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Methods for isolation of osteoclast precursor cells and inducing their differentiation into osteoclasts

a precursor cell and osteoclast technology, applied in cell culture active agents, artificial cell constructs, drug compositions, etc., can solve the problems of inability to isolate osteoclasts, unbalance between bone resorption and generation, etc., to achieve the effect of reducing bone mass and easy screening for bone metabolic diseases

Inactive Publication Date: 2005-01-06
SHIONOGI & CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0032] As “a method for screening agents for metabolic bone diseases”, (1) a method for measuring inhibitory activities on the differentiation of preosteoclasts into osteoclasts, and (2) a method for measuring inhibitory activities on the bone resorption by osteoclasts are given for example. If a candidate compound for the therapeutic agent shows a differentiation inhibitory activity against the preosteoclasts of this invention, the compound is promising as an antirheumatic drug. Futher, if a candidate compound for the therapeutic agent shows a bone resorption inhibitory activity against the osteoclasts of this invention, it is suggested that the compound is useful for treating osteoporosis caused by excessive bone resorption, decrease of bone mass due to diabetes, osteomalacia or the like. Thus, by using the preosteoclasts or the osteoclasts of this invention, the screening for bone metabolic diseases agents can easily be performed in vitro.

Problems solved by technology

However, unbalance between bone resorption and generation lead metabolic bone diseases, including osteoporosis, rheumatoid arthritis, osteoarthritis, decrease of bone quantity due to diabetes, many types of hormone abnormalities, nutritional disorder, osteopetrosis and osteomalacia.
But, they did not succeed in the isolation of osteoclasts itself.

Method used

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  • Methods for isolation of osteoclast precursor cells and inducing their differentiation into osteoclasts
  • Methods for isolation of osteoclast precursor cells and inducing their differentiation into osteoclasts
  • Methods for isolation of osteoclast precursor cells and inducing their differentiation into osteoclasts

Examples

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example

Example 1

Isolation of Human Osteoclasts from Joint Fluid of Patients with Rheumatoid Arthritis

[0059] (1) Separation of a Cellular Fraction

[0060] Joint fluids were obtained from patients with rheumatoid arthritis. The joint fluids were kept in tubes at 4° C. The following procedures were generally under sterile conditions. The joint fluid, 1 ml to several tens ml, was added to equal volume of RPMI 1640 medium (Gibco BRL, #22400 or equivalent). The mixture was centrifugated at 1,000-2,000 rpm for 5 minutes at 4° C. to obtain a cellular fraction containing granulocytes and lymphocytes.

[0061] (2) Isolation of Preosteoclasts

[0062] The obtained cellular fraction was cultured in DMEM medium (Gibco BRL, #12430-21 or equivalent) supplemented with 10% (v / v) of fetal calf serum (FCS) in 5-7% CO2-containing air at 37° C. for several weeks. During the culture period, all cells except preosteoclasts died out, and only preosteoclasts survived (FIGS. 1, 2).

[0063] (3) Preparation of Medium

[00...

example 2

Isolation of Human Osteoclasts from Peripheral Blood of Healty Donors

[0092] (1) Separation of Cellular Fraction

[0093] Fifty ml to 200 ml of peripheral blood of healthy donors were collected in the presence of heparin or an alternative anticoagulant. Peripheral blood mononuclear cells (PBMC) were obtained by a specific gravity centrifugation using a Ficoll-paque (Pharmacia Biotech). 107 cell / ml of PBMC were suspended in RPMI1640 medium containing 10% (v / v) FCS, then 1-1.5 ml / dish of the cell suspension was cultured on 60 mm culture dish at 37° C., in 5-7% CO2-containing air, for 1-2 hours. After the culture, the non-adherent cells were rinsed out from the dish with RPIM1640 medium containing 10% (v / v) of FCS at 37° C. Adherent cells to the dish were washed with cold (4° C.), serum-free RPIM1640 medium, then the cells were collected as a peripheral blood monocytes (about 3-8% of total PBMC).

[0094] (2) Isolation of Preosteoclasts

[0095] The obtained monocytes (0.5−1×106 / ml) was cult...

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Abstract

This invention relates to: a method for differentiating osteoclast precursor cells (preosteoclasts) into osteoclasts, which comprises culturing the preosteoclasts in the absence of accessory cells; a method for isolating preosteoclasts, which comprises culturing peripheral blood or joint fluid in the absence of cytokine for 1 to 3 weeks; an preosteoclasts, which is obtainable by the above method; a method for differentiating the preosteoclasts obtained by the above method into osteoclasts, which comprises culturing the preosteoclasts in the absence of accessory cell; an osteoclast, which is obtainable by the above method; a method for screening agents for treating metabolic bone diseases, which comprises using the preosteoclasts or the osteoclasts as described above; and agents for treating metabolic bone diseases, which is obtainable by the above screening method.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for the differentiation of osteoclast precursor cells (preosteoclasts) into osteoclasts, which comprises culturing the preosteoclasts in the absence of accesory cells; a method for isolating preosleoclasts; a method for screening agents for metabolic bone diseases, which comprises using the preosteoclasts or the osteoclasts; and agents for metabolic bone diseases, which is obtainable by the screening method. BACKGROUND ART [0002] Bone tissues of mammal repeat generation and resorption of bone. The tissue operates as a central point of calcium metabolism to keep the balance between bone resorption and generation in the growth period and even after the period of maturity. Bone resorption and resorption are well balanced by crosstalk between osteoclasts and osteoblasts. However, unbalance between bone resorption and generation lead metabolic bone diseases, including osteoporosis, rheumatoid arthritis, osteoarthritis, decr...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/078
CPCC12N5/0643C12N2501/21C12N2503/02C12N2501/23C12N2502/11C12N2501/22A61P19/00
Inventor MAEDA, TOMOKOSUZUKI, RYUJIOCHI, TAKAHIRO
Owner SHIONOGI & CO LTD
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