Hydrogel design

(A) Schematic top-view representation of the hydrogel, illustrating a honeycomb arrangement of 19 microwells per gel. (B) Schematic cross-sectional depiction of the microwells (not to scale). (C) CAD rendering of the hydrogel positioned within the well of a glass-bottom 96-well plate. (D) Entire glass-bottom 96-well plate with microstructured PEG-based hydrogel in each well. (E) Microwell with Mouse Embryonic Fibroblasts attached to the contact point, which is a result of the molds being in contact with the glass bottom during polymerization (top). Mean contact point diameter for two independently moulded samples. N = 16 wells (304 contact points) per sample. Statistical difference was analyzed by one-way ANOVA and Tukey’s post-hoc test where ns = not significant.

Cell seeding and data acquisition

(A) Schematic representation of protocol timeline. 200uL of the MIN6 pancreatic β-cell suspension is added to each well of the 96-well plate at hour 0. Over the next 16 hours, the cells undergo sedimentation into the microwells and spontaneously self-assemble into spheroids. Two hours prior to data acquisition, 10μM of Cal-520 is introduced for 1 hour, followed by washing and incubation in a low-glucose KREBS solution for 30 minutes (hours 17–18). At hour 19, the plates are transferred to the μCELL FDSS for fluorescence-based data acquisition. (B) Representative images of MIN6 β-cells following sedimentation within a microwell, captured 10 minutes post-seeding (top) and 16 hours post-seeding (bottom). (C) Representative fluorescence image of a section of a 96-well plate containing the Cal-520 AM-loaded MIN6 β-cell spheroids acquired using the µCELL FDSS fluorescence plate imager, distinctly showing individual spheroids arranged in a honeycomb pattern within the microwell array. (D) Histogram showing the number of spheroids still present per well at the time of the control stimulus. N = 48 wells on two independent occasions.

Oscillatory behaviour of Ca2+ signal is modulated by extracellular glucose concentrations

(A) Representative Ca²⁺ response of a single 3D MIN6 β-cell spheroid to an increase in extracellular glucose concentration (20 mM), highlighting the visual representations of the peaks and the area under the peaks. The spheroids were initially exposed to basal extracellular glucose levels (3mM) for 10 minutes, followed by an increase in extracellular glucose (20mM) for 30 minutes. The protocol ended with a control stimulus of 50mM KCl. (B) Mean frequency and the normalized area under the peaks of the fluorescent signal during the 30 minutes of exposure to increased concentrations of extracellular glucose. Peak threshold was set to 3 times the signal’s standard deviation during the initial 10 minutes of exposure to basal levels of extracellular glucose. Error bars represent the standard error of the mean (SEM). N > 50 spheroids per concentration. Signal values for each spheroid were normalized to the amplitude of the control stimulus.

KATP-agonist diazoxide inhibits oscillatory response to extracellular glucose

(A) Characteristic Ca2+ responses to 10mM extracellular glucose concentrations of individual MIN6 β-cell spheroids with and without 30 minutes of prior exposure to 100μm diazoxide. The spheroids were initially exposed to basal extracellular glucose levels for 5 minutes after which the spheroids were exposed to varying concentrations of diazoxide for 30 minutes, followed by a 30 minute period of extracellular glucose concentrations of 10mM until a control stimulus of 50mM KCl. Each signal was normalized over the amplitude of the control stimulus. (B) Average of the frequency and the normalized area under the peaks of the fluorescent signal during the 30 minutes of exposure to increased concentrations of extracellular glucose. Peak threshold was set to 3 times the signal’s standard deviation during the initial 10 minutes of exposure to basal levels of extracellular glucose. Error bars represent the standard error of the mean (SEM). N > 40 spheroids per concentration.

KATP-antagonists induces Ca2+ oscillatory behaviour with distinct potencies

(A) Characteristic Ca2+ responses of individual MIN6 β-cell spheroids to 10mM of extracellular glucose with and without prior exposure to 100nM glimepiride. The spheroids were initially exposed to basal extracellular glucose levels for 5 minutes after which the spheroids were exposed to varying levels of glimepiride for 30 minutes, followed by a 30 minute period of glimepirde + extracellular glucose concentrations of 10mM until a control stimulus of 50mM KCl. Each signal was normalized over the amplitude of the control stimulus. (B) Average of the frequency and the normalized area under the peaks of the fluorescent signal during the 30 minutes of exposure to various concentrations of glimepiride, nateglinide and tolbutamide. Peak threshold was set to 3 times the signal’s standard deviation during the initial 10 minutes of exposure to basal levels of extracellular glucose. Error bars represent the standard error of the mean (SEM). N > 50 spheroids per concentration.

