290 Chapter 11 Figure 4. Effect of cryopreservation on cardiac spheroids viability and structure. (A) Schematic representation of CSs generation, subsequent biobanking, and thawing. (B) Flow cytometry cell viability test in both fresh and cryopreserved CSs. As a positive control, a treatment with 10% Triton-X solution for 5 min was used. (n = 4 per condition). Data are represented as mean ± SD.∗∗∗∗p < 0.001; one-way ANOVA followed by Tukey’s post hoc multiple-comparisons test. (C) Calcein-AM cell viability test in fresh vs. thawed CSs after 7 days of culturing (n = 15-17 per condition, ∗∗∗∗p < 0.001, by paired t-test, scale bar, 200 μm). (D) Representative bright-field (left) and immunofluorescence staining for α-actinin and troponin T expression in fresh and thawed CSs. Immunofluorescence: Hoechst (blue), α-actinin (green), and troponin T (red). The merge pictures on the right display sarcomere striations in the CSs. Scale bar, 50 μm. Abbreviations: X = Thawing day of choice, PI = Propidium iodide, Cal-AM = calcein-AM, EthD-I = Ethidium Homodimer I. Figure 5: Calcium transients in fresh vs thawed CSs. (A) Representative calcium transient traces and time-lapse images of the CSs before cryopreservation and one week after thawing. (B-C) Beating percentage and beating rate of fresh and frozen/thawed cardiac spheroids. (D-G) Quantification of calcium transient parameters: peak value, rise time, decay time, and CTD90. Data are mean ± SD. ∗p<0.05, ∗∗∗∗p<0.001; one-way ANOVA followed by Tukey’s post hoc multiple-comparisons test. Abbreviations; CTD = Calcium transient duration, CSs = cardiac spheroids.
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