WO2002097121A1 - Method for assaying osteoclast recruitment and resorption - Google Patents

Method for assaying osteoclast recruitment and resorption Download PDF

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Publication number
WO2002097121A1
WO2002097121A1 PCT/DK2002/000361 DK0200361W WO02097121A1 WO 2002097121 A1 WO2002097121 A1 WO 2002097121A1 DK 0200361 W DK0200361 W DK 0200361W WO 02097121 A1 WO02097121 A1 WO 02097121A1
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assay
osteoclasts
migration
resorption
osteoclast
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PCT/DK2002/000361
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French (fr)
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Morten Karsdal
Michael Engsig
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Nordic Bioscience A/S
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0643—Osteoclasts
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5029—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on cell motility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/185—Osteoprotegerin; Osteoclast differentiation factor (ODF, RANKL)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/25—Tumour necrosing factors [TNF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2503/00—Use of cells in diagnostics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • This invention relates to a method for obtaining highly pure osteoclasts precursors and mature mammalian osteoclasts, to be used in migration and resorption assays.
  • This method comprises isolation of precursor cells for an initial culture period, followed by detachment of the osteoclasts or precursors from the substratum and subsequent reseeding of the osteoclasts in an appropriate recruitment or resorption assay.
  • Osteoclasts are terminally differentiated cells, which play a central role in bone resorption. Quantitative in vitro assays involving osteoclasts constitute a major asset in the development of new anti-resorptive therapies (Foged, NT et al., 1996). The migration of osteoclast precursors to the bone is a prerequisite for resorption, and therefore an essential event in the processes, which lead to bone remodeling.
  • the GPI-anchored 55 kDa glycoprotein CD 14 is expressed on monocytes/macrophages. Engagement of CD 14 by ligands like lipopolysaccharide, intact bacteria or apoptotic cells can result in either pro- or anti-inflammatory responses. Since the CD 14 molecule does not have a membrane spanning domain it cannot transmit a signal into the cell.
  • One of the unsolved problems to be solved is a safe way of manipulating the CD-14 positive cells in such a way that they can be detached from the initial substrate and reseeded on an appropriate medium such as bone slices.
  • the present invention relates to a method of using human monocytes derived osteoclasts as sources in osteoclast migration and resorption assay wherein detachment and reseeding is an essential feature.
  • the technique comprises a differentiation phase of recently purified monocytes followed by a culture period and subsequent an intermediate step in which the osteoclasts or precursors proteolytically are released and reseeded in controlled numbers. This step significantly decreases the assay variation, and therefore results in a highly robust mammalian osteoclast system in which both anti-resorptives and anti-migratory compounds can be tested.
  • the invention also relates to a kit comprising a highly robust human osteoclast system in which both anti-resorptive and anti-migratory compounds can be tested. Detailed description of the invention
  • This invention relates to a method for assaying the process of recruitment or resorption of a essential pure mammalian osteoclasts population, comprising: a) cultivating a CD-14 positive monocyte cell culture; b) detachment of the osteoclasts from the substratum; and c) subsequent reseeding of the osteoclasts in an appropriate resorption or migration assay.
  • Pursuit mammalian osteoclasts population are at least 90% pure osteoclasts derived from CD-14 positive monocytes.
  • “Differentiated osteoclasts” are mature osteoclast capable of the process of bone resorption.
  • Recruitment is the process in which osteoclasts precursors amongst other cellular events migrate in order to reach the future site of resorption as fully differentiated osteoclasts.
  • the present invention involves a new method of using a pure mammalian osteoclast population, reseeded in controlled number after the differentiation phase which thereby exhibits reduced assay variation, and therefore allows for a robust assays in which anti-migratory agents can be tested.
  • the present invention involves a new method of using a pure mammalian osteoclast population, reseeded in controlled number after the differentiation phase which thereby exhibits reduced assay variation, and therefore allows for a robust assays in which anti-resorptive agents can be tested.
  • said monocyte cell culture is essential consisting of CD-14 positive cells.
  • said cultivation conditions comprises an appropriate amount of RANKL and M-CSF in the growth medium.
  • said pure mammalian osteoclast population are reseeded in a controlled number between 10,000 and 50,000 cells per Vz square mm microtitter well .
