Novel 2D/3D Hybrid Organoid System for High-Throughput Drug Screening in iPSC Cardiomyocytes

Figures

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  Figure 1.. (a) Representative brightfield videos of traditional 2D cellular sheet, 3D organoid and a hybrid 3D/2D model, can see in supplementary files. (b–d) Total beat duration, systole duration, and diastole duration are prolonged in 2D sheets (n = 91–171). Notably, 2D sheets have visibly greater ranges for these durations, indicating less synchronicity, while cells within 3D organoids have a more compact distribution, providing evidence of improved connectivity. (e–g) Prolonged duration and increased variability amongst beat, systole, and diastole durations remains even when controlling for overall contraction time and wavelength period (n = 91–171). Cumulative overlays of cross correlation analysis for contractility (h) and fluorescence (i) for each individual video (n = 10). This analysis quantifies of how similar the signal originating from the 2D cells is to the signal from the 3D organoids in our hybrid model within each video. The strong peak at Lag = 0 indicates the signals are highly synchronous. Two-tailed t-test was used for all statistical comparisons **** p < 0.0001).

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  Figure 2.. (a) Schematic of experiment. (b) Representative videos of control and Dox organoids and cells with calcium-sensitive Rhod-2 dye, can see in supplementary files. (c) Calcium transient peak duration is prolonged in Dox group (Unpaired t-test, n = 209–342). (d) Calcium release duration is prolonged in Dox group (Unpaired t-test, n = 209–342). (e) Calcium reuptake duration is prolonged in Dox group (Unpaired t-test, n = 209–342). (f) Maximum rate of rise for calcium transient peak is lower in Dox group (Unpaired t-test, n = 209–342). (g) Area under curve for calcium transient peak, which is indicative of magnitude of calcium, is greater in Dox group (Unpaired t-test, n = 209–342). (h) The number of cells around the organoid is decreased in Dox group (Unpaired t-test, n = 7–9). (* p < 0.05, **** p < 0.0001).

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  Figure 3.. (a) Schematic of spatial segmentation to assess regional differences. (b) Representative calcium transient peak waveforms for each group. (c) Cellular concentration decreased in the further regions in Dox group. (d) Peak duration prolonged with increased distance from the organoid in Dox group (One-way ANOVA with Tukey post-hoc test, n = 23–144). (e) Calcium release duration was stable across regions in both groups (One-way ANOVA with Tukey post-hoc test, n = 23–144). (f) Calcium reuptake duration prolonged with increased distance from the organoid in Dox group (One-way ANOVA with Tukey post-hoc test, n = 23–144). (g) Maximum rate of rise decreased with increased distance from organoid in Dox group. In control group, it only decreased from organoid to first adjacent “Close” region (One-way ANOVA with Tukey post-hoc test, n = 23–144). (h–k) Heat maps of parameters analyzed in (d–g). Each dot represents an individual ROI/cell that was analyzed. The center of the graphs was normalized to the center point of each organoid. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = non-specific).

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  Figure 4.. (a) The first waveform is an example of normal calcium transient morphology. The remaining waveforms are examples of abnormal calcium transient morphology that were observed. (b) Both the control and Dox groups had abnormal calcium transients, and the proportion of cells with abnormal calcium transients increased with further distance from the organoid. Overall, Dox had a greater proportion of abnormal calcium transients. (c) Workflow for machine-learning classifier model development. (d) Confusion matrix results from the best-performing model. (e) ROC curve for best-performing model, AUC = 0.98.

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  Figure 5.. (a) Brightfield videos of control and Dox organoids and cells, can see in supplementary files. (b) Dox group had fewer beating cells, which were mostly concentrated in close region. (c) Dox had more cells with dysfunctional contractility. but in both groups, a similar percentage of dysfunctional beating can be attributed to abnormal calcium handling. (d) Total beat duration is prolonged in Dox group (Unpaired t-test, sn = 209–342. (e) Dox group has prolonged systole (Unpaired t-test, n = 209–342. (f) Dox group has prolonged diastole (Unpaired t-test, n = 209–342. (g–i) Beat, systole, and diastole durations have temporospatial changes (One-way ANOVA with Tukey post-hoc test, n = 23–144). (j–l) Heat map representation of temporospatial changes on a per-cell basis. (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = non-specific).