Isolation and Characterization of Rat Bone Marrow Stromal Cells

Bone marrow tissues were isolated from the rats’ tibia and femoral bones according to previously described procedures [3 (link)]. Briefly, animals were euthanized by intraperitoneal injection of ketamine (100 mg/mL) and xylazine (100 mg/mL) (both from Ilium Troy Laboratory, Blacktown, Australia). The central canal of the bone was flushed with 2 mL of Dulbecco Modified Eagle’s Media (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (pen/strep) (all from Gibco, Life Technologies, Carlsbad, CA, USA) to extrude the marrow tissue. Mononuclear cells were purified using Ficoll-Paque PREMIUM solution (GE Healthcare Bioscience, Uppsala, Sweden) and the extracted cells were cultured on T25 cm² flasks in a 37 °C humidified chamber with 5% CO₂. After 24 h, the non-adherent cells were removed and the attached cells were allowed to grow. Complete media for BMSC proliferation (DMEM + 20% FBS + 1% pen/strep + 1% nonessential amino acids) was used to maintain the cells. The cells were subcultured until passage 3 (P3) and were then characterized by Fluorescein isothicyanate (FITC)-conjugated CD90 mouse monoclonal antibody (1:200; Thermo Scientific, Waltham, MA, USA), a surface marker for BMSCs. Translineage differentiation was conducted using fourth generation (P4) of cells.