Alpha mem

The experimental procedures followed the rules approved by the Ethics Committee of the Luigi Vanvitelli Campania University (n. 0029471/i). Patients were informed of the research and gave permission for the use of biological samples. Bone marrow aspirate samples were obtained from eight healthy donors (age 10–18 years). We separated cells on a Ficoll (GE Healthcare) density gradient and, after centrifugation at 400g for 30 min, the mononuclear cell fraction was collected. We seeded 1–2.5 × 10⁵ cells/cm² in complete medium composed by alpha‐MEM (Microgem) containing 10% FBS (EuroClone), 4 mM l‐glutamine (EuroClone), 100 U/ml penicillin–streptomycin (HiMedia) and 3 ng/ml FGF2 (Peprotech). The cells were then incubated at 37°C in a humidified atmosphere containing 5% CO₂. After 24 h the non‐adherent cells were discarded and the adherent cells at passage 0 (P0) were washed twice in PBS (Microgem) and re‐incubated in complete medium for 7–10 days until maximum confluence was reached. We used the minimal criteria suggested by the International Society for Cellular Therapy⁷ to identify Mesenchymal Stromal Cells (MSCs). The MSCs have been expanded up to passage 4 (P4). Subsequently, the cells were used to isolate MUSE cells and an aliquot was frozen in FBS with 10% DMSO (Sigma) in liquid nitrogen.

The experimental procedures followed the rules approved by the Ethics Committee of the Luigi Vanvitelli Campania University. Patients were informed of the research and gave permission for the use of biological samples. Bone marrow was obtained from three healthy donors. Cells were separated through Ficoll density gradient (GE Healthcare, Chicago, IL, USA), and the mononuclear cell fraction was collected and washed in phosphate-buffered saline solution (PBS, Microgem, Naples, Italy). We seeded 1 to 2.5 × 10⁵cells/cm² in alpha-minimum essential medium (alpha-MEM, Microgem) supplemented with 10% fetal bovine serum (FBS, Euroclone, Pero, Italy) and 1 ng/mL beta-fibroblast growth factor (β-FGF, Prepotech, London, UK). After 72 h, non-adherent cells were discarded, and adherent cells were further cultivated to reach confluency. We verified that, under our experimental conditions, the bone-marrow stromal cultures contained MSCs that fulfilled the three criteria proposed to define MSCs [35 (link)]. All experiments were conducted on MSC cultures at passage 3 when senescence phenomena were minimal [36 (link)].