Establishment and characterization of bone cell line for blunt snout bream (Megalobrama amblycephala)

In order to study the characteristics of fish bone cells, methods for isolating and culturing bone cells from caudal vertebrae and rib of blunt snout bream Megalobrama amblycephala were explored in the present study, and the biological characters of bone cells were identified. The bone cells were cultured with tissue culture and trypsin digestion respectively by using 3-month-old M. amblycephala caudal vertebrae and ribs from tail part. The optimal conditions were as follows: 25 ng/mL epidermal growth factor (EGF) and 25 ng/mL basic fibroblast growth factor (bFGF) were added to the M-199 medium at 28 °C using tissue culture. The results of cell biology identification showed that the cultured cells had the biological activity of typical osteoblasts, with alkaline phosphatase staining, calcium nodules alizarin red staining and mineralized nodules von Kossa staining being positive. The osteocalcin content was 997.25 ng/L in the bone cells tested by the fish bone osteocalcin ELISA kit. The genes of runx2a, runx2b and osterix which regulated osteoblast differentiation, were expressed in bone cells, with the runx2b and osterix expression levels significantly higher than that in muscle cells. Results indicated that bone cells of fish have good proliferation and faster growth rate as well as stable cell biological characteristics, which is suitable for related studies in vitro.