knock-down and overexpression approaches. Knock-down of Jagn1 caused an increase in glucose-stimulated insulin secretion resulting from an increase in proinsulin biosynthesis. In contrast, overexpression of Jagn1 in insulinoma cells resulted in reduced cellular proinsulin and insulin levels. Our results identify a novel role for Jagn1 in regulating proinsulin biosynthesis in pancreatic β-cells. Under ER stress conditions Jagn1 is induced which might contribute to reducing proinsulin biosynthesis, in part by helping to relieve the protein folding load in the ER in an effort to restore ER homeostasis.

The fibroblast growth factor receptor (FGFR) family of tyrosine kinase receptors transactivate in response to ligand binding (e.g. FGF2) to initiate classical intracellular signaling cascades. Despite not having an intracellular kinase domain like canonical FGFR1, FGFR5 retains the ability to activate the MAPK signaling pathway, and particularly in the absence of ligand-receptor binding. Both receptors exhibit high affinity for FGF2, and are therefore likely to compete for ligand binding. To examine the extent of competition between FGFR1 and FGFR5 for ligand in living cells, we aimed to use the complementary techniques of fluorescence-anisotropy based homo-Forster resonance energy transfer (homoFRET) and Number and Brightness analysis (N&B). In this study, we expressed varied combinations of dark, Venus-tagged, and Cerulean-tagged FGFR5 and/or FGFR1. HomoFRET imaging using the smaller Forster distance of Cerulean-tagged receptor revealed that FGFR5 existed in pre-formed homodimers. However, the larger Forster distance of Venus-tagged FGFR5 revealed anisotropy lower than the tandem Venus-Dimer control, suggesting it more loosely formed higher order oligomers. Furthermore, N&B imaging revealed that removal of the receptor's intracellular domain (R5ΔC) resulted in higher order aggregation, suggesting FGFR5 associates through an interaction in either the transmembrane or extracellular domains. Co-expression of Cerulean-tagged FGFR5 and R5ΔC confirmed this potential alternate interaction domain. Both techniques suggest FGFR5 forms pre-formed heterodimers with canonical FGFR1. Interestingly, the addition of FGF2 ligand resulted in no change in FGFR5 aggregation state. These data suggest that FGFR5 natively exists as a higher order oligomer with the ability to complex with FGFR1. Collectively, these findings shed new light on the signaling mechanisms of FGFRs that can be used to develop novel therapeutics to treat a number of diseases.

Pancreatic islets are heavily vascularized in vivo with fenestrated endothelial cells (ECs) to facilitate blood glucose-sensing and endocrine hormone secretion. The close proximity of insulin secreting beta cells and ECs also plays a critical role in modulating the proliferation and survival of both cell types with the mechanisms governing this interaction poorly understood. Isolated islets lose EC morphology and mass over a period of days in culture prohibiting study of this interaction in vitro. The loss of ECs also limits the efficacy of islet transplant revascularization in the treatment of Type 1 diabetes. We previously showed that microfluidically driven flow positively affects beta-cell function and EC survival in culture due to enhanced transport of media into the tissue. However, holding islets stationary in media flow using a dam-wall design also resulted in reduced glucose-stimulated metabolic and Ca(2+) responses at the periphery of the tissue consistent with shear-induced damage. We have now created a device that traps islets into sequential cup-shaped nozzles. This hydrodynamic trap design limits flow velocity around the perimeter of the islet while enhancing media flow through the tissue. We demonstrate the feasibility of this device to dynamically treat and collect effluent from islets. We further show that treating islets in this device enhances EC morphology without reducing glucose-stimulate Ca(2+) responses. These data reveal a microfluidic device to study EC and endocrine cell interaction that can be further leveraged to prime islets prior to transplantation.

FGFRL1 is a newly identified member of the fibroblast growth factor receptor (FGFR) family expressed in adult pancreas. Unlike canonical FGFRs that initiate signaling via tyrosine kinase domains, the short intracellular sequence of FGFRL1 consists of a putative Src homology domain-2 (SH2)-binding motif adjacent to a histidine-rich C terminus. As a consequence of nonexistent kinase domains, FGFRL1 has been postulated to act as a decoy receptor to inhibit canonical FGFR ligand-induced signaling. In pancreatic islet beta-cells, canonical FGFR1 signaling affects metabolism and insulin processing. This study determined beta-cell expression of FGFRL1 as well as consequent effects on FGFR1 signaling and biological responses. We confirmed FGFRL1 expression at the plasma membrane and within distinct intracellular granules of both primary beta-cells and βTC3 cells. Fluorescent protein-tagged FGFRL1 (RL1) induced a significant ligand-independent increase in MAPK signaling. Removal of the histidine-rich domain (RL1-ΔHis) or entire intracellular sequence (RL1-ΔC) resulted in greater retention at the plasma membrane and significantly reduced ligand-independent ERK1/2 responses. The SHP-1 phosphatase was identified as an RL1-binding substrate. Point mutation of the SH2-binding motif reduced the ability of FGFRL1 to bind SHP-1 and activate ERK1/2 but did not affect receptor localization to insulin secretory granules. Finally, overexpression of RL1 increased cellular insulin content and matrix adhesion. Overall, these data suggest that FGFRL1 does not function as a decoy receptor in beta-cells, but rather it enhances ERK1/2 signaling through association of SHP-1 with the receptor's intracellular SH2-binding motif.

