**2. Materials and methods**

#### **2.1 Preparation and culture of MSCs**

For isolation of rat MSCs; female Sprague-Dawley rats (weighing 200-250 g) with the approval from the Institute for Animal Care were obtained from the Animal Center, Faculty of Medicine, Guilan University of Medical Sciences. Rats were killed by intraperitoneal administration of a lethal dose of sodium pentobarbital. The femurs and tibias were carefully dissected away from attached soft tissue as previously reported with modification (1). The ends of the bones were cut, and the bone marrow was aseptically extruded with 5 ml PBS solution by using a syringe with a 21G needle and flushing the shaft ten times. The marrow tissue was dissociated by pipetting. The cell suspension was then centrifuged at 500 × g for 5 minutes and the supernatant was discarded. Bone marrow mesenchymal stem cells (BMSCs) were then mechanically dispersed into a single-cell suspension so that the density of BMSCs reached 106 cells/ml. At this point, marrow cells were plated in a 25 cm² plastic flask in Dulbecco's modified eagle medium (DMEM) containing 20% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin. All cells were incubated at 37 °C, in an atmosphere of 5 % humidified CO2. After 48 hours incubation, the nonadherent cell populations were removed and the medium was added and replaced every three or four days for about two weeks. When the cells grew to 80% confluency they were harvested with 0.25% trypsin and 1 mM EDTA (Gibco, UK) for 5 minutes at 37°C, replated and diluted 1:3 on a 25 cm² plastic flask, again cultured to the next confluency and harvested. Prior to their use in inducing differentiation and vitrification MSCs that were passaged approximatly 15 times were morphologically evaluated.
