Improved methods for testing antiresorptive compounds in human osteoclast cultures.
We cultured human bone marrow-derived stem cells on bovine bone slices in 96-well plates in the presence of M-CSF and RANKL, allowing them to differentiate into osteoclasts. Secreted TRACP 5b was a useful endpoint measurement to demonstrate effects of inhibitors of osteoclast differentiation in the culture system, reflecting accurately the number of formed osteoclasts. Inhibitors of osteoclast activity were added into the cultures after the differentiation period, and the cultures were continued to allow the formed osteoclasts to resorb bone. CTX values obtained after the resorption period were normalized with TRACP 5b values obtained after the differentiation period, before adding the inhibitors. This normalization prevents false results that could be obtained from the presence of different amounts of osteoclasts in different wells before adding the inhibitors. These results demonstrate that the use of TRACP 5b and CTX allows rapid and reliable testing of antiresorptive compounds in human osteoclast cultures.
Rissanen
Jukka P
JP
Pharmatest Services Ltd., Itäinen Pitkäkatu 4 C, 20520 Turku, Finland.
Ylipahkala
Hannele
H
Fagerlund
Katja M
KM
Long
Clive
C
Väänänen
H Kalervo
HK
Halleen
Jussi M
JM
D000135:Acid Phosphatase / Q000378:metabolism; D000818:Animals; D050071:Bone Density Conservation Agents / Q000494:pharmacology* / Q000627:therapeutic use; D001862:Bone Resorption / Q000188:drug therapy / Q000503:physiopathology; D002417:Cattle; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000071681:Tartrate-Resistant Acid Phosphatase
D050071:Bone Density Conservation Agents; D024042:Collagen Type I; D007527:Isoenzymes; C000605510:ACP5 protein, human; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase