Mesenchymal Stem Cells Isolated From Rat by Whole Bone Marrow Adherent Culture:An In Vitro Study

【Objective】To evaluate the biological properties of mesenchymal stem cells (MSC) isolated from rat by whole bone marrow adherent culture. 【Methods】 The MSCs were collected from bone marrow of a 4 weeks old male SD rat, and amplified using adherent culture. The growth and morphologic change of MSCs was dynamic observed by inverted phased fluorescence microscope. The passage 3 MSCs were used for experiment, and MSCs surface antigens were analyzed by flow cytometry. Cell viability was evaluated by the exclusion of trypan blue dye. Cell proliferation was measured by cell counting Kit-8 (CCK-8) assay and the growth curve was drawed. Hoechst 33258 staining were used to determine cell apoptosis. Multipotent capacity of MSCs was proven by inducing differentiation into adipocytes or osteocytes. 【Results】 Adherent cells appeared 24 h after innoculation. Nonadherent cells were removed by replacing the medium, and MSCs were effective purified by subculture. The MSCs were spindle-shaped or polygonal-shaped with irregular processes, arranged in spiral-shaped or fish-like. The passaged cells growth faster than the original cells. Flow cytometry revealed that the MSCs were rich in CD29 , CD44 and low in CD45, CD11b positivity. The MSCs had good cell viability, strong proliferative ability, a low apoptosis rate and capability of differentiation into osteogenic and adipogenic phenotypes. 【Conclusions】 Whole bone marrow adherent culture is a convenient and effective method to gain purified MSCs with strong proliferative ability, and capability of multi-directional differentiation.