Human primary osteoclasts: in vitro generation and applications as pharmacological and clinical assay

Journal of Translational Medicine

Rainer Gamse¹  Natasa Zamurovic¹  David Cappellen¹  Ngoc-Hong Luong-Nguyen¹  Mira Susa¹ 

[1] Arthritis and Bone Metabolism Disease Area, Novartis Institutes for BioMedical Research Basel, Novartis Pharma AG, CH-4002 Basel, Switzerland

关键词: estrogen;    cathepsin K;    bisphosphonate;    screening;    osteoporosis;    method;    osteoclast;   

Others  :  1208556
DOI  :  10.1186/1479-5876-2-6

 received in 2004-02-02, accepted in 2004-03-16,  发布年份 2004

【 摘 要 】

Osteoclasts are cells of hematopoietic origin with a unique property of dissolving bone; their inhibition is a principle for treatment of diseases of bone loss. Protocols for generation of human osteoclasts in vitro have been described, but they often result in cells of low activity, raising questions on cell phenotype and suitability of such assays for screening of bone resorption inhibitors. Here we describe an optimized protocol for the production of stable amounts of highly active human osteoclasts. Mononuclear cells were isolated from human peripheral blood by density centrifugation, seeded at 600,000 cells per 96-well and cultured for 17 days in α-MEM medium, supplemented with 10% of selected fetal calf serum, 1 μM dexamethasone and a mix of macrophage-colony stimulating factor (M-CSF, 25 ng/ml), receptor activator of NFκB ligand (RANKL, 50 ng/ml), and transforming growth factor-β1 (TGF-β1, 5 ng/ml). Thus, in addition to widely recognized osteoclast-generating factors M-CSF and RANKL, other medium supplements and lengthy culture times were necessary. This assay reliably detected inhibition of osteoclast formation (multinucleated cells positive for tartrate-resistant acid phosphatase) and activity (resorbed area and collagen fragments released from bone slices) in dose response curves with several classes of bone resorption inhibitors. Therefore, this assay can be applied for monitoring bone-resorbing activity of novel drugs and as an clinical test for determining the capacity of blood cells to generate bone-resorbing osteoclasts. Isolation of large quantities of active human osteoclast mRNA and protein is also made possible by this assay.

【 授权许可】

   
2004 Susa et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.

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