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. 2021 Jun 11;9:674260. doi: 10.3389/fbioe.2021.674260

FIGURE 1.

FIGURE 1

Optimization of suspension differentiation protocol. (A) Schematic of the overall process and differentiation timeline for cardiac spheroid production pursued in this paper. Monolayer cultured iPSCs were seeded into 3D culture where they were differentiated followed by multimodal biomolecular and functional analysis. After seeding on day-7 cells are cultured in TeSR E8 3D for 7 days with a single passage on day-4. Cell density is calculated on day 0 and spheroids are then cultured in RPMI1640/B27- supplemented with CHIR on day 1 and IWR on day 3. Metabolic purification is initiated on day 9 using RPMI 1640 without glucose supplemented with B27+ and d-lactate. On day 12 and every 3 days thereafter, media is partially changed with fresh RPMI1640/B27+. Optimization studies analyzed cells at point A and all characterization experiments used cells at point B. (B) Flow cytometric analysis of cTnT positive cells for optimization of differentiation conditions including CHIR concentration, cell density on day 0, and shaker speed. (C) Total cardiomyocytes produced in each differentiation condition during optimization. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 (n = 4).