In vitro cultivation of bone marrow mesenchym stem cells.

Objective To establish a feasible method which separate and purify bone marrow mesenchymal stem cells(MSCs)and provide cell source for cell engineering science.Methods ①Separation and culture of MSCs:Myeloid cells of limbs bone were collected in Wistar male rat at the age of 3～4 weeks in asepsisc condition.MSCs were separated by splitting red cells with adherent culture.The adherent difference between MSCs and other adherent cells was used to purify MSCs by strictly controlling the quantity of enzyme and the time of digestion during the subculture.②ldentification of MSCs:Passage 4 MSCs were detected surface antigen CD44,CD45 and CD90 by immunofluorescence stainting.Results ①The bone marrow monocytes separated by splitting red cells with adherent culture had more shape,nonuniform distribution,in which there were also many mixed cells among them.After many times of replacing the liquid and passage,the cellular purity was increased,the cells had identical shape,uniform distribution,and the floating cells and mixed cells were rarely observed after passage 4;②The result of immunofluorescence stainting show that positive expression emerged on CD44 and CD90.and negative expression on CD45.Conclusion split red cells culture combined with adherent culture method to separate and purify MSCs can lead to MSCs with preferrable activity and purity.