Figure 3.

Concomitant modulation of cell shape and stiffness by PAAm-co-PAAc hydrogel influences the structure of isolated single hiPSC-CMs. Representative images of hiPSC-CMs stained for cTnT, α-actinin and Cx-43 on (A) the flat control surface and (B) inside the 3D adult-like microwell of physiologic substrate stiffness (9.83 kPa). Yellow outline demonstrates the perimeter of cell. (C) Sarcomere length showed a significantly higher spacing when cultured on physiologic stiffness than on pathologic surfaces (112 kPa) (2-way ANOVA, *p < 0.05). (D) Quantification of area occupied by Cx-43 on 3D microwells and flat surface controls, showing increased presence of Cx-43 in cells residing inside 3D microwells (2-way ANOVA, *p< 0.05, **p < 0.01). (E) Analysis of CM sarcomeric directionality, measured against X-axis, demonstrates capacity of the 3D microwells for inducing cell alignment. Representative images of Cx-40 and Cx-45 staining on (F) the flat control surface and (G) 3D microwell of physiologic stiffness. (H) Cell membrane stiffness measured using AFM, demonstrating increased stiffness under pathologic condition versus physiologic condition, as well as increased membrane rigidity of isolated single hiPSC-CMs residing inside the 3D microwells (2-way ANOVA, **p < 0.01). Scale bars: 10 µm. Data shown as mean ± SEM, N ⩾ 3. (9.83 F = physiologic stiffness flat control, 9.83 W = physiologic stiffness 3D microwell, 112 F = pathologic stiffness flat control, 112 W = pathologic stiffness 3D microwell).