Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Patent
1997-11-17
2000-07-25
Wityshyn, Michael G.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
435325, 435347, 435372, 435373, 435375, 435384, 435 405, 435 4052, 435383, C12Q 100, C12N 500, C12N 502
Patent
active
060935332
DESCRIPTION:
BRIEF SUMMARY
DESCRIPTION OF THE INVENTION
This invention relates to a method of producing a population of cells which is highly enriched in its content of osteoclast precursor cells, and to the cell populations so produced.
BACKGROUND OF THE INVENTION
Osteoclasts are terminally differentiated cells which play a key role in bone resorption. Due to the low number of mammalian osteoclasts and the difficulty to isolate them from bone tissue, and from other cells, their characterization has been limited primarily to immunohistochemistry or anatomical and physiological measurements on single cells.
Several attempts have been made, with limited success, to identify, isolate and establish in culture, cells capable of differentiating into osteoclasts from either bone marrow (Billecocq et al., 1990 Pro. Nat. Acad. Sci. USA 87:6470-6474; Prallet et al., 1992, J. Bone Min. Res. 7:405-414; and Chambers et al., 1993, Proc. Nat. Acad. Sci. USA 90:5578-5582) or leukemic and promyelocytic cell lines (Yoneda et al., 1991, Endocrinology 129:683-689; Gattei et al., 1992 J. Cell Biol. 116:437-447). Recently, cells obtained from human giant cell tumors of bone were reported to form osteoclasts in culture (Grano et al., 1994 J. Bone Min. Res. 9:1013-1020; Grano, et al., 1994 Exp. Cell Res. 212:209-218).
To obtain enriched osteoclast preparations, Akatsu et al., 1992, J. Bone Miner. Res. 7:1297-1306; cultured the osteoblastic and bone marrow cells on collagen gel-coated dishes, and released the cells from the collagen matrix using collagenase. However, the yield of osteoclastic cells was quite low. Recently, Shioi et al., 1994, Calcif. Tissue Int. 55:387-394; using a similar co-culture system reported the enrichment of generated osteoclasts by treating the cultured cells with bacterial collagenase to remove the stromal supporting cells. By this procedure they obtained 60% pure tartrate resistant acid phosphate positive (TRAP.sup.+) cells, however the osteoclasts were not available except as attached cells to either dishes or bone slices.
Oursler et al., 1991, J. Bone Min. Res. 6:375-385 cultured avian osteoclasts and further purified the osteoclasts by density gradient centrifugation. This method yielded an enriched population of TRAP.sup.+ cells capable of bone resorption.
To date, however, the only mammalian osteoclasts obtained in high yields (300,000 per rabbit) and purity (98%) are rabbit osteoclasts (Tezuka et al., 1992, Biochem. Biophys. Res. Comm. 186: 911-917). These cells, however were attached to the plastic culture dishes and cannot be removed.
DESCRIPTION OF THE INVENTION
This invention relates to a method of obtaining a highly enriched population of osteoclast precursor cells comprising co-cultivation of osteoblastic cells and bone marrow cells and treatment of the culture with an integrin .alpha..sub.v .beta..sup.3 receptor ligand to release a population which is highly enriched with precursor osteoclasts. This invention also comprises the process of obtaining a highly enriched population of mature osteoclast cells comprising reseeding the population of highly enriched precursor osteoclast cells so produced on osteoblast cells for a time sufficient for the osteoclast precursor cells to fuse into mature multinucleated osteoclasts.
This invention also relates to the enriched populations of mammalian precursor osteoclasts. Yet another aspect of this invention is a population which is highly enriched with mature osteoclasts. Further aspects of this invention is a suspension culture comprising mammalian osteoclast precursor cells, and to suspension cultures comprising mature osteoclast cells.
A further aspect of this invention is an assay for bone resorption activity comprising exposing a population of manunalian cells which is highly enriched in osteoclasts, but also comprising osteoblasts, to a bone, in the presence of Vitamin D3 or a biologically active derivative of Vitamin D3 and measuring the bone-resorption which occurs.
In accordance with this invention, it was observed that integrin .alpha..sub.v .beta..sub.3 recepto
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Rodan Gideon A.
Rodan Sevgi B.
Wesolowski Gregg
Cocuzzo Ann L.
Giesser Joanne M.
Kerr Janet M.
Merck & Co. , Inc.
Tribble Jack L.
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