  • said pure mammalian osteoclast population are reseeded in a controlled number between 15,000 and 30,000 cells per Vi square mm microtitter well.
  • the present invention relates to a assay for detecting the inhibitory or stimulatory effect of a test substance on mammalian osteoclast resorption comprising: a) placing differentiated osteoclasts in a migration assay in the presence of differentiation factors or a biologically active derivative thereof and the test substance, and b) measuring the amount of bone resorption.
  • the amount of resorption measured is compared to a result obtained in the absence of a test substance.
  • the present invention relates to a assay for detecting the inhibitory or stimulatory effect of a test substance on mammalian osteoclast recruitment/migration comprising: a) placing differentiated osteoclasts in a migration assay in the presence of differentiation factors or a biologically active derivative thereof and the test substance, and b) measuring the amount of bone recruitment/migration.
  • the amount of recruitment measured is compared to a result obtained in the absence of a test substance.
  • the migration measured is compared to a l o result obtained in the absence of a test substance.
  • said pure mammalian osteoclast population is reseeded in an assay.
  • said pure mammalian osteoclast population is reseeded in a modified Boyden chamber migration-assay.
  • said pure mammalian osteoclast population is reseeded in a pericellular collagenolysis assay (PCA).
  • PCA pericellular collagenolysis assay
  • the mammalian osteoclasts are human.
  • this invention is an easy procedure to obtain a separation of the 25 differentiation process and the process of migration of mammalian osteoclasts comprising detachment from the substratum and subsequent reseeding of the osteoclasts in an appropriate assay.
  • the cultures of this invention comprise a population of pure osteoclast precursors.
  • One important distinguishing feature of this invention is that the differentiated osteoclasts of the population are reseeded and are thus able to be further manipulated by the researcher. This invention thus makes it possible to study the biochemistry of pure differentiated osteoclasts, the regulation of genes expressed by these cells and other characteristics.
  • the present invention relates to a kit for assaying migration and/or resorption of mammalian osteoclasts comprising one or more of the essential components as described above.
  • said kit comprises a combination of the above described osteoclast differentiation, detachment, reseeding, resorption and or migration procedures.
  • said kit comprises reseeding in a pericellular collagenolysis assay (PCA).
  • PCA pericellular collagenolysis assay
  • said kit comprises essentially a pericellular collagenolysis assay (PCA).
  • PCA pericellular collagenolysis assay
  • the culture processes of this invention can be used with any species of mammals, particularly those of bovines, rodents (such as mouse and rat), and primates (such as human and monkey). Particularly preferred are human, and mammalian species such as rat and mouse or other animals whose osteoclasts share biochemical characteristics with human osteoclasts.
  • This method provides an assay to monitor recruitment and/or migration of human osteoclast without the support of other cells e.g. stromal cells.
  • PMBC peripheral blood monocyte cells
  • Human peripheral blood was diluted with 1 to 1 with PBS.
  • the PBS/blood suspension was carefully laired over a Ficoll-plaque (vol to vol). This suspension was centrifuged at 290 RCF (relative centrifugation force) for 20 min. The upper and inter phase were collected, and washed in cold PBS (kept on ice). The cells were centrifuged at 380 RCF (relative centrifugation force) for 20 min. The pellet was dissolved and washed once more with PBS. Again the pellet was dissolved and centrifuged at 380 RCF (relative centrifugation force) for 20 min.
  • the cells were resuspended with cold PBS containing 2% FCS (10ml PBS for 50ml PBS/blood starting volume). The cells were counted, with exclusion of platelets that look like very tiny cells. The entire procedure was preformed on ice.
  • osteoclasts were seeded at a density of 20000 cells/bone slice in a 96 well culture plate, and cultured for 7 days in the presence of 25ng/ml M-CSF and RANK-L. Description of the figures
  • FIG. 1 Monocytes were isolated from peripheral blood as previously described and cultured for 14 days in the presence of both RANKL and M-CSF. At day 14, the newly 5 . formed pre-osteoclasts were detached by trypsin treatment and cell scraping followed by replating collagen type I coated transSwells in the presence of both RANKL and M-CSF. Cells were cultured for 3 days in the presence of different concentrations of the matrix metalloprotease inhibitor GM6001, and stained for TRAP activity. Total cell number was counted and expressed in percentage in the total number of cells that had penetrated the o collagen gel and expressed in the graph as percent of cells migrating.
