89 Massive Expansion and Cryopreservation of Beating Human iPSC-derived Cardiomyocytes 4 METHODS Before You Begin Timing: 30 min In a sterile hood, dilute Matrigel to a concentration of 1.2 mg/ml in 4°C RPMI-1640 in a 50 ml tube on ice. Prepare aliquots of 1 ml in 15 ml tubes on ice and store at -20°C. Remove one Matrigel aliquot (1 ml) from the freezer, and add 11 ml of cold RPMI-1640 to a final concentration of 0.1mg/mL (empirically up to 5x lower concentrations of Matrigel are working). Gently mix the Matrigel solution to thaw and dissolve the Matrigel. Immediately add 1 ml/ well Matrigel in RPMI-1640 for 6-well plates, 1,5 ml/flask for T25s, 2,5 ml/flask for T75, or 5 ml/ flask for T175 flasks. Allow the Matrigel to set for 30 minutes at 37°C before use. The Matrigelcoated plates and flasks can be stored at 4 °C for up to 3 weeks. Note: Keep Matrigel on ice to avoid solidification during the coating procedure. Boxes with pipet tips can be pre-chilled at -20°C. MATERIALS AND EQUIPMENT • hiPSC E8 complete medium: In a sterile environment, mix E8 basal medium, E8 supplement (2% (vol/vol) and penicillin/streptomycin (1% (vol/vol)). Note: We recommend to aliquot the E8 supplement (-20°C) and medium (4°C) and prepare weekly aliquots of the complete medium by pre-warming the required amount. The basal medium can be stored at –20°C for up to 1 year. After preparation, hiPSC E8 complete medium can be stored at 4°C for up to 1 month. • 10 mM stock Y27632 (Y): In a sterile environment, add 31.2246 mL DMSO to 100 mg Y27632. Note: Prepare aliquots of 2-10 µL and store at −80°C for up to 6 months. After thawing the aliquot can be stored at 4°C for 2 weeks. • E8 + 5 µM Y-27632: In a sterile environment, add 0.05% Y-27632 (1:2000 dilution of the 10mM stock) to the hiPSC E8 complete medium. Note: After preparation, this medium can be stored at 4°C for up to 1 week. • 40 mM stock CHIR99021 (CHIR): In a sterile environment, add 1.2455 mL DMSO to 25 mg CHIR. Note: Prepare aliquots of 2-10 µL and store at −80°C for up to 1 year. Avoid re-freezing of the aliquots. • CM differentiation medium (B27): In a sterile environment, mix RPMI 1640, 2% B-27 minus insulin (1:50) and 1% penicillin/streptomycin (1:100). • CM differentiation medium + 7 μM CHIR: In a sterile environment, add 0.0267% CHIR (1:3750 dilution of the 40mM stock) into CM differentiation medium. Note: After preparation, do not store this medium to avoid repeated freeze-thaw cycles of CHIR.
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