Fig. S1.
Development, optimization, and characterization of Heart-Dyno (supporting data for Fig. 1). (A) Photomicrographs of Heart-Dyno culture inserts with hCOs in a 96-well plate. (B) Force of contraction responses to known repressors/activators of force of contraction; n = 5. (C) Proarrhythmogenic compounds cause prolonged relaxation time in hCO as expected; n = 5. (D) Validation of Ki-67 marker activation using whole-mount immunostaining; n = 5–7 from two experiments. hCOs treated with 0.05% DMSO and 5 µm CHIR 99021. (E) Lactate dehydrogenase (LDH) levels in response to a full factorial glucose (0, 0.5, 1, and 5.5 mM) and palmitate (0, 1, 10, and 100 μM) screen in the presence of insulin. LDH levels were assessed after 5 d of serum-free culture after 2 d of hCO formation in CTRL medium; n = 8–9 from three experiments (HES3-derived hCOs). (F) Cardiac troponin (CTNI) levels for serum-free conditions. CTNI levels were assessed after 5 d of serum-free culture after 2 d of hCO formation in CTRL medium; n = 2–3 from one experiment (HES3-derived hCOs). −VE, negative control; +VE, positive control (mechanically crushed hCO). (G) Whole-mount images of hCOs stained with α-actinin that mechanically failed from broken arms (Upper) and necking (Lower) issues after 5 d of serum-free culture after 2 d of hCO formation in CTRL medium. (Scale bars: 200 μm.) (H) Force heat map in response to a full factorial glucose (0, 0.5, 1, and 5.5 mM) and palmitate (0, 1, 10, and 100 μM) screen. Force was assessed after 5 d of serum-free culture after 2 d of hCO formation in CTRL medium; n = 8–12 tissues from three experiments (H9-derived hCOs). (I) Whole-mount MLC2v expression in response to a full factorial glucose (0, 0.5, 1, and 5.5 mM) and palmitate (0, 1, 10, and 100 μM) screen. MLC2v expression was assessed after 5 d of serum free culture after 2 d of hCO formation in CTRL medium; n = 8–12 from three experiments (H9-derived hCOs). MLC2v expression is relative to control serum-free conditions (5.5 mM glucose, no palmitate). (J) TGFβ-1 is detrimental to hCO function. Force heat map in response to TGFβ-1 concentration (0, 1, 3, and 10 ng/mL) and duration (days 2–5, 2–7, and 2–9). Force was assessed after 10 d of serum-free culture after 2 d of hCO formation in CTRL medium; n = 13–16 from three experiments (H9-derived hCOs). (K) Increased collagen I and Matrigel improve tissue viability. Tissue viability was assessed after 10 d of serum-free culture following 2 d of hCO formation in CTRL medium. n = 18 from three experiments (H9-derived hCOs). Low Col = 1.6 mg/mL collagen matrix, High Col = 3.2 mg/mL collagen matrix, High Col + MG = 2.6 mg/mL collagen plus 9% (vol/vol) Matrigel matrix. SF, Serum -free medium (1 mM Glucose, 10 μM Palmitate with insulin). (L) Influence of the amount and composition of ECM on the force of hCO. Tissue viability was assessed after 10 d of serum free culture after 2 d of hCO formation in CTRL medium; n = 6–15 from three experiments (H9-derived hCOs). Data are mean ± SEM. High Col, 3.2 mg/mL collagen matrix; High Col + MG, 2.6 mg/mL collagen plus 9% (vol/vol) Matrigel matrix; Low Col, 1.6 mg/mL collagen matrix; SF, serum-free medium (1 mM glucose, 10 μM palmitate with insulin). *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001, one-way ANOVA plus Dunnet’s posttest relative to baseline or 0.2 mM Ca2+ (B and C); t test (D) with Dunnet’s posttest relative to no TGFβ-1 (J); or using ANOVA comparing either CTRL medium or SF groups only with Tukey’s posttest (L).