Piao Zhang

BMSCs from SD rats (4 weeks,60-80g) were isolated and harvested as described previously[11].In brief, bone marrow tissues were acquired by flushing the cavities of femurs and tibias using a syringe and 22-gauge needle into culture medium (Dulbecco’s modified Eagle’s medium, Gibco, Carlsbad, CA, USA ; 10% fetal bovine serum, Hyclone, Logan, UT, USA; 2 mM L-glutamine; 10,000 U/L penicillin, and10 mg/L streptomycin, Gibco BRL, Life Technologies, Paisley, United Kingdom). The whole volume of the flushing fluid was made into the single-cell suspension and seeded into 15 ml culture flasks with culture medium. Cells were cultured at 37°C in a humidified environment with 5% CO2. Non adherent cells were removed 24 hours later, and adherent cell colonies were washed three times with phosphate-buffered saline solution. Fresh complete medium was added and changed every 3-4 days. Cells were subcultured 1:2 or 1:4 when they reached 80-90% confluence. Cells used in this experiment were all harvested from three passages. Cell surface markers CD44 were detected by cell immunohistochemistry for identification of BMSCs.

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