287 Generation, high throughput screening, and biobanking of human iPSC-derived cardiac spheroids 11 but significantly dropped in Wk6-CSs (Figure 2C). The beating rate was significantly reduced at Wk3 compared to Wk1 and, similar to the percentage of beating CSs, dramatically dropped at Wk 6 (Figure 2D). At Wk6, CSs deterioration was observed, which could explain the drop in both beating rate and number of beating CSs. Measurement of calcium transient parameters indicated a significantly higher peak value at Wk2 (Figure 2E), while the rise time, decay time, and CTD90 were significantly increased at Wk3 compared to Wk1 (Figure 2F-H). Taken together, these results show that hiPSC-CM-derived spheroids are functional optimal round weeks 2 and 3 post-generation. Figure 1: Generation of cardiac spheroids. (A) Schematic representation of Wnt-based directed cardiac differentiation, the subsequent expansion of hiPSC-CMs, and the generation of CSs. Created with biorender. com. (B) Bright-field images at different time points of CSs culturing. Scale bar, 200 μm. (C) Representative immunofluorescence images for cardiac sarcomeric proteins α-actinin and troponin-T in 3-week-old CSs. Immunofluorescence: Hoechst (blue), α-actinin (green), and troponin T (red). The merged picture on the right displays the sarcomere organization. Scale bar, 50 μm. (D) Flow cytometry quantification of α-actinin positive cells before (day 0) and 3 weeks after the formation of CSs. (n= 14-23 per condition. Data are represented as mean ± SD). Figure 3 shows the effect of spheroid size on the beating rate and calcium handling. CSs were generated by seeding 2.5, 5, 10, and 20x104 hiPSC-CMs in a well of a 96-well plate, for a total of 24 CSs/wells per condition (Figure 3A). As expected, the spheroid size increased as the number of cells used increased, ranging from 178±36 to 351±65 μM (Figure 3A, right panel). Ca2+ transients were measured in 3-week-old CSs at the four different seeding densities (Figure 3B). Measurements of beating CSs indicated that only about 50% of the smaller sizeCSs (2.5K- and 5K-CSs) were beating, while the percentage of bigger size-beating CSs (10K-
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