91 Massive Expansion and Cryopreservation of Beating Human iPSC-derived Cardiomyocytes 4 • Wash buffer: This buffer contains 50 ml of PBS and 1% BSA. Note: After preparation, all buffers can be stored at 4°C for up to 1 month. Alternatives: • 6-Well cell culture plates: Greiner, cat. no. 657160. • T25 cell culture flasks: Greiner, cat. no. 690170. • T75 cell culture flasks: Greiner, cat. no. 658170. • T175 cell culture flasks: Greiner, cat. no. 660160. • Centrifuge tubes: 15 ml, Greiner, cat. no. 188280. • Room temperature CoolCell cell freezing container (-1 °C /minute cell freezing). • Cryovials: VWR, cat. no. 479-6846. • Counting slides: Countess, Cat. no C10228. • BSL2 biosafety cabinet: e.g., Telstar EF. • Cell Counter: e.g, Countess II FL Automated Cell Counter from Life Technologies. • Cell culture incubator (37°C, 5% CO2, 21% O2): e.g. Panasonic MCO 170. • 37°C water bath: e.g., Julabo Pura. Key Resources Tables Table 1. key culture format cultivation table STEPS Culture format #Culture formats Start amount of cells (104 / cm2) Total amount of cells after each step (104) Volume of medium/format Timing hiPSC culturing 6 wells plate 1 well ~2 ~300 2 mL per wells 4 d Cardiac differentiation 6 wells plate 1 well ~30 ~700 3 mL per wells 11 d Passage 1 T75 flask 1x ~2.5 ~2100 7 mL per T75 flask ~4 d Passage 2 T175 flask 3x ~4 ~7000 21 ml per T175 flask ~5 d Passage 3 T175 flask 8x ~5 ~20000 21 ml per T175 flask ~6 d Passage 4 T175 flask 14x ~8 ~35000 21 ml per T175 flask ~7 d Passage 5 T175 flask 20x ~10 ~50000 21 ml per T175 flask ~8 d
RkJQdWJsaXNoZXIy MTk4NDMw