title: Establishment of a rapid bone resorption in vitro assay using previously frozen mouse unfractionated bone cells pretreated with parathyroid hormone.
abstract: We established a useful assay system for evaluating osteoclast-mediated bone resorption based on the use of unfractionated bone cells obtained from 10- to 11-day-old mice. When cells from 10 to 11 mice were treated for 7 days with rat parathyroid hormone (rPTH, 10(-8) M), a total of 4 to 5 x 10(7) cells could be obtained from the culture by treatment with 0.05% trypsin and 0.02% EDTA in PBS. These harvested cells contained about 20% tartrate-resistant acid phosphatase (TRAP)-positive mononuclear cells. When the harvested cells were cultured on dentine slices without rPTH, after 1 day, they formed TRAP-positive multinucleate cells that were active in bone resorption. Eel calcitonin (eCT) decreased the number of pits in a dose-dependent manner, and its half maximal inhibition dose (ID50) was 1.08 x 10(-11) M. Even after having been frozen in liquid nitrogen for 5 months, upon thawing, these cells were capable for forming pits; and this pit formation was inhibited by eCT. Since no appropriate osteoclastic cell line for evaluating bone resorption is available at present, this system can provide a useful, practical means for assaying osteoclastic bone-resorbing activity.
authors.affiliation: Laboratory for Cell Biology, Pharma Research Laboratories, Hoechst Japan Limited, Saitama.
authors.forename: K
authors.initials: K
authors.lastname: Kitamura
authors.forename: M
authors.initials: M
authors.lastname: Katoh
authors.forename: O
authors.initials: O
authors.lastname: Komiyama
authors.forename: H
authors.initials: H
authors.lastname: Kitagawa
authors.forename: F
authors.initials: F
authors.lastname: Matsubara
authors.forename: M
authors.initials: M
authors.lastname: Kumegawa
mesh_terms: D000135:Acid Phosphatase / Q000097:blood; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D001862:Bone Resorption / Q000473:pathology*; D002478:Cells, Cultured; D005615:Freezing; D007958:Leukocyte Count; D007963:Leukocytes, Mononuclear / Q000201:enzymology; D051379:Mice; D008813:Mice, Inbred ICR; D010010:Osteoclasts / Q000187:drug effects*; D010281:Parathyroid Hormone / Q000494:pharmacology*; D015203:Reproducibility of Results; D013644:Tartrates; D013997:Time Factors
chemical_list: D010281:Parathyroid Hormone; D013644:Tartrates; D000135:Acid Phosphatase; C029768:tartaric acid