Bone marrow-derived mesenchymal stem cells (Third Military Medical University)

Bone marrow (30 mL) was obtained from the iliac crest and subjected to Percoll (1.073 g/mL) density gradient centrifugation. Then, the mononuclear cell layer was harvested and cultured in flasks containing alpha-modified minimum essential medium (α-MEM) supplemented with 10% autologous serum. On day 1, non-adherent cells were discarded, and adherent cells were washed with phosphate-buffered saline and then cultured in α-MEM with 10% autologous serum. The medium was replaced every 2–3 days. At approximately 70–80% confluence, the cells were resuspended in trypsin–EDTA and the cell density was adjusted to approximately 4000 cells per cm2. The cells were passaged once every 4–5 days.