286 Chapter 11 17. Remove OWB leaving 200 μL in the well. 18. Add 200 μL of OWB with secondary antibodies, conjugated antibodies, and dyes 2x concentrated (e.g., DAPI [1ug/mL], mouse-AF488 [1:500], rabbit-AF568 [1:500], for results in Figure 1C and 4D and incubate overnight at 4 °C while mildly rocking/shaking. 19. The next day, repeat steps 13 and 14 for two more times. 20. Carefully transfer the spheroids to a 1.5 mL tube and spin down at 70xg for 3 min. 21. Remove as much as possible the OWB by pipetting without disrupting the spheroids. 22. Add FUnGI (at least 50 μL, RT) using a 200 μL tip with the end cut off and resuspend gently to prevent bubble formation. Incubate at RT for 20 min. 23. In the meantime, create a square container on a glass slide with either nail polish or silicone sealant. 24. Cut off the end of a 200 μL tip and transfer the spheroids in FUnGI to the middle of the square container. 25. Place a square coverslip on top. To minimize trapped air bubbles, place the left side of the coverslip first, then slowly lower the coverslip from left to right until there is no trapped air and then release the coverslip. 26. Gently apply pressure on all edges of the coverslip to firmly attach it to the silicone sealant. 27. Leave the slide overnight on RT. The next day, the slide is ready for imaging. NOTE: Optical clearing by FUnGI may cause minor tissue shrinkage. This will not affect the general morphology of monolayered and multilayered spheroids. The protocol can be paused here and samples can be stored at 4 °C (for at least 1 week) or at -20 °C (for at least 6 months). Representative Results The protocol shown in Figure 1A describes the generation of CSs from previously expanded hiPSC-CMs. The CSs acquire a 3D structure by day 1 post-seeding in ultra-low attachment round-bottom plates and can be cultured for up to 6 weeks (Figure 1B). As assessed by immunofluorescence staining, the majority of the cells in 3-week-old CSs expressed sarcomeric proteins such as alpha-actinin and troponin T, and displayed regular sarcomere organization (Figure 1C). For quantification of α-actinin-positive cells, flow cytometry analysis was performed. In accordance with the immunofluorescence results, the flow cytometry data demonstrated comparable high levels of α-actinin in both day 0 (76.9±16.6%) and 3 weekold CSs (71.1±22.7%) (Figure 1D), indicating a constant and highly pure cellular composition during culturing. Subsequently, the functional properties of CSs including beating rate and Ca2+ handling were assessed at different time points (Figure 2). Calcium transient parameters such as rise time, peak time, decay time, and calcium transient duration (CTD90) were evaluated as indicated in Figure 2A-B. The percentage of beating CSs is similar in the first 3 weeks post-generation
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