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

Abstract

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.

Podosome organization drives osteoclast-mediated bone resorption

“…First, the resorption tracks made by OC cultured on bone, dentine, ivory, or hydroxyapatite slides consist of several individual resorption lacunae that partially overlap to collectively form what is referred to as trails of resorption pits. [125][126][127][128] Second, in vivo optical imaging of mature OCs has demonstrated the presence of two distinct states of the OC: motile and non-motile. 129 Based on non-overlapping data provided by the literature, these observations imply two completely independent states of OC functions.…”

Aptamer-functionalized lipid nanoparticles targeting osteoblasts as a novel RNA interference–based bone anabolic strategy

“…Rat pre-osteoclasts (B-Bridge International) and human osteoclasts (PriCells) were cultured in DMEM containing 10% FBS, 100 units ml −1 penicillin-streptomycin, macrophage colony-stimulating factor (M-CSF) and receptor for activation of nuclear factor kappa B ligand (RANKL). Phenotypes of osteoblasts and osteoclasts were confirmed by real-time PCR as described previously 28–31 . The primers are listed in Supplementary Table 3.…”

NFAM1 signaling enhances osteoclast formation and bone resorption activity in Paget's disease of bone

“…Human peripheral blood mononuclear cells (PBMC) were isolated as described [20]. All human samples were obtained following the IRB-approved protocol at the Medical University of South Carolina.…”