Abstract: BACKGROUND: Using conditioned medium and human pulp stem cells, we will initially explore methods to rapidly proliferate human dental pulp stem cells to provide a research basis for future cell treatment and expansion of high-quality seed cells.  
OBJECTIVE: To preliminarily explore the effect of conditioned medium from different sources on the proliferation of human dental pulp stem cells.   
METHODS: Human dental pulp stem cells were isolated in vitro and identified by flow cytometry. Fetal bovine serum ultra-high-speed cryogenic centrifugal products, urine ultra-high-speed cryogenic centrifugal products of fracture patients, in vitro culture supernatants of bone marrow-derived dendritic cells, human skin fibroblast cultures and low-glucose DMEM medium were configured into conditioned medium. After co-culture with human pulp stem cell cells, images of cell growth in each group were taken continuously for 72 hours using a cell label-free culture observation device (BioStation-T). The cells were dynamically monitored using a real-time label-free cell analyzer for 150 hours. Differences in normalized cell index values were compared between each group of cells.
RESULTS AND CONCLUSION: (1) Human dental pulp stem cells are typical of mesenchymal stem cells. Expression of the mesenchymal stem cell surface markers CD90 and CD105 was found, but CD34 and CD45 expression was not observed. (2) Cell label-free culture observation device when dynamically monitoring found that the absorption time in the group was significantly earlier than in other experimental and blank control groups. (3) When detected on a real-time label-free cell analyzer, compared with the blank control group, the normalized cell index values of the fetal bovine serum ultra-high-speed cryogenic centrifugal group, the bone marrow-derived dendritic cell in vitro culture supernatant group, and the human skin fibroblast culture supernatant group were significantly increased (P < 0.001). The normalized cell index value of fracture patients of the urine ultra-high-speed cryogenic centrifugal group was significantly decreased (P < 0.001). (4) The results show that the conditioned medium configured by ultra-high-speed centrifugation, in vitro culture supernatant of bone marrow-derived dendritic cells, and human skin fibroblasts can promote the proliferation of human dental pulp stem cells. Among them, the conditioned medium configured by the fetal bovine serum ultra-high-speed cryogenic centrifugal products also had some protective effects on the cells. 

Key words: human dental pulp stem cell, ultra-high-speed cryogenic centrifugation, conditioned medium, cell proliferation, normalized cell index, tissue culture method