B 27 supplement minus insulin

Hep-Orgs used in this study were previously generated from human fetal livers that were obtained from Leiden University Medical Centre (MC) under the approval of the Dutch Ethical Medical Council (Leiden University MC) (Hu et al., 2018 (link); Artegiani et al., 2020 (link)). All organoids were grown in Cultrex reduced growth factor basement membrane extract (BME), type 2 (#3533-001; R&D Systems). Culturing medium was refreshed every 2 to 3 days, and organoids were split 1:6 or 1:8 every 7 days as previously described for Chol-Orgs (Broutier et al., 2016 (link)) and Hep-Orgs (Hu et al., 2018 (link)). Expansion culture medium components are listed in Table S1. Differentiation medium was prepared by adding 1 µM dexamethasone (#D4902; Sigma-Aldrich) and 10 ng/μl oncostatin M (#295-OM; R&D Systems) to Hep-Org expansion medium as described previously (Hu et al., 2018 (link)). In experiments testing the effect of insulin, insulin (#I9278; Sigma-Aldrich) was added for 1 day to Hep-Org expansion medium with a final concentration of 20 µg/ml, and medium without insulin was added for 7 days and was made by replacing the B-27 Supplement minus vitamin A of the expansion medium by B-27 Supplement minus insulin (#A1895601; Gibco). Since B-27 Supplement minus insulin contains vitamin A, B-27 Supplement (#17504044; Gibco) was used as a control in these experiments.

In this study, human induced pluripotent stem cells (hiPSC) expressing GCaMP (kindly gifted by Prof. Bruce Conklin, Gladstone Institute, USA) were cultured on Matrigel coated plates with mTeSR‐1 medium (StemCell Technologies). hiPSC were passaged by using 0.5 × 10⁻³m ethylenediaminetetraacetic acid (Gibco) every 4–5 days. Differentiation of hiPSCs to cardiomyocytes was based on a previously described protocol.^[68] Briefly, at 80–90% confluence, the medium was exchanged with a differentiation medium composed of RPMI‐1640, 2% B27 supplement minus insulin (ThermoFisher Scientific), 1% penicillin/streptomycin, and supplemented with 6 × 10⁻⁶m CHIR99021 (Stemgent) for 2 days. Medium was then exchanged into RPMI/B27 supplemented with 2 × 10⁻⁶m Wnt‐C59 (Selleck Chemicals) for another 2 days. After the 5th day, cells were cultured with RPMI/B27 medium alone. Cardiomyocytes (after 10–14 days of differentiation) were enzymatically dissociated using TrypLE express (Gibco). For bioprinting experiments, 8 million cardiomyocytes were suspended in 1 mL fibrin bioink.