  • GM6001 matrix metalloprotease inhibitor
  • FIG. 2 Monocytes were isolated from peripheral blood as previously described and cultured for 14 days in the presence of both RANKL and M-CSF. At day 14, the newly formed pre-osteoclasts were detached by trypsin treatment and cell scraping followed by replating on collagen type I coated cover slips and cultured for 24 hours in the presence of 5 RANK-L and M-CSF in the presence of 10 MM of the matrix metalloprotease inhibitor
  • GM6001 GM6001.
  • the pericellular area that was cleared by proteolysis was visualised by stained with an anti-collagen antibody. Total area of the migration path and osteoclast area were quantified.
  • FIG. 3 Monocytes were isolated from peripheral blood as previously described and o cultured for 14 days in the presence of both RANKL and M-CSF. At day 14, the newly formed pre-osteoclasts were detached by trypsin treatment and cell scraping followed by replating on bovine cortical bone slices in the presence of both RANKL and M-CSF. Cells were plated at a density of 20000 osteoclasts pr. Bone slice in a 96 well culture plate. After 7 days culture period in the presence of the carbonic anhydrase inhibitor, ethoxyzolamide, 5 the release amount of total pit formation was measured by pit staining with mayer hematoxylin followed by pit counting.
  • the carbonic anhydrase inhibitor ethoxyzolamide
  • Ethoxyzolamide inhibited human osteoclastic bone resorption with an IC50 value of approx. 10 ⁇ M.
  • Figure 4 Monocytes were isolated from peripheral blood as previously described and cultured for 14 days in the presence of both RANKL and M-CSF. At day 14, the newly formed pre-osteoclasts were detached by trypsin treatment and cell scraping followed by replating on bovine cortical bone slices in the presence of both RANKL and M-CSF. Cells were plated at a density of 20000 osteoclasts pr. Bone slice in a 96 well culture plate.
  • Ethoxyzolamide inhibited human osteoclastic bone resorption with an IC50 value of approx. 10 ⁇ M.
  • FIG. 5 Monocytes were isolated from peripheral blood as previously described and cultured for 14 days in the presence of both RANKL and M-CSF. At day 14, the newly formed pre-osteoclasts were detached by trypsin treatment and cell scraping followed by replating on bovine cortical bone slices in the presence of both RANKL and M-CSF. Cells were plated at a density of 20000 osteoclasts pr. Bone slice in a 96 well culture plate. After 7 days culture period in the presence of the carbonic anhydrase inhibitor, ethoxyzolamide, the release of collagen fragments from the bone slices were measured by the CrossLaps for culture kit and stained by Mayer's hematoxylin to visualise pit formation. Levels are expressed in percent of control, and show strict correlation of the to measurements.
  • the carbonic anhydrase inhibitor ethoxyzolamide
  • This method provides an assay to monitor migration of human osteoclast without the support of other cells e.g. stromal cells.
  • This method provides a Pericellular Collagenolysis Assay (PCA) to monitor migration of human osteoclast without the support of other cells e.g. stromal cells.
  • PCA Pericellular Collagenolysis Assay
  • Monocytes were isolated from peripheral blood as previously described and cultured for 14 days in the presence of both RANKL and M-CSF.
  • the newly formed pre- osteoclasts were detached by trypsin treatment and cell scraping followed by replating on collagen type I coated cover slips and cultured for 24 hours in the presence of RANK-L and M-CSF in the presence of 10 -M of the matrix metalloprotease inhibitor GM6001.
  • the pericellular area that was cleared by proteolysis was visualised by stained with an anti-collagen antibody. Total area of the migration path and osteoclast area were quantified. Inhibition of osteoclast migration compared to control treatment is shown in figure 2.
  • This method includes a simple quantification of bone resorption through ELIS A measurement of CTX collagen fragments released into the conditioned medium by the human osteoclasts or by counting the total resorption area made by the osteoclasts.
  • Monocytes were isolated from peripheral blood as previously described and cultured for 14 days in the presence of both RANKL and M-CSF. At day 14, the newly formed pre- osteoclasts were detached by trypsin treatment and cell scraping followed by replating on bovine cortical bone slices in the presence of both RANKL and M-CSF. Cells were plated at a density of 20,000 osteoclasts pr. Bone slice in a 96 well culture plate.