Type 2 diabetes is characterized by a loss of normal pancreatic beta-cell function and mass. In beta-cells, tyrosine kinase Fibroblast Growth Factors Receptors (FGFRs) modulate insulin processing, fatty acid metabolism and cell survival. However, a deeper understanding of mechanisms that regulate function of these receptors will be necessary to use this pathway therapeutically. We have recently identified beta-cell expression of Fibroblast Growth Factor Receptor-like 1 (FGFRL1), a newly-identified member of the FGFR family. FGFRL1 shares the canonical extracellular domain of FGFRs but uniquely exhibits a short C-terminal histidine-rich zinc-binding domain rather than intracellular catalytic kinase domains. Zinc is a second messenger normally found at picomolar concentration in the cytosol that mediates phosphatase activity to regulate Mitogen-Activated Protein Kinase (MAPK) signaling. We determined that FGFRL1 co-localizes with insulin secretory granules where zinc accumulates at micromolar concentrations. We therefore postulate that FGFRL1 alters beta-cell MAPK signaling by chelating zinc and regulating its intracellular concentration. To measure intercellular zinc, we imaged living murine beta-cells expressing full-length and truncated fluorescent protein variants of FGFRL1 co-labeled with cell-permeable zinc indicators FluoZin-3 and RhodZin-3. Our data confirm that FGFRL1 reduces free intracellular zinc via the unique histidine-rich region. We further show that zinc induces FGFRL1 receptor dimerization at the cell membrane using homo-fluorescence resonance energy transfer (Homo-FRET) imaging. Dimerization of FGFRL1 and association with insulin secretory granules suggest receptor activity is tightly regulated by zinc and likely associated with glucose-stimulated insulin secretion. Elucidating novel signaling mechanisms that regulate FGFR-activity in beta-cells will improve our understanding of how this pathway can be used therapeutically to treat diabetes.

Pancreatic islets are metabolically active tissue that can be studied ex vivo with genetically encoded fluorescent sensors. Adenoviral transduction is currently the most widely practiced method to express sensor in islets. However transduction is limited to a subset of cells on the periphery of the tissue (reaching ∼15-30% of cells) significantly biasing and reducing the statistical power of imaging. To improve transduction efficiency, we created a silicone-based microfluidic device designed for enhanced transport of adenoviral particles deeper into the tissue compared to conventional transduction protocols. This device features hydrodynamic traps arranged in parallel to segregate flow to individual islets. The device was also engineered to provide similar fluidic resistance and pressure drop across each trap, resulting in an analogous viral load being applied to each islet. Adenoviral particle penetration was physically modeled using fluorescently labeled gold nanoparticles of varying size (5 to 100 nm) and subsequently measured using GFP-expressing adenovirus (∼100 nm). Despite significant media exchange to the center of islets trapped in the device, gold nanoparticles >10 nm in diameter were excluded from the tissue. Consistently, nanoparticle and adenovirus penetration necessitated a transient increase in interstitial volume by reversibly blocking Ca2+-dependent cell adhesion using EDTA. Using this treatment combined with flow, we achieved ∼75% transduction efficiency in the core of the islets. The islets exhibited normal glucose stimulated NAD(P)H and Ca2+-responses, indicating that transient tissue expansion and fluid flow did not adversely affect beta cell function. Finally, we validated the utility of this platform by expressing eROGFP, an ER-targeted redox sensor. Even expression of eROGFP throughout islets revealed depth-dependent heterogeneity in ER redox potential. These data reveal a platform to transduce islets throughout and suggest a similar strategy could be adopted on other tissues.

