280 Chapter 11 PROTOCOL hiPSC-CMs used in this study were generated according to previously described hiPSC culturing and CM differentiation protocols[26,27]. Optionally, the hiPSC-CMs can be expanded and cryopreserved as recently published before starting the CSs protocol[10]. 1. Preparation of Media 1. Prepare basal RPMI medium Equilibrate pen/strep and the basal medium (RPMI 1640) to room temperature (RT). Ensure that the supplement has thawed completely. Mix 500 mL of the basal medium and 5 mL of Pen/strep. Store at 4 °C. 2. Prepare RPMI + B27. 1. Equilibrate the B27 supplement and the basal medium (RPMI 1640) to RT. Ensure that the supplement has thawed completely. Mix 485 mL of the basal medium, 5 mL of Pen/ strep, and 10 mL of the 50x supplement. Store at 4 °C for up to 2 weeks, equilibrate to 37 °C before use. 3. Prepare CM re-plating media (RM) 1. Add Rho-associated, coiled-coil containing protein kinase (ROCK) inhibitor (2 μM final concentration) and Knockout Serum Replacement (Knockout SR, 10% final concentration) to RPMI + B27 media. Store at 4 °C for up to 1 week, equilibrate to 37 °C before use. 4. Preparez CM thawing media 1. Add Revitacell (100X) and Knockout SR (20% final concentration) to RPMI + B27 media and equilibrate to 37 °C before use. 5. Prepare maturation media 1. Equilibrate the B27 supplement, Knockout SR, pen/strep, maturation medium[28], and the basal DMEM no-glucose medium to RT. Ensure that the supplement has thawed completely. Mix 435 mL of the basal medium and 10 mL of the 50X B27 supplement, 5 mL of pen/strep, 5 mL Knockout SR, and 45 mL of maturation medium aliquot as previously described[28] and filter using a 0.22 μm vacuum-driven filter. Store at 4 °C for up to 2 weeks, equilibrate to 37 °C before use. 6. Prepare fluor bright medium 1. Equilibrate, pen/strep, and the basal DMEM Fluorobrite medium to RT. Ensure that the supplement has thawed completely. Mix 500 mL of the basal medium and 5 mL of pen/ strep. Store at 4 °C, equilibrate to 37 °C before use.
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