DMEM, no glucose, no glutamine, no phenol red

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hCO Fabrication. CTRL medium: α-MEM GlutaMAX (ThermoFisher Scientific), 10% fetal bovine serum (FBS) (ThermoFisher Scientific), 200 μM l-ascorbic acid 2 phosphate sesquimagnesium salt hydrate (Sigma) and 1% Penicillin/Streptomycin (ThermoFisher Scientific). For each hCO, 5 × 104 cardiac cells in CTRL medium were mixed with collagen I to make a 3.5-μl final solution containing 2.6 mg/ml collagen I and 9% Matrigel. The bovine acid-solubilized collagen I (Devro) was first salt-balanced and pH-neutralized using 10X DMEM and 0.1 M NaOH, respectively, prior to mixing with Matrigel and cells. The mixture was prepared on ice and pipetted into the Heart-Dyno. The Heart-Dyno was then centrifuged at 100 × g for 10 s to ensure the hCO form halfway up the posts. The mixture was then gelled at 37°C for 30 min prior to the addition of CTRL medium to cover the tissues (150 μl/hCO). The Heart-Dyno design facilitates the self-formation of tissues around in-built PDMS exercise poles (designed to deform ∼0.07 μm/μN). The medium was changed every 2-3 days (150 μl/hCO). hCOs were cultured in CTRL medium for formation and then changed to serum-free media as indicated for experiments. For all screening experiments, after hCO formation, hCOs were cultured in serum-free conditions comprising DMEM without glucose, glutamine, and phenol red (ThermoFisher Scientific) supplemented with 4% B27 (with or without insulin) (ThermoFisher Scientific), 1% GlutaMAX (ThermoFisher Scientific), 200 μM l-ascorbic acid 2 phosphate sesquimagnesium salt hydrate and 1% Penicillin/Streptomycin (ThermoFisher Scientific). Additions to the medium included glucose, palmitic acid (conjugated to bovine serum albumin within B27 by incubating for 2h at 37°C, Sigma), or TGFβ-1 (Peprotech). A timeline of the finalized hCO fabrication, culture and maturation protocol can be found in Fig. 1M.

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