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Bone marrow derived mesenchymal stem cells (BM-MSCs) represent a promising source for cell therapy, As they can differentiate into bone, cartilage, fat, tendon and many other organ progenitor cells. Although the culture of MSCs has been studied for over 30 years. The identification of standard protocol for isolation and characterization have yet to be developed. A comparison study was made on three different isolation techniques, which include: direct plating, red blood lysis buffer strategies, and density gradient (DG) method (using two different gradient media: Ficoll and Percoll) to find the optimal isolation and culture condition for adequate amount of MSCs for clinical use.The results demonstrate that direct plating of whole bone marrow (BM) suspension provides a suitable alternative protocol for isolation of BM-MSCs with minimal requirements, which mainly based on the frequent medium change in primary culture. The BM crud cell suspension were isolated by aspiration from albino mice and cultured in fresh complete isolation media (CIM) RPMI-1640/10%FBS. Adherent cells from BM cells suspension were MSCs which then expanded by complete expansion media (CEM) α-MEM/ 10% FBS. By concluding, direct plating of whole bone marrow represent the alternative method for isolating of mesenchymal stem cells from small specimen of bone marrow. By concluding, the present study was designed to investigate the optimal technique for the isolation of BM-MSCs for use in tissue engineering and regenerative medicine.