289 Generation, high throughput screening, and biobanking of human iPSC-derived cardiac spheroids 11 Finally, the beating activity and Ca2+ handling were measured in both fresh and cryopreserved CSs (Figure 5). The percentage of beating CSs was measured at different time points after thawing, respectively at 2, 5 and 7 days. While most of the fresh CSs showed beating activity over time, clearly the cryopreserved CSs needed up to a week of culturing in order to recover their beating activity (Figure 5B). There was no significant change in the beating rate of thawed CSs versus fresh, however no spontaneous beating activity was observed in some frozen CSs (Figure 5C). Although peak values were significantly reduced in frozen/thawed CSs compared to fresh (Figure 5D), no significant changes were observed in rise time, decay time and the CTD90 of frozen/thawed CSs compared to fresh (Figure 5E-G). These data indicate that, after thawing, it is important to let the CSs recover in the incubator for at least 1 week before measuring beating activity and Ca2+ transient. Figure 3: Beating rate and calcium handling in CSs generated using different cell seeding densities. (A) Bright-field imaging (left) and size measurements (right) of CSs generated using different numbers iPSC-CM. Scale bar, 200 μm. (B) Representative calcium transients traces and time-lapse images of the 2.5K-20K-CSs. (C-D) Beating percentage and beating rate of 2.5K-20K-CSs. (E-H) Peak value, rise time, decay time, and CTD90 in 2.5K-20K-CSs. Data are mean ± SD. Biological replicates = 3, technical replicates = 28-39. ∗p < 0.05, ∗∗∗∗p < 0.001; one-way ANOVA followed by Tukey’s post hoc multiple-comparisons test. Abbreviations: CTD = Calcium transient duration, wk = week, k = x1000 cells, CSs = Cardiac spheroids. Taken together, these results show that cryopreservation of hiPSC-CM-derived spheroids, using PSC Cryopreservation Kit, preserves cardiomyocyte viability, the sarcomeric structure and their functional characteristics such as the spontaneous beating activity and calcium handling. Thus, hiPSC-CM-derived spheroids represent a suitable model to accurately recapitulate cardiac electrophysiology in vitro.
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