Human Peripheral Blood Monocyte Cells Induced To Osteoclast-like Cells In Vitro And Identification

Object: The study is to find a feasibility method of obtaining human osteoclast-like cells for the purpose of further research.Method: Mononucleated cells were isolated by means of density gradient centrifugation from 20ml peripheral blood which collected from healthy normal donor. The mononucleated cells were planted on the glass coverships or bone slices and cocultured whith UMR 106 cell line, DMEM which contented 1,25 dihydroxyvitamin D3, M-CSF and dexamethasone was used as the culture medium. The control group used the same culture medium but did not co-culture with UMR 106. The culture medium was replaced every 3 or 4 days with fresh medium supplemented with cytokines and hormones. After culture for 20 days, the cells on glass coverships were fixed and stained for tartrate resistant acid phosphatase (TRAP). TRAP-positive cells containing more than three nuclei were counted as osteoclast-like cells. The cells on the bone slices were identified with toluidine blue stain and electrical microscope scan.Result: Most of the peripheral blood cells adhereced to the wall when cultured to the third days. At the 7th days, the peripheral blood monocyte cells stretched, the shape of some cells became multi-angle, anachromasis granula appeared in some cells. The changes of the shape did not relative with the appearance of anachromasis granula. At the 10th days, most of the peripheral blood cells had not change than before in the trial group, but in the cells which had had changes that we mentioned before, the volume enlarged obviously, and the granula increased obviously too, a few cells had more than one nuclear, In the control group, the same phenomenons were not found besides the shape changing in a few cells. At the 13th days, the volume and the number of granula increased further than before in the cells which had had changes in the trial group, the other cells had no changes neither in the shape nor in the granula number . There were no changes in the control group. At the 20th days, the cells which had changes shown TRAP-positive. Bone absorb pits were found in the bone slice in the investigation of toluidine blue stain and electrical microscope scan, polynuclear macrophage with pseudopodium were found in electric microscope scan, In control group, we did not found the same result.Conclusion: Our study showed that about 3% mononucleated cells of human peripheral blood could be induced to osteoclast-like cells which have the ability of bone resorption and show TRAP-positive when coculture with UMR106 in the presence of 1,25 dihydroxyvitamin D3, macrophage-colony stimulating factor(M-CSF) and dexamethasone.