Isolation and Expansion of Human Wharton's Jelly Mesenchymal Stem Cells

Human WJ-MSCs were isolated from umbilical cords of 3 healthy donor mothers after informed consent as previously described [67 (link)]. The study was approved by the Local Ethical Committee of Bologna (IRCCS St. Orsola-Malpighi University Hospital Ethical Committee, protocol n° 2481/2017, ref n° 68/2017/U/Tess). Cells were cultured in Dulbecco’s Modified Eagle’s Medium—1 g/L of glucose (L-DMEM; Corning Incorporated, Corning, NY, USA) supplemented with 10% Fetal Bovine Serum (FBS; Gibco, Waltham, MA, USA) and antibiotics (1% Penicillin-Streptomycin Solution; Thermo-Fisher Scientific, Waltham, MA, USA) and were maintained in standard culture conditions at 37 °C with 5% carbon dioxide (CO₂) in a humidified atmosphere. The non-adherent cells were removed, and medium was changed twice a week. When cells reached 80% confluence, they were detached using a trypsin-EDTA solution (Sigma-Aldrich Co., St. Louis, MO, USA) and were seeded for maintenance and/or expansion. For experiments, cells were seeded at a density of 5000 cells/cm² (except where otherwise noted) in adequate plastic support (Corning Incorporated, Corning, NY, USA) and incubated in standard condition for 24 h before treatment. Experiments were performed using hWJ-MSCs at the 3rd–6th culture passages and were repeated in biological triplicate (n = 3).