1711-P: A 3D High-Content Islet Imaging Toolbox

Previously, we optimized hanging-drop based, scaffold-free and high-throughput reaggregation of primary islet cells to obtain uniform islets that display long-term and robust function. Using our model, we reported type 1 and 2 diabetes relevant stress models that rely on analysis of insulin secretion and insulin and ATP contents. While these biochemical assays effectively assess islet function and viability, they lack the ability to report at a single-cell resolution. Here, we showcase multiple 3D high-content confocal microscopy applications that provide single-cell insights into pancreatic cell function, identity, survival and proliferation. During islet culture, inflammatory stress was induced using proinflammatory cytokines, activated peripheral blood mononuclear cells or islet-specific cytotoxic T lymphocytes. For metabolic stress, glucolipotoxic culture conditions were used. Gene expression was modified via adeno-associated viral transduction during reaggregation. Islet function and viability were analyzed through basal and stimulated insulin secretion, insulin and ATP contents. 3D confocal imaging protocols of key diabetes-relevant targets were established by optimization of islet fixation, permeabilization, blocking, antibody staining, clearing, and high-content imaging followed by high-resolution, multicolor 3D microscopy. In addition, cell tracker dyes were utilized to observe longitudinal real-time islet-immune cell interactions. Images were analyzed with custom CellPathFinder pipelines. These established methods enable quantitative analysis of islet hormone content, amyloid deposition, HLA expression, transduction efficiency, T-cell infiltration, and β- and non-β-cell death, survival, mass and proliferation. In summary, by combining cutting-edge spheroid and 3D high-content imaging technologies, we developed unique tools for quantitative, high-throughput, and automated phenotypic characterization of pancreatic islets in parallel with a variety of functional parameters. Disclosure A.C. Title: Other Relationship; Biomea Fusion, Inc., Boehringer-Ingelheim, Novo Nordisk, Merck Boehringer-Ingelheim, Merck Boehringer-Ingelheim, Novo Nordisk, Merck Biomea Fusion, Inc., Boehringer-Ingelheim, Merck Biomea Fusion, Inc., Novo Nordisk, Boehringer-Ingelheim, Merck Novo Nordisk, Boehringer-Ingelheim, Biomea Fusion, Inc., Merck & Co., Inc.