Methods to Isolate, Culture, and Study Osteoblasts

Abstract

Primary cells are often desirable over clonal cell lines, because they are more likely to retain the presence and activity of certain enzymes and proteins that are often lost in clones. In addition, primary cells more closely resemble the cell in the actual animal than clones do. An often-cited drawback of primary cells is the heterogeneity inherent in them. Primary osteoblast cultures, for example, consist of preosteoblasts, osteoblasts, and osteocytes (if the culture is old enough), and possibly even a few fibroblasts Such a mix of osteoblastic cells, however, could be deemed beneficial and more realistic. The most detrimental cell contaminant is the fibroblast. Fibroblasts, if present in the culture, will eventually take over. We have never had this problem with our cultures Osteoblastic cultures isolated by the method described in this chapter have been shown to be osteoblastic in nature by exhibiting extensive alkaline phosphatase activity (1–3), by forming nodules that stain positive for mineral by a Von Kossa stain (1–5, and by showing a characteristic increase (three-fold) in intracellular cAMP in response to PTH stimulation (2.7×10⁻⁸ M for 15 mm) (1).