  • CTX collagen fragments

Abstract

This invention relates to a method for separation of the differentiation process and the process of recruitment of mammalian osteoclasts comprising an initial culture period, cultivating a CD-14 positive monocyte cell culture, followed by detachment of the osteoclasts from the substratum and subsequent reseeding of the osteoclasts in an appropriate recruitment assay, in the presence of differention factors as RANKL and M-CSF.

Description

METHOD FOR ASSAYING OSTEOCLAST RECRUITMENT AND RESORPTION
Field of the invention
This invention relates to a method for obtaining highly pure osteoclasts precursors and mature mammalian osteoclasts, to be used in migration and resorption assays. This method comprises isolation of precursor cells for an initial culture period, followed by detachment of the osteoclasts or precursors from the substratum and subsequent reseeding of the osteoclasts in an appropriate recruitment or resorption assay.
Background of the invention
Osteoclasts are terminally differentiated cells, which play a central role in bone resorption. Quantitative in vitro assays involving osteoclasts constitute a major asset in the development of new anti-resorptive therapies (Foged, NT et al., 1996). The migration of osteoclast precursors to the bone is a prerequisite for resorption, and therefore an essential event in the processes, which lead to bone remodeling.
Until recently it has not been possible to propagate these highly specialized cells in culture, without the support of stromal cells. As a consequence the source of material for quantitative in vitro assay has been restricted to cultures of cells isolated from long bones and long bone marrow of rodent's and rabbits. Human origin has likewise only been possible through the support of stromal cells arid additionally by an osteoclastoma derived osteoclast cell line (James, IE et al., 1999, Fujikawa, Y et al., 1996).
With the recent discovery of RANKL (a.k.a. osteoclast differentiating factor, ODF) it has been possible to differentiate monocytes separated from whole peripheral blood into mature osteoclasts in a pure preparation yielding near 100% pure osteoclasts (Matsuzaki, K et al., 1998), thereby eliminating the need for supporting cells e.g. stromal cells. Furthermore by the discovery that almost 100% of all osteoclast originate from CD 14 precursor cells (Massey, HM et al., 1999) substantial populations of both osteoclast precursors and mature osteoclasts can be obtained in vitro.
The GPI-anchored 55 kDa glycoprotein CD 14 is expressed on monocytes/macrophages. Engagement of CD 14 by ligands like lipopolysaccharide, intact bacteria or apoptotic cells can result in either pro- or anti-inflammatory responses. Since the CD 14 molecule does not have a membrane spanning domain it cannot transmit a signal into the cell.
One of the unsolved problems to be solved is a safe way of manipulating the CD-14 positive cells in such a way that they can be detached from the initial substrate and reseeded on an appropriate medium such as bone slices.
Furthermore, since recruitment and resorption phases have been inseparable it has not been possible to study the direct effect of tested compounds on each of these phases.
Short description of the invention
The present invention relates to a method of using human monocytes derived osteoclasts as sources in osteoclast migration and resorption assay wherein detachment and reseeding is an essential feature. Basically, the technique comprises a differentiation phase of recently purified monocytes followed by a culture period and subsequent an intermediate step in which the osteoclasts or precursors proteolytically are released and reseeded in controlled numbers. This step significantly decreases the assay variation, and therefore results in a highly robust mammalian osteoclast system in which both anti-resorptives and anti-migratory compounds can be tested.
The invention also relates to a kit comprising a highly robust human osteoclast system in which both anti-resorptive and anti-migratory compounds can be tested. Detailed description of the invention
This invention relates to a method for assaying the process of recruitment or resorption of a essential pure mammalian osteoclasts population, comprising: a) cultivating a CD-14 positive monocyte cell culture; b) detachment of the osteoclasts from the substratum; and c) subsequent reseeding of the osteoclasts in an appropriate resorption or migration assay.
As used throughout the specification and claims, the following definitions will apply:
"Pure mammalian osteoclasts population" are at least 90% pure osteoclasts derived from CD-14 positive monocytes.
"Differentiated osteoclasts" are mature osteoclast capable of the process of bone resorption.
"Recruitment" is the process in which osteoclasts precursors amongst other cellular events migrate in order to reach the future site of resorption as fully differentiated osteoclasts.
