C57BL/6 mouse bone marrow monocytes: isolation, cultivation, purification and differentiation into osteoclasts

doi:10.3969/j.issn.2095-4344.2015.06.021

Abstract

Abstract:

BACKGROUND: Isolation and extraction of bone marrow monocytes is mostly realized via osteoclast differentiation induced by RAW264.7 cells as reported at home and abroad.
OBJECTIVE: To explore a new method for the isolation, culture, purification and identification of C57BL/6 mouse bone marrow monocytes and to observe the growth features and osteoclast differentiation of mouse bone marrow monocytes in vitro.
METHODS: The femurs and tibias of C57BL/6 mice were aseptically removed to extract bone marrow cells that were cultured overnight (> 16 hours). During the extraction process, erythrocyte lysate was used to remove red blood cells. At day 2, suspension cells were isolated and cultured with the medium containing macrophage colony-stimulating factor to harvest adhered mouse bone marrow monocytes. The medium was changed to purify and amplify the mouse bone marrow monocytes. Cell morphology was observed, the growth curve was tested and using flow cytometry, the cell surface antigen of primary mouse bone marrow monocytes was detected. Macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand were added to
differentiate the cells into osteoclasts.
RESULTS AND CONCLUSION: The newly separated mouse bone marrow monocytes were circular and feelers were extended from both ends. After 5 days of culture, the cells were oval-shaped with more obvious feelers, and cell proliferation relied on macrophage colony-stimulating factor. Under the flow cytometry, isolated mouse bone marrow monocytes had high purity and could be induced to differentiate into osteoclasts. It is effective to isolate mouse bone marrow monocytes that grow stably by vigorously promoting the proliferation of monocytes by using different adherent abilities of mesenchymal stem cells and monocytes as well as macrophage colony-stimulating factor. Cultured mouse bone marrow monocytes are phenotypically stable and can be used for further research.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: Monocytes, Osteoclasts, Macrophage Colony-Stimulating Factor, RANK Ligand

CLC Number:

R394.2

Zhou Long, Chen Xi, Luo Zong-ping, Yang Hui-lin, He Fan. C57BL/6 mouse bone marrow monocytes: isolation, cultivation, purification and differentiation into osteoclasts[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(6): 940-944.

Figures/Tables 4

References

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