288 Chapter 11 and 20K-CSs) was significantly higher (about 85%) (Figure 3C). 5K-, 10K- and 20K-CSs showed similar beating rate (about 28 bpm) which was significantly higher compared to 2.5K-CSs (Figure 3D). The peak values of calcium images were similar in all tested conditions (Figure 3E), however, rise time (Figure 3F), decay time (Figure 3G), and CTD90 (Figure 3H) were significantly increased in bigger size-CSs (10K-, and 20K-CSs) compared to the smaller ones (2.5K- and 5K-CSs). Taken together, these results show that hiPSC-CM-derived spheroids are optimal for calcium handling screening when a seeding density between 10K- and 20K hiPSCCMs/well is used. Figure 2: Beating rate and Calcium handling in CSs at different weeks post generation. (A) Examples of calcium transient parameters calculated by the Vala sciences analysis algorithm in Cyteseer Software. (B) Representative calcium transient traces and time-lapse images of the CSs at different time points (weeks) post-generation. Scale bar, 200 μm. (C) Time course quantification of spontaneous beating activity is expressed as the percentage of beating CSs. (D) Beating rate of CSs during culturing time. (E-H) Quantification of the calcium transients showing peak value, rise time, decay time, and CTD90. Data shown are mean ± SD. Biological replicates = 3 technical replicates = 38,50,66 and 7, respectively. ∗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, CSs= human cardiac spheroids. Next, we evaluated the impact of cryopreservation on CS’s viability and function. Before analysis, thawed CSs were maintained in culture for 1 week (Figure 4A). As shown by both flow cytometry (Figure 4B) and Calcein-AM (Figure 4C) cell viability tests, cryopreservation did not affect cell viability within the CSs. Additionally, thawed CSs showed similar expression levels of sarcomeric proteins as compared to the fresh age-matched CSs (Figure 4D). These data indicate that CSs can be efficiently cryopreserved for subsequent cardiac function analysis and high throughput screening.
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