H9c2 cell culture model.

H9c2 cardiomyoblasts were purchased from the American Type Culture Collection and maintained in cell culture flasks. Once ~80% confluency was reached, cells were cultured in 100-mm² tissue culture plates (5 × 10⁵ cells/plate; passage number 8–9) and in 8-chamber polystyrene vessel tissue culture glass slides (10,000 cells/chamber; Falcon) using DMEM supplemented with 10% FBS, glutamine, NaP, and P/S for 48 h. Cells were assigned into six different study groups: control (only medium), ES-Exos (baseline control), MEF-Exos (baseline control), Dox (2-µM concentration, 24-h incubation), Dox + ES-Exos (10-µg concentration, 24-h incubation), and Dox + MEF-Exos (10-µg concentration, 24-h incubation; used as a cell line control). Cells were treated with a 2-µM concentration of Dox (Sigma) as reported by other investigators (45, 60, 66, 75) for 24 h to induce cell death. Cells were cultured for an additional 24 h, followed by replacing medium with fresh cell culture medium and or medium containing ES-Exos or MEF-Exos in different groups (79).