99 Massive Expansion and Cryopreservation of Beating Human iPSC-derived Cardiomyocytes 4 44. Transfer the cell suspension to the 15-ml tube, drop by drop and swirl the tube while adding the cell suspension. The dropwise addition of medium to the cell suspension is critical to minimize osmotic shock. 45. Centrifuge the cell suspension at 200xg for 3 min at room temperature. 46. Remove the supernatant and resuspend the hiPSC-CMs in the desired volume of CM thawing medium. 47. Count the cells within the cell suspension. 48. Aspirate the liquid from the Matrigel-coated flasks, and seed the CMs into the Matrigelcoated flasks, according to the format amount in Table 1. 49. 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. 50. Replace the thawing medium with CM expansion medium the next day. The hiPSC-CMs should be attached, beating, and can be used for expansion (step 19) or downstream assays. (Please see Figure 3 for representative viability, morphology, and proliferation rate after thawing of hiPSC-CM) Flow cytometry analyses of dissociated hiPSC–CMs Timing: 1.5 h, plus 30 min for dissociation of CMs 51. Dissociate the CMs as described in the ‘Replating of differentiated hiPSC-CMs’ section. 52. After counting the hiPSC-CMs, collect 100.000 CMs from the cell suspension in 1,5 ml tubes. 53. Centrifuge the cell suspension at 200xg for 3 min at room temperature. 54. Discard the supernatant and add 50 μl 4% PFA. 55. Incubate 10 minutes at room temperature. 56. Centrifuge the cell suspension at 200xg for 3 min at room temperature. 57. Discard the supernatant and add 1 ml PBS. Pause Point: The fixed hiPSC-CMs can be stored at 4°C for up to 4 weeks.
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