283 Generation, high throughput screening, and biobanking of human iPSC-derived cardiac spheroids 11 6. Thawing of cardiac spheroids 1. Prepare 20 mL of 37°C-preheated RPMI medium in a 50-mL tube. 2. Collect the cell plate with CSs from the liquid nitrogen and place it in the incubator for 15 min. NOTE: Do not thaw more than one plate at a time to ensure a quick thawing process. 3. Remove the supernatant till 50 µL remains and resuspend each well in RPMI medium. Use 200 µL medium per well. 4. Centrifuge for 3 min at 70xg. 5. Repeat steps 3 and 4. 6. Remove the supernatant till 50 μL remains and resuspend each well in CM thawing medium. Use 200 μL medium per well. 7. Place the plate of spheroids on an orbital shaker at 70 rpm in a 37°C incubator for 24 h. The incubator conditions should be set to 37°C, 5% CO2, 21% O2, and 90% humidity. 8. Aspirate 50 µL of medium from each well and add 100 µL RPMI + B27 medium per well for the first 48 h. 9. Aspirate 100 µL of medium from each well and add 100 µL maturation medium per well. Maintain cells in maturation media, changing media every 2-3 days until ready for functional analysis. 7. Assessment of Intracellular Ca2+ Transients 1. After 1 week of culture, the thawed CSs are optimal for calcium handling optical imaging. NOTE: As indicated previously, the spheroids are in culture for the optimal timing of 3 weeks, two weeks before freezing, and one week after thawing. The ‘fresh’ controls are age-matched. 2. Treat them with 100 μL per well of calcium dye medium and incubate at 37 °C for 60 min. NOTE: Cal520AM is light-sensitive. Perform all loading procedures and experiments in the dark. 3. Prepare the calcium acquisition and analysis system. 1. Power the Leica Thunder microscope system ensuring the environmental control option is on. 2. Adjust the camera and framing aperture dimensions to minimize background area. NOTE: Here, the Leica Thunder DMi8 microscope was used, but other microscope systems might be applicable as well, considering that it allows a frame rate of over 30 frames/ second. 4. Begin recording of a video collecting a consistent stream of 2−10 peaks within 10 seconds and scan across the 96wells plate, initially moving to the left, then downwards in a zig-zag fashion to cover the whole plate. 5. Once the Ca2+ transients are acquired, analyze the data with the fluorescence traces analysis software according to the manufacturer's instructions.
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