2.3.1. hMDSC isolation and expantion

Adult human skeletal muscle tissue samples were obtained during anterior cruciate ligament reconstruction procedure performed on a 53-year-old male, after informed consent for the biopsy material to be used in laboratory research. The ethical consent was approved by the Regional Medical Ethics Committee (No. BE-2-22). The samples were processed within a few hours after surgery and hMDSCs were isolated by strictly following the previously reported pre-plate technique [28]. In the last cell fraction, the cells that were slowest to adhere to the collagen-coated flasks, i.e. pre-plate 6 (pp6), were expanded up to 6–10 passages and used for further differentiation experiments. The cells were cultivated in a monolayer on a collagen-coated surface using Dulbecco's modified Eagle's medium (DMEM) with 4.5 g glucose/L (Gibco), supplemented with 10% fetal bovine serum (FBS) (Gibco), 10% horse serum (HS) (ThermoFisher Scientific), 0.5% chicken embryo extract (CEE) (Life Science Production), and 1% penicillin/streptomycin (P/S (10,000 U/L) (Gibco)) at 37°C, 5% CO₂, in a humidified Binder C150 incubator, changing the culture medium every 2–3 days.

For visualization and live cell staining, the cells on hydrogels were incubated with Calcein AM reagent (Invitrogen). Dye was diluted to a final 5 μM working concentration with serum free DMEM and incubated with cells for 90 min. Images were recorded using an Olympus BX 51 upright microscope.