In a first aspect, the present invention involves a new method of using a pure mammalian osteoclast population, reseeded in controlled number after the differentiation phase which thereby exhibits reduced assay variation, and therefore allows for a robust assays in which anti-migratory agents can be tested.
In a second aspect, the present invention involves a new method of using a pure mammalian osteoclast population, reseeded in controlled number after the differentiation phase which thereby exhibits reduced assay variation, and therefore allows for a robust assays in which anti-resorptive agents can be tested.
In one embodiment of the present invention, said monocyte cell culture is essential consisting of CD-14 positive cells.
In another embodiment of the present invention, said cultivation conditions comprises an appropriate amount of RANKL and M-CSF in the growth medium.
In a preferred embodiment of the present invention, said pure mammalian osteoclast population are reseeded in a controlled number between 10,000 and 50,000 cells per Vz square mm microtitter well .
In a more preferred embodiment of the present invention, said pure mammalian osteoclast population are reseeded in a controlled number between 15,000 and 30,000 cells per Vi square mm microtitter well.
In another aspect the present invention relates to a assay for detecting the inhibitory or stimulatory effect of a test substance on mammalian osteoclast resorption comprising: a) placing differentiated osteoclasts in a migration assay in the presence of differentiation factors or a biologically active derivative thereof and the test substance, and b) measuring the amount of bone resorption.
In one embodiment of the present invention the amount of resorption measured is compared to a result obtained in the absence of a test substance.
In another aspect the present invention relates to a assay for detecting the inhibitory or stimulatory effect of a test substance on mammalian osteoclast recruitment/migration comprising: a) placing differentiated osteoclasts in a migration assay in the presence of differentiation factors or a biologically active derivative thereof and the test substance, and b) measuring the amount of bone recruitment/migration.
5
In one embodiment of the present invention the amount of recruitment measured is compared to a result obtained in the absence of a test substance.
In another embodiment of the present invention, the migration measured is compared to a l o result obtained in the absence of a test substance.
In a preferred embodiment of the present invention, said pure mammalian osteoclast population is reseeded in an assay.
15 In a more preferred embodiment of the present invention, said pure mammalian osteoclast population is reseeded in a modified Boyden chamber migration-assay.
In a most preferred embodiment of the present invention, said pure mammalian osteoclast population is reseeded in a pericellular collagenolysis assay (PCA).
20
In another preferred embodiment of the present invention, the mammalian osteoclasts are human.
Accordingly, this invention is an easy procedure to obtain a separation of the 25 differentiation process and the process of migration of mammalian osteoclasts comprising detachment from the substratum and subsequent reseeding of the osteoclasts in an appropriate assay. The cultures of this invention comprise a population of pure osteoclast precursors. One important distinguishing feature of this invention is that the differentiated osteoclasts of the population are reseeded and are thus able to be further manipulated by the researcher. This invention thus makes it possible to study the biochemistry of pure differentiated osteoclasts, the regulation of genes expressed by these cells and other characteristics.
In another aspect the present invention relates to a kit for assaying migration and/or resorption of mammalian osteoclasts comprising one or more of the essential components as described above.
In one embodiment said kit comprises a combination of the above described osteoclast differentiation, detachment, reseeding, resorption and or migration procedures.
In a preferred embodiment, said kit comprises reseeding in a pericellular collagenolysis assay (PCA).
In a more preferred embodiment, said kit comprises essentially a pericellular collagenolysis assay (PCA).
The culture processes of this invention can be used with any species of mammals, particularly those of bovines, rodents (such as mouse and rat), and primates (such as human and monkey). Particularly preferred are human, and mammalian species such as rat and mouse or other animals whose osteoclasts share biochemical characteristics with human osteoclasts.
This method provides an assay to monitor recruitment and/or migration of human osteoclast without the support of other cells e.g. stromal cells.
In accordance with this invention we have used the passage method for the monocytes derived human osteoclast to assay human osteoclast migration in a pure culture system. Human osteoclast and their precursors are isolated by the following procedure:
Preparation of peripheral blood monocyte cells (PMBC)
Human peripheral blood was diluted with 1 to 1 with PBS. The PBS/blood suspension was carefully laired over a Ficoll-plaque (vol to vol). This suspension was centrifuged at 290 RCF (relative centrifugation force) for 20 min. The upper and inter phase were collected, and washed in cold PBS (kept on ice). The cells were centrifuged at 380 RCF (relative centrifugation force) for 20 min. The pellet was dissolved and washed once more with PBS. Again the pellet was dissolved and centrifuged at 380 RCF (relative centrifugation force) for 20 min. The cells were resuspended with cold PBS containing 2% FCS (10ml PBS for 50ml PBS/blood starting volume). The cells were counted, with exclusion of platelets that look like very tiny cells. The entire procedure was preformed on ice.
