Isolation and Characterization of Highly Enriched, Prefusion Mouse Osteoclastic Cells

• Published in: Experimental cell research 1995-08, Vol.219 (2), p.679-686
• Main Authors: Wesolowski, Gregg, Duong, Le T., Lakkakorpi, Päivi T., Nagy, Rose M., Tezuka, Ken-Ichi, Tanaka, Hirofumi, Rodan, Gideon A., Rodan, Sevgi B.
• Format: Article
• Language: English
• Subjects: Animals; Bone Marrow Cells; Cell Separation - methods; Cells, Cultured; Integrins - biosynthesis; Mice; Osteoclasts - cytology; Osteoclasts - metabolism; Osteopontin; Peptides - pharmacology; Sialoglycoproteins - biosynthesis; Viper Venoms - pharmacology
• ISSN: 0014-4827; 1090-2422
• DOI: 10.1006/excr.1995.1279

This study describes the isolation and characterization of highly enriched mammalian osteoclast precursors, released by the "disintegrin" echistatin, from an osteoclast formation culture. Incubation of a 6-day coculture of mouse bone marrow cells and mouse osteoblastic cells (MB1.8) with echistatin (30 n M), an RGD-containing snake venom, for 20 min yielded an 88-95% pure population of tartrate-resistant acid phosphatase-positive cells, 1.5 × 10 5 cells per 150 cm 2 culture dish. These cells were mostly mononucleated and based on the following characteristics are considered to be prefusion osteoclasts (pOC cells): (i) presence of calcitonin (CT) receptors documented by 125I-sCT auto-radiography and cAMP generation in response to salmon calcitonin; (ii) expression of mRNAs for α vβ 3 integrin, osteopontin, 92-kDa type IV collagenase (matrix metalloproteinase 9), carbonic anhydrase II, OC-2 (an "osteoclastic" cysteine proteinase), and protein tyrosine phosphatase ϵ; and (iii) high level expression of pp60 c-src protein. The pOC cells resorb bone (form "pits" on bone slices) but only in the presence of osteoblastic MB1.8 cells and 1,25(OH) 2D 3. Resorption was inhibited by CT. In conclusion, we describe a rapid, reproducible procedure to isolate virtually pure mammalian prefusion osteoclasts, which should help in the study of osteoclast formation, composition, and function.