Pancreatic islet β-cells metabolically sense nutrients to maintain blood glucose homeostasis through the regulated secretion of insulin. Long-term exposure to a mixed supply of excess glucose and fatty acids induces β-cell dysfunction and type II diabetes in a process termed glucolipotoxicity. Despite a number of documented mechanisms for glucolipotoxicity, the interplay between glucose and fatty acid oxidation in islets remains debated. Here, we develop confocal imaging of electron transfer flavoprotein (ETF) autofluorescence to reveal the dynamics of fatty acid oxidation in living pancreatic islets. This method further integrates microfluidic devices to hold the islets stationary in flow, and thus achieve ETF imaging in the β-cells with high spatial and temporal resolution. Our data first confirm that ETF autofluorescence reflects electron transport chain (ETC) activity downstream of Complex I, consistent with a response directly related to fatty acid metabolism. Together with two-photon imaging of NAD(P)H and confocal imaging of lipoamide dehydrogenase (LipDH) autofluorescence, we show that the ETC predominantly draws electrons from LipDH/NADH-dependent Complex I rather than from ETF/FADH(2)-dependent ETF:CoQ oxidoreductase (ETF-QO). Islets stimulated with palmitate also show increased ETF redox state that is dose-dependently diminished by glucose (10 mM). Furthermore, stimulation with a glucose bolus causes a two-tier drop in the ETF redox state at ∼5 and ∼20 min, suggesting glucose metabolism immediately increases ETC activity and later decreases fatty acid oxidation. Our results demonstrate the utility of ETF imaging in characterizing fatty acid-induced redox responses with high subcellular and temporal resolution. Our results further demonstrate a dominant role of glucose metabolism over fatty acid oxidation in β-cells even when presented with a mixed nutrient condition associated with glucolipotoxicity.

A number of genetically targetable biosensors are based on the allosteric binding of a ligand to a protein, inducing changes in the molecular distance between two spectrally overlapping fluorescent proteins (e.g. Cerulean and Venus). Ligand concentration is reflected in FRET efficiency, which is typically determined by emission wavelength or fluorescent lifetime. Due to the number of correction factors required for this technique, biosensor design has been largely limited to intramolecular FRET. In nature, however, protein homo-dimerization is a fairly common event. Therefore, the ability to reliably detect intermolecular FRET would open a much larger design space. To that end, we are investigating FRET between identical fluorophores (homoFRET) as a method of detecting intermolecular homo-dimerization through changes in fluorescence anisotropy. To address an ongoing need for measuring cellular NADPH in the field of diabetes, we have developed an NADP+-sensor based on the dimerization of Glucose-6-Phosphate Dehydrogenase. We describe the various stages of sensor development including: linker optimization, enzymatic inactivation and a confirmation of dimerization using Number and Brightness analysis. We further demonstrate other advantages of homoFRET sensor design, such as the ability to swap between fluorescent protein colours and an improved capacity for multiparametric imaging. Functionally, we show that the sensor responds consistently with two-photon excitation NAD(P)H autofluorescence (which cannot distinguish between NADPH and NADH), but could also capture previously inaccessible events unique to NADPH. Finally, the sensor was applied to study nutrient-stimulated changes in NADP+ in beta-cells, where we found significant NADPH depletion in the mitochondria, but not in the cytoplasm, after fatty acid treatment. Overall, this project demonstrates the utility of intermolecular biosensors using homoFRET thus revealing a new paradigm for biosensor design.

Diabetes is a global epidemic that is associated with the deterioration of pancreatic beta-cell mass and function. Beta-cell function within the islet has previously been studied ex vivo and the role of specific proteins is commonly investigated with overexpression models using viral transfection. However, poor penetration and reduced transfection efficiency limits the application of this technology in whole ex vivo islets. To improve transfection efficiency and maintain islet structure, we aim to develop a method to bring viral particles deeper into the tissue. Improving upon a previous microfluidic device design featuring hydrodynamic traps (nozzles) in series, a spiral-shaped microfluidic device with parallel nozzles was created to trap, transfect and deliver nutrients to islets. The device was engineered to provide sufficient resistance and pressure drop along the nozzles, allowing for homogeneous transfection throughout captured islets. Computer-generated models were used to optimize our design parameters to minimize pressure drop variations along with shear induced damage of islets. Through quantitative fluorescence microscopy, we have measured the media exchange rate through islets and have determined a range of operating flow-rates. We have also shown successful culturing of the loaded islets in our microfluidic device. Beta-cell-specific transfection in whole islets with desired genes will facilitate the investigation of islets in a more physiologically relevant environment. Therefore, we have developed a beta-cell specific shuttle vector in order to deliver fluorescent protein-tagged genes via adenoviral particles. Currently viral penetration efficiency is being tested in this device through fluorescence microscopy and western immunoblotting. We aim to create a multi-purpose platform that is not limited to the study of beta-cell biology in islets, but may be translated to the investigation of other tissue models and diseases.