Insulin is released from pancreatic islets in a biphasic and pulsatile manner in response to elevated glucose levels. This highly dynamic insulin release can be studied in vitro with islet perifusion assays. Herein, a novel platform to perform glucose‐stimulated insulin secretion (GSIS) assays with single islets is presented for studying the dynamics of insulin release at high temporal resolution. A standardized human islet model is developed and a microfluidic hanging‐drop‐based perifusion system is engineered, which facilitates rapid glucose switching, minimal sample dilution, low analyte dispersion, and short sampling intervals. Human islet microtissues feature robust and long‐term glucose responsiveness and demonstrate reproducible dynamic GSIS with a prominent first phase and a sustained, pulsatile second phase. Perifusion of single islet microtissues produces a higher peak secretion rate, higher secretion during the first and second phases of insulin release, as well as more defined pulsations during the second phase in comparison to perifusion of pooled islets. The developed platform enables to study compound effects on both phases of insulin secretion as shown with two classes of insulin secretagogs. It provides a new tool for studying physiologically relevant dynamic insulin secretion at comparably low sample‐to‐sample variation and high temporal resolution.

Restoration of β-cell mass through the induction of proliferation represents an attractive therapeutic approach for the treatment of diabetes. However, intact and dispersed primary islets suffer from rapidly deteriorating viability and function ex vivo, posing a significant challenge for their experimental use in proliferation studies. Here, we describe a novel method for the assessment of compound effects on β-cell proliferation and count using reaggregated primary human islets, or islet microtissues (MTs), which display homogeneous size and tissue architecture as well as robust and stable functionality and viability for 4 weeks in culture. We utilized this platform to evaluate the dose-dependent short- and long-term effects of harmine on β-cell proliferation and function. Following compound treatment and EdU incorporation, islet MTs were stained and confocal-imaged for DAPI (nuclear marker), NKX6.1 (β-cell marker), and EdU (proliferation marker), allowing automated 3D-analysis of number of total cells, β-cells, and proliferating β- and non-β-cells per islet MT. In parallel, insulin secretion, intracellular insulin and ATP contents, and Caspase 3/7 activity were analyzed to obtain a comprehensive overview of islet MT function and viability. We observed that 4-day harmine treatment increased β- and non-β-cell proliferation, NKX6.1 expression, and basal and stimulated insulin secretion in a dose-dependent manner, while fold-stimulation of secretion peaked at intermediate harmine doses. Interestingly, 15-day harmine treatment led to a general reduction in harmine’s proliferative effects as well as altered dose-dependent trends. The described methodology provides a unique tool for in vitro high-throughput evaluation of short- and long-term changes in human β-cell proliferation, count and fraction along with a variety of functional parameters, in a representative 3D human islet model.

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Modification of gene expression in pancreatic islets can be a powerful strategy for understanding the pathology of diabetes and developing novel therapeutic strategies against it. However, amenability of the isolated islets to genetic manipulation has been limited to only a subset of cells at the periphery due to poor penetration of transduction particles. To address this issue, we developed a standardized islet model, produced by optimized dissociation and controlled scaffold-free reaggregation of primary human islet cells. This process allowed for an ideal experimental window for accessing and manipulating the pancreatic endocrine cells at their single cell state, while enabling production of uniform islet microtissues displaying long-term (>28 days) and robust function. We used an adenovirus that allows tracking of transduced total cells, endocrine cells and beta cells by labeling them with three specific fluorescent reporters expressed from a single back-bone. To define the optimal transduction conditions, we introduced the virus at various titers during three different production stages; after islet dispersion, during and post reaggregation. We quantified transduction efficiency and viral penetration via 3D confocal microscopy followed by assessment of insulin secretory function, insulin content, and cell viability of transduced islet microtissues. Highly efficient (>75%) and uniform transduction was achieved when the virus was added after cell dispersion and during reaggregation. Approximately 80-95% of transduced cells were endocrine cells, of which 50-63% corresponded to β-cells. Although highly transduced islet microtissues displayed decreased chronic (35-50%), basal (55-62%) and stimulated (65-75%) insulin secretion, a significant fold induction of insulin secretion and unaltered insulin/ATP content was observed. Here we present efficient genetic manipulation of functional reaggregated islets by viral transduction as a novel tool for diabetes research. Disclosure B. Yesildag: None. J. Mir-Coll: Employee; Self; InSphero. Employee; Spouse/Partner; Roche Pharma. A. Neelakandhan: None. F. Forschler: Employee; Self; InSphero. A. Biernath: None. I.B. Leibiger: Consultant; Self; Biocrine AB. Consultant; Spouse/Partner; Biocrine AB. B. Leibiger: Consultant; Self; Biocrine AB. Consultant; Spouse/Partner; Biocrine AB. P. Berggren: None. T. Moede: None. C. Ammala: None.

Type 1 diabetes is a heterogeneous group of disorders majorly characterized by autoimmune destruction of pancreatic β-cells, resulting in absolute insulin deficiency. Current research models lack many functions critical for understanding the onset and progression of this disease in humans. Although isolated primary islets are considered the standard tool for diabetes research, their experimental use is challenging due to inherent heterogeneity in size, cellular composition and purity, as well as rapid decline in their functionality and viability ex vivo. Here we investigate a novel co-culture platform of peripheral blood mononuclear cells (PBMCs) and a uniform 3D islet model. Human islet microtissues, produced by optimized dissociation and controlled reaggregation of primary islet cells, were cultured in a one-islet per well format in 96-well plates. They displayed uniform, long-term (>28 days), and robust viability and function enabling high-throughput and longitudinal study of immune cell-endocrine cell interactions and β-cell function. PBMCs in their naïve form or following T-cell specific activation, were combined in various ratios with healthy or stressed islets - preconditioned with a mild cytokine cocktail. Co-culturing of naïve PBMCs and islet microtissues had only a minor impact on β-cell function. Whereas, combination of activated PBMCs and islet microtissues resulted in a PBMC-number dependent decline in islet health shown by increased basal insulin release and decreased glucose-stimulated insulin secretion, insulin content and PDX-1 positive nuclei within each microtissue. The observed destruction correlated with the amount of CD3+ cells infiltrating the microtissues and was significantly augmented by cytokine preconditioning. The established islet-PBMC co-culture platform represents a novel in vitro model to study the autoimmune component of T1D and to screen for compounds that could prevent immune-cell mediated destruction of pancreatic islets. Disclosure B. Yesildag: None. N. Perdue: None. J. Mir-Coll: Employee; Self; InSphero. Employee; Spouse/Partner; Roche Pharma. A. Biernath: None. A. Neelakandhan: None. F. Forschler: Employee; Self; InSphero. M.G. von Herrath: None. J.D. Wesley: Employee; Self; Novo Nordisk A/S.