Optimization of Isolation and Cultivation of Rat Bone Marrow Mesenchymal Stem Cells

Objective:To establish and optimize the condition of isolation and cultivation of rat bone marrow mesenchymal stem cells(MSCs),in order to acquire pure MSCs with good differential capability.Methods:Bone marrow cells were collected from rats of different age.Bone marrow mononuclear cells were isolated by density gradient centrifugation under different concentration of Percoll. Using 60% low-glucose DMEM/40%MCDB201 as basic culture medium,the suspending cells were discarded after 24h incubation. MSCs were then subcultured with different inoculation density,and the osteogenic differentiation and adipogenic differentiation of MSCs were investigated by alkaline phosphatase staining and oil red staining respectively.Results:The isolation effect under percoll of 57% was better than under percoll of 70%.The quality and quantity of the isolated bone marrow cells could reach the highest by using rats of 6 weeks(weighed about 180g).Discarding the suspended cells after 24h incubation and inoculating the cells with the cell density of 5×10~3/cm~2 were helpful to acquire MSCs with strong reproductive capability and active function.Experiments of osteogenic differentiation and adipogenic differentiation revealed that MSCs remained well pluripotential differentiation potentials.Conclusion:The optimization of rats' age,the density of percoll,the conditions and methods of cell cultivation conduced to obtaining pure MSCs with good pluripoten- tial differentiation potentials.