97 Massive Expansion and Cryopreservation of Beating Human iPSC-derived Cardiomyocytes 4 33. Remove the Matrigel solution from new flasks and immediately add the cell solution to the prepared culture flasks/plates. 34. Put the flask into the incubator and distribute the cells in the well by moving the flask in short side-to-side and back-and-forth motions. Incubator conditions should be set to 37°C, 5% CO2, 21% O2, and 90% humidity. 35. Refresh the expansion medium every other day in the volume according to table 1. After 5-8 days, the hiPSC-CMs will reach 70-80% confluency and should be passaged to inhibit cell-cell contact. Repeat step 26-34 for passag e 2-5. By every passage, split the hiPSC-CMs in a lower splitting ratio and flasks according to table 1. Note: By higher passage numbers hiPSC-CMs proliferation gradually slows down and further passaging then P4-5 usually does not result in increased cell numbers. (Please see Figure 2 for representative morphologies during hiPSC-CM passaging) CRITICAL: Replacement of medium is recommended for every other day in order to provide fresh nutrients and stable concentrations of CHIR for expanding hiPSC-CMs. For weekend free culturing it is possible to stretch the medium changing interval accordingly while increasing the medium volume in table 1 with 1.5X. 36. After substantial expansion of the CM population, CHIR withdrawal from the medium results in the decrease of hiPSC-CMs proliferation, and subsequent regular CM culture medium change intervals can be extended for long term culturing or downstream assays. PAUSE POINT: After passage 1 or passage 2, the expanded hiPSC-CMs can be efficiently frozen for biobanking and thawing upon analysis (See freezing of hiPSC-CMs; Steps 35-40, Figure 2). Freezing of hiPSC-derived CMs Timing: 30 min Note: Volumes are given for one cryovial. 35. Count the CMs after the dissociation as described in the ‘Replating of differentiated hiPSCCMs’ section. CRITICAL: Use hiPSC-CMs in passage 1-2 for the cryopreservation and biobanking to generate viable and expandable cultures. 36. Transfer the desired cell suspension for freezing to a 15-ml tube. 37. Centrifuge the cell suspension at 200xg for 3 min at room temperature. 38. Remove the supernatant and resuspend the CM in STEMdiff™ Cardiomyocyte Freezing Medium. Use 0.5 ml medium for 0.5-2 x 106 CMs and 1 ml of freezing medium for 2-8 x 106 CMs per vial. Note: Keep the cell suspension on ice. 39. Freeze the vials at −80 °C overnight in a CoolCell or 2-propanol-filled freezing container. 40. Transfer the vials to liquid nitrogen or −150 °C for long-term storage.
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