Isolation of CD 14 positive cells from the PMBC fraction The vial containing the beads was shaken vigorously (no vortex) to bring beads in suspension. One hundred twenty five W (10xl0e6 beads/ml final cone.) beads were transferred into a 15ml-tube. Five ml of cold PBS were added followed by gently mixing. The tube was placed on a magnetic device (DYNAL Biotech, Oslo, Norway) for 1 minute. The supernatant was removed. This washing procedure was repeated twice. Five ml of PBMC at 50-60x10e6 cells/ml were added to the beads in the 15 ml tube. The beads and cells were shaken gently. The tube was placed on the magnetic device for 2 minutes, after which the supernatant removed. Five ml of cold PBS+ 10 % FCS was added and pipetted gently up and down to resuspend the beads. The tube was placed on the magnetic device for 2 minutes and the supernatant was removed. This washing step was repeated twice. The cells were finally resuspended in 5 ml «-MEM+10%o FCS (GIBCO Invitrogen Corporation, California, USA). The cells were counted. Finally the cells were seeded at a density of 20000/well in 96 well plates and incubated with RANK-L and M-CSF (R&D Diagnostics, Holargos, Greece) for the indicated time. The cells were counted. Finally cells were seeded at a density of 10 million cells in a 75 cm2 flask and cultured 14 days in the presence of 25 ng/ml M-CSF and 25 ng/ml RANK- L.
The intermediate manipulation step.
After this initial cultures period, at day 14, the newly formed pre-osteoclasts were detached by trypsin treatment and cell scraping followed by re-plating in the appropriate assay, please refer to option 1 through 3 below.
1. Modified Boyden chamber: Invasion assay
For the invasion/migration assay human osteoclast were seeded on collagen coated transwells in the presence of both RANKL and M-CSF. Cells were cultured for 3 days, and stained for TRAP activity. Total cell number was counted and expressed in percentage in the total number of cells that had penetrated the collagen gel and expressed in the graph as percent of cells migrating.
2. Pericellular collagenolysis assay: Migration assay
For the migration/ non-invasive assay human osteoclast were plated on collagen coated cover slips and cultured for 24 hours in the presence of RANK-L .and M- CSF in the presence and absence of test compound. During the culture the osteoclast degrade collagen in the path of its migration. For the assessment of human osteoclast migration cover slips were stained with an anti-collagen type I antibody to visualize the path in which the osteoclast have migrated. Total collagen free area and osteoclast area were quantified.
Bone slice assay: Resorption assay:
For the resorptive assay osteoclasts were seeded at a density of 20000 cells/bone slice in a 96 well culture plate, and cultured for 7 days in the presence of 25ng/ml M-CSF and RANK-L. Description of the figures
Figure 1: Monocytes were isolated from peripheral blood as previously described and cultured for 14 days in the presence of both RANKL and M-CSF. At day 14, the newly 5 . formed pre-osteoclasts were detached by trypsin treatment and cell scraping followed by replating collagen type I coated transSwells in the presence of both RANKL and M-CSF. Cells were cultured for 3 days in the presence of different concentrations of the matrix metalloprotease inhibitor GM6001, and stained for TRAP activity. Total cell number was counted and expressed in percentage in the total number of cells that had penetrated the o collagen gel and expressed in the graph as percent of cells migrating.
Figure 2: Monocytes were isolated from peripheral blood as previously described and cultured for 14 days in the presence of both RANKL and M-CSF. At day 14, the newly formed pre-osteoclasts were detached by trypsin treatment and cell scraping followed by replating on collagen type I coated cover slips and cultured for 24 hours in the presence of 5 RANK-L and M-CSF in the presence of 10 MM of the matrix metalloprotease inhibitor
GM6001. For the assessment of human osteoclast migration on cover slips, the pericellular area that was cleared by proteolysis was visualised by stained with an anti-collagen antibody. Total area of the migration path and osteoclast area were quantified.
