95 Massive Expansion and Cryopreservation of Beating Human iPSC-derived Cardiomyocytes 4 20. Aspirate the supernatant and resuspend the cell suspension in 1 ml/flask of 37°C-preheated of the CM replating medium. 21. Count the cell suspension. 22. Re-plate passage 1 (P1) of hiPSC-CMs in the culture system of choice in a culture surface split ratio of 1:10-20 and add 37°C-preheated CM replating medium in the volume according to table 1. Note: If Animal-free and chemical defined is not desired, the Knock-out serum can be replaced with 10% FBS in the replating medium. 23. Remove the Matrigel coating from the flasks and immediately add the cell solution to the prepared culture flasks/plates. 24. Put the flask into the incubator and distribute the cells by moving the flask in short sideto-side and back-and-forth motions. Incubator conditions should be set to 37°C, 5% CO2, 21% O2, and 90% humidity. Proceed to step 25. hiPSC-CM expansion Timing: 30 min 25. Day 1: After attachment and recovery of passage 1 (P1) hiPSC-CM for 24 hours start the expansion process by replacing the replating medium with 37°C-preheated cardiac expansion medium according to Table 1 every other day or once every three days. For most cell lines 2 µM CHIR induces efficient expansion of hiPSC-CMs. CRITICAL: Even though we found that 2 μM of CHIR is the optimal concentration for the expansion of hiPSC-CMs from three different donors that we tested, it should be noted that cell line-to-line and molecular batch-to-batch variabilities are possible. Therefore, it is recommended to optimize CHIR concentrations between 1-3 µM. Note: The protocol for the generation of hiPSC-CMs can be variable. Here, the differentiation methods; B273, Heparin4, and CDM32 are used to produce CMs, which can be efficiently expanded (Please see Figure 1). 26. After 3-5 days, the hiPSC-CMs will reach 70-80% confluency and should be passaged to inhibit cell-cell contact. Aspirate the medium, wash with PBS. Add 1 ml of room temperature warm TrypLE per 25-cm2 growth surface and incubate the hiPSC-CMs for 10–15 min at 37°C. 27. After incubation for ~15 minutes, detach the hiPSC-CMs with a 5 or 10 ml tip filled with 2 ml per 25-cm2 37°C-preheated RPMI-1640 and dispense over the surface of the plate well until all the cells are detached and gently mix 2-5 times using the 5 ml pipette. Note: The dissociation of expanding hiPSC-CMs will be more quickly and easier than the dissociation of d11 differentiated hiPSC-CMs, which sometimes requires prolonged incubation with TrypLE™ Select (10X).
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