Gibco rpmi

For maintenance, HiTCA and non-gene edited wild type (WT) SC cells were cultured in media composed of 88% Gibco RPMI (Thermo Fisher Scientific, Waltham, MA, USA), 10% FBS, 1% Sodium Pyruvate (100 mM), and 1% Pen-Strep antibiotic. The cell cultures were propagated in 25 cm² canted neck cell flasks with vented caps at a density between 2.0 × 10⁶ and 2.0 × 10⁷ cells/mL. The flasks were incubated at 37 °C and 5% CO₂. While maintaining the number of cells in culture, they were passaged every 2–3 days by decanting half of the culture media and adding an equal volume of fresh culture media. Cells were tested periodically for viability with trypan blue and counted with a Bio-Rad TC20 Cell Counter (Bio-Rad Laboratories, Hercules, CA, USA) to ensure viability over 90%.

Blood samples (2 tubes of 10 mL) from patients were collected at different times after myocardial infarction: at the time of angiography (H0), and 4 h (H4), 24 h (H24), 48 h (H48), 1 month (M1) after reperfusion for the Hibiscus-STEMI cohort, and 3 days (D3), 6 months (M6), 12 months (M12) after reperfusion for the CARIM cohort. Blood samples from healthy donors were obtained by the Etablissement Français du Sang (French blood transfusion agency).
Even if collected after AMI, PBMC cultured without stimulation did not secrete the cytokines of interest (IL-1β, IL-6, IL10, IFN-γ, TNF-α). Cells were then stimulated using phorbol myristate acetate (PMA) and ionomycin. Fresh PBMC were deposited in 2 wells of a 96-well plate (2 × 10⁵ PBMC/well) with 200µL of Gibco™ RPMI (Roswell Park Memorial Institute medium, ThermoFisher Scientific, Waltham, MA, USA) complete medium (RPMI with 10% FBS, and 1% penicillin–streptomycin solution), and incubated with (condition well) or without (control well) PMA and ionomycin at a final concentration of 50 ng/mL and 1 µg/mL respectively. Supernatants were collected after overnight incubation and stored at − 20 °C until use.
Plasma was collected after tubes centrifugation (800 g for 12 min) and PBMC using Lymphosep™ (Lymphocyte separation medium, Dutscher, Belgium), and frozen in 90% fetal bovine serum (FBS) and 10% DMSO at − 80 °C after isolation.

Cell cultures were performed using primary human cardiac fibroblasts (HCF) (HCF-av, ScienCell Research Laboratories, San Diego, CA, USA) (3 to 5 passages) and PBMC collected from healthy donors and from patients at different times after myocardial infarction (H0, H24, H48, M1). According to the manufacturer’s instructions, HCF were cultured with complete medium (Fibroblast Medium 2 (FM-2) with 5% FBS, 1% Fibroblast Growth Supplement-2 (FGS-2), and 1% penicillin–streptomycin solution, ScienCell Research Laboratories, San Diego, CA, USA) in a 37 °C, 5% CO₂ atmosphere.
The first day, 3 × 10⁵ HCF were seeded in a 6-well plate and grown in FM-2 complete medium. PBMC were thawed and cultured with Gibco™ RPMI (Roswell Park Memorial Institute medium, ThermoFisher Scientific, Waltham, MA, USA) complete medium (RPMI with 10% FBS, and 1% penicillin–streptomycin solution) in a separate 6-well plate during 24 h in the incubator. The medium of HCF was then replaced by DMEM complete medium, and inserts with 0.4 µm pores were placed in each well. 1.5 × 10⁶ PBMC were distributed in the upper chamber of inserts. For each insert containing PBMC (“PBMC well”), a “control well” (insert without PBMC) was performed. After 24 h of coculture, supernatants were collected and stored at − 20 °C, and fresh HCF were stained and analyzed by flow cytometry.