Pluronic f68 solution

Human adenocarcinoma alveolar basal epithelial (A549) cells (ATCC, VA, USA) and Madin-Darby Canine Kidney (MDCK) cells (ATCC, VA, USA) were maintained at 37°C at 5% CO₂ in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), 100U/mL penicillin-streptomycin (Gibco), 2mM GlutaMAX supplement (ThermoFisher). Expi293 cells (Gibco) were grown in Expi293 Expression Medium (ThermoFisher) at 37°C at 8% CO₂ with regular agitation (150RPM). Trichoplusia ni High Five (ATCC) insect cells were maintained in HyClone SFX insect cell culture media (Cytiva/Fisher Scientific) at 28°C with regular agitation (70RPM). Spodoptera frugiperda Sf9 insect cells (ATCC) were maintained as adherent cultures in Roux flasks in full TNM-FH insect cell media (Sigma) supplemented with 10% FBS, 1% penicillin-streptomycin (Gibco) and 0.1% Pluronic F68 solution (Gibco) at 27°C.

Petri dishes (BioLite Cell Culture Treated Dishes, Thermo Scientific) were coated with 5% pluronic F-68 solution (Gibco) and incubated overnight at 4 °C; then, the pluronic solution was removed, and a sterile, modified Delrin frame (1 cm $\times$ 2 cm) was secured to the dish with 2% agarose. hCMPs were assembled in three layers over a three-day period (one layer per day). Each layer was generated by depositing 600 μL of an hiPSC-CM–containing fibrin solution (Gao et al., 2018 (link); Pretorius et al., 2020 (link), 2021 (link)) (10 × 10⁶ cells/mL) into the frame (Pretorius et al., 2020 (link), 2021 (link)); then, after fibrin polymerization, the layer was cultured in STEMdiff Cardiomyocyte Support Medium with 2 mg/mL ε-aminocaproic acid overnight at 37 °C and 5% CO₂; the procedure was repeated twice to form the second and third layers. After the third cell layer was generated, the frame containing the engineered tissue was lifted off of the petri dish and placed on a custom-cut polydimethylsiloxane (PDMS) platform; then, the hCMP was cultured in fresh culture medium consisting of 2% fetal bovine serum (FBS), 2% B27⁺ (Gibco), and 2 mg/mL ε-aminocaproic acid in RPMI (Gibco) for one week before the maturation protocols were initiated.