Isolation and Expansion of Rat Bone Marrow-Derived Mesenchymal Stem Cells

Rat BM-MSCs were isolated from the tibias and femurs of donner male rats under sterile aseptic condition. Briefly, after euthanasia, long bones were dissected free of soft tissues, shaved by surgical blades, and cut open at both ends with scissors. Then, bone marrow was harvested by flushing out from the bone cavity with DMEM (Lonza, Basel, Switzerland) using a 22-gauge syringe. Bone marrow was vigorously pipetted repeatedly to produce a single-cell suspension, followed by filtration through a 70 μm nylon mesh filter. After being centrifuged at 1800 rpm for 10 min, the supernatant of the single-cell suspension was discarded. Cell pellets were suspended with complete media and cultured in T25 flasks (CELLSTAR-Greiner bio-one) containing complete media; composed of 89% DMEM, 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin (PS) (1:1) (Lonza, Verviers, Belgium). The medium was refreshed every 2–3 days. All cultures were incubated at 37 °C in a 5% CO₂ under humidified environment. The non-adherent cells were removed by changing the culture medium. When the adherent cells grew to 85–95% confluence, the culture was trypsinized by 0.25% trypsin/EDTA (Gibco, Grand Island, NY, USA) and expanded through three passages (P1–P2). Cells of P2 were used for characterization and transplantation [53 (link)].