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

Susa M, Luong-Nguyen NH, Cappellen D, Zamurovic N, Gamse R - J Transl Med (2004)

Bottom Line: 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 alpha-MEM medium, supplemented with 10% of selected fetal calf serum, 1 microM dexamethasone and a mix of macrophage-colony stimulating factor (M-CSF, 25 ng/ml), receptor activator of NFkappaB ligand (RANKL, 50 ng/ml), and transforming growth factor-beta1 (TGF-beta1, 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Arthritis and Bone Metabolism Disease Area, Novartis Institutes for BioMedical Research Basel, Novartis Pharma AG, CH-4002 Basel, Switzerland. mira.susa_spring@pharma.novartis.com

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 alpha-MEM medium, supplemented with 10% of selected fetal calf serum, 1 microM dexamethasone and a mix of macrophage-colony stimulating factor (M-CSF, 25 ng/ml), receptor activator of NFkappaB ligand (RANKL, 50 ng/ml), and transforming growth factor-beta1 (TGF-beta1, 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.

No MeSH data available.


Related in: MedlinePlus

The effects of several bone resorption inhibitors on human osteoclast and resorption pit morphology. Human osteoclasts were generated as described under Materials and Methods, with or without the presence of bone resorption inhibitors: zoledronic acid (Z.A., 50 nM), selective estrogen receptor modulator (SERM) raloxifene (Ral., 100 nM), non-specific cathepsin K inhibitor E-64 (100 nM), and selective cathepsin K inhibitor AAR494 (AAR, 100 nM). Microphotographs show TRAP-stained cell morphology at culture day 17 (left panels) and pit morphology on dentin (control, zoledronic acid and raloxifene) or bovine bone (E-64 and AAR494). C, control. The activities (IC50 values) of E-64 and AAR494 on cathepsin K, S and L were (E-64: 1.4; 4.1; 2.5 nM, and AAR494: 3.4; 3700; >10000 nM, respectively).
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Figure 4: The effects of several bone resorption inhibitors on human osteoclast and resorption pit morphology. Human osteoclasts were generated as described under Materials and Methods, with or without the presence of bone resorption inhibitors: zoledronic acid (Z.A., 50 nM), selective estrogen receptor modulator (SERM) raloxifene (Ral., 100 nM), non-specific cathepsin K inhibitor E-64 (100 nM), and selective cathepsin K inhibitor AAR494 (AAR, 100 nM). Microphotographs show TRAP-stained cell morphology at culture day 17 (left panels) and pit morphology on dentin (control, zoledronic acid and raloxifene) or bovine bone (E-64 and AAR494). C, control. The activities (IC50 values) of E-64 and AAR494 on cathepsin K, S and L were (E-64: 1.4; 4.1; 2.5 nM, and AAR494: 3.4; 3700; >10000 nM, respectively).

Mentions: The effects of several known bone resorption inhibitors were measured in the human osteoclast assay. We used representative compounds for bisphosphonates (zoledronic acid), selective estrogen receptor modulators (SERM, raloxifene) and cathepsin K inhibitors (E-64 and AAR494). These compounds have different mechanisms of action and it was interesting to evaluate whether the assay would detect these differences. The control osteoclast cultures at day 17 contained only TRAP-positive cells and were almost confluent (Fig. 4, TRAP). Osteoclasts were of various sizes: very large cells, smaller multinucleated cells, and small mononucleated cells (Fig. 4, TRAP, C). In the control cultures on bone slices, pits covered most of the bone slice surface and formed characteristic resorption tracks, resulting from osteoclast migration (Fig. 4, PIT, C). The bisphosphonate zoledronic acid (50 nM) and selective estrogen receptor modulator (SERM) raloxifene (100 nM) reduced osteoclast number, leaving the culture largely depleted from mononuclear and smaller multinuclear cells (Fig. 4, TRAP, Z.A., Ral.). At these concentrations both compounds partially inhibited pit formation (Fig. 4, PIT, Z.A., Ral.). Two cathepsin K inhibitors were tested, a non-selective cysteine protease inhibitor E-64 and a selective inhibitor AAR494. Both compounds did not affect osteoclast formation, but partially inhibited pit formation (Fig. 4, E-64, AAR). AAR494 in particular induced changes in cell morphology, evident as densely TRAP-stained regions within cells. In addition, the remaining pits after treatment with this compound did not have a track appearance, but were cluster-like, containing small round pits.


