Background and aim: Mesenchymal stem cells are multipotent with self-renewal potential that isolated from different tissues. The appropriate and efficient method of cultivating these cells may be important for various researches. Methods: Rat bone marrow was harvested using 5-ml syringe and flashed with low glucose DMEM medium containing Penicillin/Streptomycin (100u/ml)/(0/1mg/ml). Cells were collected and washed three times with PBS and added to the flask containing DMEM-low glucose medium with 15 and 20% FBS. Cell medium was replaced three times every 12 hours. Results: According to this culture method, the cells attached to the flask in a fibroblastic-like after three days and filling about 60% of the flask surface. After one week, the density of mesenchymal stem cells reached 80-90%. The cells were passaged at 80% confulancy after three days. The first passage of mesenchymal stem cells was frozen with 10% dimethyl sulfoxide. Conclusion: The method of multi-passages mesenchymal stem cell culture can solve the problems that include the long time to reach the desired density in-vitro proliferation and maintenance of these cells.
