Abstract: BACKGROUND: There is still controversy whether human bone marrow mesenchymal stem cells can maintain their biological characteristics, energy metabolism patterns, and multidirectional differentiation potential after long-term expression in vitro. Further comprehensive and systematic research is needed. 
OBJECTIVE: To evaluate the effects of long-term expansion in vitro on the biological characteristics of mesenchymal stem cells. 
METHODS: Human bone marrow mesenchymal stem cells cultured to passage 5, 10, and 15 in vitro. MTT assay was used to detect cell proliferation ability. Flow cytometry was used to detect cell cycle. The multi-differentiation potential of mesenchymal stem cells was detected by inducing to adipogenic, osteogenic and chondrogenic differentiation. Cell migration and invasion abilities were detected by scratch test and Transwell assay. The mitochondrial oxidative phosphorylation and glycolysis function were analyzed using energy metabolism analyzer. The cell senescence was detected by senescence-associated β-galactosidase staining. The expression levels of p21, p16, and p53 proteins were detected by western blot assay. 
RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cells at passages 5, 10, and 15 grew adherently; the volume of passage 15 mesenchymal stem cells increased and its proliferation ability decreased; the percentage of S-phase cells decreased (P < 0.05). With the increase of culture passages, the migration and invasion abilities decreased gradually (P < 0.05). There was no significant difference in the differentiation potential, demonstrated by adipogenic, osteogenic and chondrogenesis induction. The ability of oxidative phosphorylation of mitochondria and glycolysis decreased gradually (P < 0.05). The number of senescence-associated β-galactosidase-positive cells increased with the increase of passages (P < 0.05), and the expression of senescence protein p21, p16, and p53 increased gradually (P < 0.05). The results indicated that the biological characterization of mesenchymal stem cells changed after long-term in vitro expansion. Mesenchymal stem cells cultured over 10 passages may have a reduced activity due to increasing senescence. Therefore, bone marrow mesenchymal stem cells cultured less than 10 passages are suitable for clinical research/therapy.

Key words: bone marrow mesenchymal stem cell, passage, long-term culture, energy metabolism, senescence