Abstract:

Abstract:Objective
To establish a high-performance induction culture system for Raw264.7 cells to differentiate into osteoclasts（OC）　in vitro　 by improving the cell culture program.Methods The Raw264.7 cells were cultured with α-MEM medium containing 50 μg•L^-1 M-CSF,100 μg•L^-1 RANKL,and 1×10-8  mol•L-1  1α,25-（OH）2D₃  in 5% CO₂ for 12 d at 37℃. The cells were digested transiently every time before the medium was changed after every three days.The morphologic changes of the Raw264.7 cells were observed by  inverted microscope.The maturation and phagotrophic function of OC were identified by HE,TRAP,FITC-phalloidin staining and immunofluorescence.Results The cells remained to grow in single layers all the time in most areas of the well during the whole induction by the improved culture program.The observation results of inverted microscope and HE staining showed that the growth area of the polykaryotic OC reached to 70% of the well on day 12. FITC-phalloidin staining showed that in the maturation of the OC,the cluster-shaped podosomes in the pseudopodia gradually transformed into rings,which finally fused to form a large belt surrounding the periphery of the cytoplasm.The calcitionin receptor (CTR)  expressed by OC was markedly enhanced compared with the precursor cells by  immunofluroescence staining,and a large number of red granules appeared in the cytoplasm of OC with TRAP staining on day 12.  These results comfirmed that the obtained  OC were maturated and owned phagotrophic function.  Conclusion A high-performance induction culture system for Raw264.7 cells to differentiate into OC in vitro induced by combination of 50 μg•L^-1 M-CSF,100 μg•L^-1 RANKL, and 1×10^-8  mol•L^-1 1α,25-(OH)2D3 is established by improving the cell culture program.

Key words: Raw264.7 cell；osteoclast；macrophage colony-stimulating factor；receptor activator for nuclear factor-&kappa, B ligand； 1α,25-dihydroxyvitamin D3