Glimepiride and diazoxide impact glucose dependent insulin release by MIN6 spheroids

(A) Characteristic Ca2+ responses of individual MIN6 β-cell spheroids. The spheroids were initially exposed to basal extracellular glucose levels for 10 minutes after which the spheroids were exposed to either 8mM of extracellular glucose, or 8mM of extracellular glucose supplemented with 10nM glimepiride or 10uM diazoxide followed by a control stimulus of 50mM KCl. Each signal is normalized over the maximum value of the control stimulus (B) Average of the frequency and the normalized area under the peaks of the fluorescent signal during the 60 minutes of exposure to the various modulators. Peak threshold was set to 3 times the signal’s standard deviation during the initial 10 minutes of exposure to basal levels of extracellular glucose. Error bars represent the standard error of the mean (SEM). N > 50 spheroids per concentration. (C) Corresponding insulin concentrations present in the supernatant of the wells at the 1-hour timepoint quantified using ELISA. Data is presented as means ± SEM (n = 4 wells per condition). Statistical difference was analyzed by one-way ANOVA and Tukey’s post-hoc test where ns = not significant, ** p < 0.01, **** p < 0.0001.

PS induces Ca2+-oscillatory behaviour and increases insulin secretion.

(A) Characteristic Ca²⁺ responses of individual MIN6 β-cell spheroids exposed to an extracellular glucose concentrations of 8mM, with and without 50μM PS. Spheroids were initially exposed to a baseline extracellular glucose concentration of 3mM for 10 minutes, followed by a 1-hour incubation with varying glucose concentrations, with or without 50 μM PS. After 60 minutes, 15 μL of supernatant was collected for insulin level analysis, concluding with a control stimulus of 50 mM KCl. (B) Quantitative analysis of Ca²⁺ oscillation parameters. The frequency and normalized area under the peaks were analysed for spheroids exposed to glucose concentrations of 3mM, 8mM, and 20mM, with or without 50μM PS. Peak threshold was set at 3 times the signal’s standard deviation during the initial 10 minutes of exposure to basal levels of extracellular glucose. (C) Corresponding insulin concentrations present in the supernatant of the wells at the 1-hour timepoint quantified using ELISA. Data is presented as means ± SEM (n = 4 wells per condition). (D) Characteristic smoothed Ca²⁺ responses of individual MIN6 β-cell spheroids exposed to an extracellular glucose concentrations of 8mM + 50μM PS, with and without 10μM isosakurantin (ISN). Spheroids were initially exposed to a baseline extracellular glucose concentration of 3mM for 5 minutes, followed by a 1-hour incubation with 8mM of extracellular glucose + 50μM PS with and without 10μM ISN. After 60 minutes, 15 μL of supernatant was collected for insulin level analysis, concluding with a control stimulus of 50 mM KCl. (E) Quantitative analysis of Ca²⁺ oscillation parameters. The frequency and normalized area under the peaks were analysed for spheroids exposed to a glucose concentration of 8mM with or without 50μM PS, supplemented with or without 10μM ISN. Peak threshold was set at 5 times the signal’s standard deviation during the initial 10 minutes of exposure to basal levels of extracellular glucose. (F) Corresponding insulin concentrations present in the supernatant of the wells at the 1-hour timepoint quantified using ELISA. Data is presented as means ± SEM (n = 4 wells per condition). Statistical difference was analyzed by one-way ANOVA (E&F) or two-way ANOVA (B&C) and Tukey’s post-hoc testm where ns = not significant, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Ca2+ signal of MIN6 2D monolayer to extracellular glucose

(A) Characteristic Ca2+ responses of wells containing 20 MIN6 β-cell monolayers exposed to an range of extracellular glucose concentrations. The monolayers were initially exposed to basal extracellular glucose levels (3mM) for 10 minutes, followed by an increase in extracellular glucose for 30 minutes. Between minutes 40 and 50, the liquid dispensing head was used to aspirate supernatant from the wells. The protocol ended with a control stimulus of 50mM KCI. (B) The number of pekas and the normalized area under the peaks of the fluorescent signal during the 30 minutes of exposure to increased concentrations of extracellular glucose. Peak threshold was set to 3 times the signal’s standard deviation during the initial 10 minutes of exposure to basal levels of extracellular glucose. Error bars represent the standard error of the mean (SEM). N = 8 wells.