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Traditional high-throughput screening methods typically rely on 2D \u03b2-cell cultures, but such cultures do not mimic the 3D organization and cell-to-cell communication of \u03b2-cells in pancreatic islets of Langerhans. Existing 3D \u03b2-cell culture models are hindered by high costs, technical complexity, and limited compatibility with high-throughput screening platforms. In this work, we developed an approach for generating 19 homogeneously shaped pancreatic \u03b2-cell spheroids in each well of a 96-well plate, using micropatterned polyethylene glycol (PEG)-based hydrogels and murine mouse insulinoma 6 (MIN6) insulinoma cells. The uniform shape and positioning of the individual spheroids enabled the simultaneous, real-time imaging of Ca\n                    <jats:sup>2+<\/jats:sup>\n                    signals in up to 1824 independent spheroids in response to glucose and various test compounds. Using this approach, we show that increasing glucose causes concentration-dependent Ca\n                    <jats:sup>2+<\/jats:sup>\n                    oscillations in individual spheroids, that these Ca\n                    <jats:sup>2+<\/jats:sup>\n                    oscillations are sensitive to modulators of ATP-sensitive K\n                    <jats:sup>+<\/jats:sup>\n                    channels, and that the frequency of Ca\n                    <jats:sup>2+<\/jats:sup>\n                    oscillations correlates with insulin secretion. Finally, we demonstrate that the neurosteroid pregnenolone sulfate, an agonist of the cation channel TRPM3, increases the frequency of glucose-induced Ca\n                    <jats:sup>2+<\/jats:sup>\n                    oscillations and enhances insulin release; the TRPM3 antagonist isosakuranetin inhibited these responses. In conclusion, we established a cost-effective and scalable 3D \u03b2-cell platform for high-throughput screening of insulin release-modifying compounds, with potential applications in drug development and personalized medicine for the management of diabetes mellitus.\n                  <\/jats:p>","DOI":"10.7554\/elife.110428","type":"journal-article","created":{"date-parts":[[2026,3,4]],"date-time":"2026-03-04T14:33:53Z","timestamp":1772634833000},"update-policy":"https:\/\/doi.org\/10.7554\/elife.110428","source":"Crossref","is-referenced-by-count":0,"title":["A high-throughput assay for the measurement of Ca2+ oscillations and insulin release from uniformly sized mouse \u03b2-cell (MIN6) 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