100 Chapter 4 Frozen P1 P2 P3 P4 P5 1 10 100 1000 10000 Total Cell number (106) Fresh Thawed 100 Frozen C A Fresh B 72% 94% % PIFresh Frozen 0 20 40 60 80 100 %PIP=0,0669 0 P=0.0669 Fresh Total Cell number (106) D P1 thaw +1 P1 +4 P2 +5 P3 +6 P4 +7 P5 +8 Figure 3: Cryopreservation and subsequent expansion of beating hiPSC-CMs. (A) Schematic representation of small molecule-based Wnt-modulated directed cardiac differentiation and subsequent cryopreservation and expansion. hiPSC-CMs can be stored in liquid nitrogen after passage 1 and after thawing expanded for 5 passages (P) before downstream assays. Abbreviations: TM; CM thawing medium, EM; CM expansion medium, SM; CM splitting medium, P; passage. (B) Left panels are flow cytometer detection of dead cells with propidium iodide (PI) after detaching or after thawing of cardiomyocytes, cryopreserved on day 15 of differentiation. Right is the quantification of fresh vs thawed PI % (n=5 per condition, P<0.05, paired T-test.). (C) Representative images of the thawed hiPSC-CMs before starting the expansion protocol and during passaging (scale bar: 100 μm). (D) Quantification of cardiomyocyte expansion curve of fresh vs thawed hiPSC-CMs, n=7, n=5 respectively from thawed P10 to P5. 58. Transfer the cell suspension to a FACS tube. 59. Centrifuge the cell suspension at 4 °C for 3 min at 200xg and discard the supernatant. 60. Resuspend 1 × 105 cells in 50 μl of permeabilization buffer containing 5% BSA and 0.3% Triton-X-100. 61. Incubate the cells for 30 min at 4 °C. 62. Resuspend in 50 μl of flow cytometry buffer containing the α-actinin antibody (1:300 dilution) and in another FACS tube resuspend 1 × 105 cells in 50 μl of flow cytometry buffer with the respective isotype control (e.g., FITC mouse IgM, κ isotype (1:200 dilution)) and 1 × 105 cells in 50 μl of flow cytometry buffer for negative control. 63. Incubate the cells for 30 min at 4°C.
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