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

Susa M, Luong-Nguyen NH, Cappellen D, Zamurovic N, Gamse R - J Transl Med (2004)

The effects of several bone resorption inhibitors on human osteoclast and resorption pit morphology. Human osteoclasts were generated as described under Materials and Methods, with or without the presence of bone resorption inhibitors: zoledronic acid (Z.A., 50 nM), selective estrogen receptor modulator (SERM) raloxifene (Ral., 100 nM), non-specific cathepsin K inhibitor E-64 (100 nM), and selective cathepsin K inhibitor AAR494 (AAR, 100 nM). Microphotographs show TRAP-stained cell morphology at culture day 17 (left panels) and pit morphology on dentin (control, zoledronic acid and raloxifene) or bovine bone (E-64 and AAR494). C, control. The activities (IC50 values) of E-64 and AAR494 on cathepsin K, S and L were (E-64: 1.4; 4.1; 2.5 nM, and AAR494: 3.4; 3700; >10000 nM, respectively).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC394349&req=5

Figure 4: The effects of several bone resorption inhibitors on human osteoclast and resorption pit morphology. Human osteoclasts were generated as described under Materials and Methods, with or without the presence of bone resorption inhibitors: zoledronic acid (Z.A., 50 nM), selective estrogen receptor modulator (SERM) raloxifene (Ral., 100 nM), non-specific cathepsin K inhibitor E-64 (100 nM), and selective cathepsin K inhibitor AAR494 (AAR, 100 nM). Microphotographs show TRAP-stained cell morphology at culture day 17 (left panels) and pit morphology on dentin (control, zoledronic acid and raloxifene) or bovine bone (E-64 and AAR494). C, control. The activities (IC50 values) of E-64 and AAR494 on cathepsin K, S and L were (E-64: 1.4; 4.1; 2.5 nM, and AAR494: 3.4; 3700; >10000 nM, respectively).
Mentions: The effects of several known bone resorption inhibitors were measured in the human osteoclast assay. We used representative compounds for bisphosphonates (zoledronic acid), selective estrogen receptor modulators (SERM, raloxifene) and cathepsin K inhibitors (E-64 and AAR494). These compounds have different mechanisms of action and it was interesting to evaluate whether the assay would detect these differences. The control osteoclast cultures at day 17 contained only TRAP-positive cells and were almost confluent (Fig. 4, TRAP). Osteoclasts were of various sizes: very large cells, smaller multinucleated cells, and small mononucleated cells (Fig. 4, TRAP, C). In the control cultures on bone slices, pits covered most of the bone slice surface and formed characteristic resorption tracks, resulting from osteoclast migration (Fig. 4, PIT, C). The bisphosphonate zoledronic acid (50 nM) and selective estrogen receptor modulator (SERM) raloxifene (100 nM) reduced osteoclast number, leaving the culture largely depleted from mononuclear and smaller multinuclear cells (Fig. 4, TRAP, Z.A., Ral.). At these concentrations both compounds partially inhibited pit formation (Fig. 4, PIT, Z.A., Ral.). Two cathepsin K inhibitors were tested, a non-selective cysteine protease inhibitor E-64 and a selective inhibitor AAR494. Both compounds did not affect osteoclast formation, but partially inhibited pit formation (Fig. 4, E-64, AAR). AAR494 in particular induced changes in cell morphology, evident as densely TRAP-stained regions within cells. In addition, the remaining pits after treatment with this compound did not have a track appearance, but were cluster-like, containing small round pits.

Bottom Line: 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 alpha-MEM medium, supplemented with 10% of selected fetal calf serum, 1 microM dexamethasone and a mix of macrophage-colony stimulating factor (M-CSF, 25 ng/ml), receptor activator of NFkappaB ligand (RANKL, 50 ng/ml), and transforming growth factor-beta1 (TGF-beta1, 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Arthritis and Bone Metabolism Disease Area, Novartis Institutes for BioMedical Research Basel, Novartis Pharma AG, CH-4002 Basel, Switzerland. mira.susa_spring@pharma.novartis.com

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 alpha-MEM medium, supplemented with 10% of selected fetal calf serum, 1 microM dexamethasone and a mix of macrophage-colony stimulating factor (M-CSF, 25 ng/ml), receptor activator of NFkappaB ligand (RANKL, 50 ng/ml), and transforming growth factor-beta1 (TGF-beta1, 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.

No MeSH data available.


Related in: MedlinePlus