Preparation and characterization of a mouse osteoclast-like multinucleated cell population

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Abstract
We have reported that numerous tartrate-resistant acid phosphatase-positive osteoclast-like multinucleated cells (TRAP+ MNCs) are formed when mouse osteoblastic cells and spleen cells are cocultured in the presence of 1α25-dihydroxyvitamin D3 [1α,25-(OH)2D3] (Endocrinology 123:2600, 1988). In this study, we prepared a TRAP+ MNC population using a modified coculture system and examined its osteoclastic properties. TRAP+ MNCs were formed in cocultures of mouse osteoblastic cells and marrow cells on 10 cm collagen gelcoated dishes. The TRAP+ MNC population was prepared by treating the dishes with 0.2% bacterial collagenase followed by density gradient centrifugation. The yield of TRAP+ MNCs was 20,000–40,000 cells per dish, much higher than that of osteoclasts (OCLs) isolated from neonatal rat bones (∼ 1000 cells per head). The purity of TRAP+ MNCs was 5.6 ± 0.6% in cell number and about 30% in the number of nuclei. The recovery of TRAP+ MNCs after density gradient centrifugation was 30–40%. Acid production by MNCs was demonstrated by vital staining with acridine orange. Numerous resorption pits were formed when the MNC population was cultured for 48 h on bone slices. Autoradiography using [125I]salmon calcitonin (CT) showed abundant CT binding in most TRAP+ MNCs. Saturation analysis of [125I]salmon CT indicated a dissociation constant Kd for TRAP+ MNCs of 8.9 ± 0.7 × 1010 M and 16.5 ± 1.5 ± 106 binding sites per cell. These results were similar to the value (3.5 × 10−10 M) and the number of binding sites (3.3 × 106 per cell) in isolated rat OCLs. Displacement curves for [125I]salmon CT with unlabeled salmon and human CT were similar in MNC and OCL preparations. Salmon and human CT increased cAMP production (maximal response: salmon CT at 10−10 M, human CT at 10−8 M; ED50: salmon CT, 2.2 × 10−11 M, human CT, 1.3 × 10−9 M) in the MNC preparation. These results indicate that a large number of mouse TRAP+ MNCs possessing OCL characteristics can be easily prepared from in vitro cultures. This procedure will facilitate examination of mammalian OCL functions.
Funding Information
  • Ministry of Science, Education and Culture of Japan (01480437, 01870078)