Figure 3: Monocytes were isolated from peripheral blood as previously described and o cultured for 14 days in the presence of both RANKL and M-CSF. At day 14, the newly formed pre-osteoclasts were detached by trypsin treatment and cell scraping followed by replating on bovine cortical bone slices in the presence of both RANKL and M-CSF. Cells were plated at a density of 20000 osteoclasts pr. Bone slice in a 96 well culture plate. After 7 days culture period in the presence of the carbonic anhydrase inhibitor, ethoxyzolamide, 5 the release amount of total pit formation was measured by pit staining with mayer hematoxylin followed by pit counting. Ethoxyzolamide inhibited human osteoclastic bone resorption with an IC50 value of approx. 10 μM. Figure 4: Monocytes were isolated from peripheral blood as previously described and cultured for 14 days in the presence of both RANKL and M-CSF. At day 14, the newly formed pre-osteoclasts were detached by trypsin treatment and cell scraping followed by replating on bovine cortical bone slices in the presence of both RANKL and M-CSF. Cells were plated at a density of 20000 osteoclasts pr. Bone slice in a 96 well culture plate. After 7 days culture period in the presence of the carbonic anhydrase inhibitor, ethoxyzolamide, the release of collagen fragtments from the bone slices were measured by the CrossLaps for culture kit. Ethoxyzolamide inhibited human osteoclastic bone resorption with an IC50 value of approx. 10 μM.
Figure 5: Monocytes were isolated from peripheral blood as previously described and cultured for 14 days in the presence of both RANKL and M-CSF. At day 14, the newly formed pre-osteoclasts were detached by trypsin treatment and cell scraping followed by replating on bovine cortical bone slices in the presence of both RANKL and M-CSF. Cells were plated at a density of 20000 osteoclasts pr. Bone slice in a 96 well culture plate. After 7 days culture period in the presence of the carbonic anhydrase inhibitor, ethoxyzolamide, the release of collagen fragments from the bone slices were measured by the CrossLaps for culture kit and stained by Mayer's hematoxylin to visualise pit formation. Levels are expressed in percent of control, and show strict correlation of the to measurements.
The invention is illustrated hereinafter with reference to some unlimitative examples.
Examples
Example 1: Human osteoclast migration
This method provides an assay to monitor migration of human osteoclast without the support of other cells e.g. stromal cells.
Migration of purified rabbit osteoclasts through a collagen gel has previously been described (Sato, T et al., 1998). Here we have used the passage method for the monocytes derived human osteoclast to assay human osteoclast migration in a pure culture system. Monocytes were isolated from peripheral blood and cultured for 14 days in the presence of both RANKL and M-CSF (25 ng/ml both). At day 14, the newly formed pre-osteoclasts were detached by trypsin treatment and cell scraping followed by replating collagen coated transwells (modified Boyden chambers) in the presence of both RANKL and M-CSF. Cells were cultured for 3 days, and stained for TRAP activity. Total cell number was counted and expressed in percentage in the total number of cells that had penetrated the collagen gel and expressed in the graph as percent of cells migrating.
Example 2: PCA assay monitoring migration path of human osteoclast
This method provides a Pericellular Collagenolysis Assay (PCA) to monitor migration of human osteoclast without the support of other cells e.g. stromal cells. This assay is a novel method for the description of the path of migration of osteoclasts, and thus provides for an easy and reliable measurement for compounds effect on migration.
Monocytes were isolated from peripheral blood as previously described and cultured for 14 days in the presence of both RANKL and M-CSF. At day 14, the newly formed pre- osteoclasts were detached by trypsin treatment and cell scraping followed by replating on collagen type I coated cover slips and cultured for 24 hours in the presence of RANK-L and M-CSF in the presence of 10 -M of the matrix metalloprotease inhibitor GM6001. For the assessment of human osteoclast migration on cover slips, the pericellular area that was cleared by proteolysis was visualised by stained with an anti-collagen antibody. Total area of the migration path and osteoclast area were quantified. Inhibition of osteoclast migration compared to control treatment is shown in figure 2.
