282 Chapter 11 4. Cardiac spheroid generation 1. Three weeks before performing the Ca2+ transient measurements or any functional analysis, add 1 mL of sterile TrypLE Select Enzyme to each well. Incubate the plate at 37 °C for 15 min. 2. Using a 5 mL pipette, mechanically dissociate the cells by flushing with 2 mL warm basal RPMI medium so that single cells can be seen when observed under a microscope. 3. Transfer the cells to a sterile 15 mL conical tube. Centrifuge for 3 min at 300xg. 4. Aspirate the supernatant and resuspend the cells in CM replating media (RM). 5. Using a 1,000 mL pipette tip, mechanically dissociate the cell pellet until the solution appears homogeneous. 6. Transfer 10,000 cells in 100 μL RM to each ultra-low attachment round bottom 96-wells well. Place the plate of CSs on an orbital shaker at 70 rpm in a 37°C incubator for 24 h. NOTE: For more CSs/well; seed 1 million CMs in a 6wells-format ultra-low attachment plate with 2 mL RM. 7. Aspirate 50 μL of medium from each well and add 100 μL RPMI + B27 medium per well for the first 48 h. NOTE: Always keep 50 μL of the medium in the 96-well to avoid accidental aspiration and spheroid rupture. 8. 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. 5. Cryopreservation of cardiac spheroids Cardiac spheroids can be cryopreserved for long-term storage. Cryopreservation can be performed from day 3 after the generation of CSs. CSs can be cryopreserved directly in the wells of a 96 wells plate or as a CSs suspension in cryovials. 1. Pre-chill the plate to 4°C by placing the plate on ice for 10 min. 2. Centrifuge spheroid plate for 3 min at 70xg. 3. Remove the supernatant till 50 µL remains and resuspend each well in STEMdiff hiPSC Freezing Medium. Use 200 μL medium per well. NOTE: Keep the cell suspension on ice. NOTE: In case of a 6 wells plate with spheroids; freeze one well in 500 μL freezing medium cryovial. 4. Freeze the plate at −80°C for a minimum of 4 h in a CoolCell. 5. Transfer the plate to liquid nitrogen or −150°C for long-term storage.
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