Example 3: Human osteoclast resorption
This method includes a simple quantification of bone resorption through ELIS A measurement of CTX collagen fragments released into the conditioned medium by the human osteoclasts or by counting the total resorption area made by the osteoclasts. Monocytes were isolated from peripheral blood as previously described and cultured for 14 days in the presence of both RANKL and M-CSF. At day 14, the newly formed pre- osteoclasts were detached by trypsin treatment and cell scraping followed by replating on bovine cortical bone slices in the presence of both RANKL and M-CSF. Cells were plated at a density of 20,000 osteoclasts pr. Bone slice in a 96 well culture plate. After a 7-day culture period, cells stained positive for TRAP and most were multinucleated. Their osteoclastic phenotype was further confirmed by immunohistochemistry and zymography showing their expression of calcitonin receptor, αvβ3, MMP-9 and actin ring formation.
When replated osteoclasts were cultured in the presence of the carbonic anhydrase inhibitor, ethoxyzolamide for the described 7-day culture period, both pit foraiation Fig 3 and CTX Fig 4 release was inhibited to the same degree, with IC50 values of approx. 10 μM.
The collagen fragments (CTX) release into the culture medium was measured by the CrossLaps for Culture ELISA and compared to the total plan area of resorption pits, Fig 5. The release of CTX fragments correlated strictly with the total area resorbed measured by pit counting.
References
Foged NT, Delaisse M, Hou P, Lou H, Sato T, Winding B, Bonde M., J Bone Miner Res 1996 Feb;l l(2):226-37.
James IE, Lark MW, Zembryld D, Lee-Rykaczewski EV, Hwang SM, Tomaszek TA, Belfϊore P, Gowen M., J Bone Miner Res 1999 Sep; 14(9): 1562-9.
Fujikawa Y, Quinn JM, Sabokbar A, McGee JO, Athanasou NA., Endocrinology 1996 Sep;137(9):4058-60.
Matsuzaki K, Udagawa N, Takahashi N, Yamaguchi K, Yasuda H, Shima N, Morinaga T, Toyama Y, Yabe Y, Higashio K, Suda T., Biochem Biophys Res Commun 1998 May 8;246(l):199-204.
Sato T, Foged NT, Delaisse JM., J Bone Miner Res 1998 Jan;13(l):59-66.
Massey HM, Flanagan AM., Br J Haematol 1999 Jul;106(l):167-70.

Claims

Claims:
1. A method for assaying the process of recruitment or resorption of a pure mammalian osteoclasts population, comprising: a) cultivating a CD-14 positive monocyte cell culture; 5 b) detachment of the osteoclasts from the substratum; and c) subsequent reseeding of the osteoclasts in an appropriate resorption or migration assay.
2. A method according to claim 1, wherein said monocyte cell culture is essential o consisting of CD- 14 positive cells.
3. A method according to claim 1, wherein said cultivation conditions comprises an appropriate amount of RANKL and M-CSF in the growth medium.
5 4. A method according to claim 1, wherein said pure mammalian osteoclast population are reseeded in a controlled number between 10,000 and 50,000 cells per micro titter well.
5. An assay for detecting the migration inhibitory or stimulatory effect of a test substance on mammalian osteoclast resorption comprising: 0 a) placing differentiated osteoclasts in a resorption assay in the presence of differentiation factors or a biologically active derivative thereof and the test substance, and b) measuring the amount of bone resorption.
5 6. An assay for detecting the inhibitory or stimulatory effect of a test substance on mammalian osteoclast recruitment/migration comprising: a) placing differentiated osteoclasts in a migration assay in the presence of differentiation factors or a biologically active derivative thereof and the test substance, and 0 b) measuring the amount of bone recruitment/migration.
7. An assay according to the claims 5 and/or 6, wherein the effect of the test substance is compared to the effect obtained in the absence of a test substance.
8. An assay according to claim 6, wherein said pure mammalian osteoclast population is reseeded in a modified Boyden chamber migration-assay.
9. An assay according to claim 6, wherein said pure mammalian osteoclast population is reseeded in a pericellular collagenolysis assay (PCA).
10. An assay according to claim 1, wherein the mammalian osteoclasts are human.
11. A kit for assaying migration and/or resorption of mammalian osteoclasts comprising a reseeding procedure and one or more of the essential components according to the preceding claims.
12. A kit according to claim 11 comprising essentially a pericellular collagenolysis assay (PCA).
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