Optimisation of primary osteoblast cell culture from suckling mouse

Abstract

Objective To develop a suitable medium and optimize culture time for the primary osteoblast culture from suckling mouse, so to provide an improved experimental protocol for primary osteoblast culture in vitro. Methods Primary osteoblasts were collected from skull of CD1 suckling mouse by interrupted enzyme digestion. The purified osteoblasts were harvested by differential centrifugation. The incubation time, concentration of fetal bovine serum(FBS),β-glycerophosphate sodium and dexamethasone were tested and optimized. The change of osteoblast maturation marker was examined by Western blot(WB) and immunofluorescence staining (IF). The osteogenic activity was determined by alkaline phosphatase staining, alizarin red staining and ultrastructure. Results Primary osteoblast were obtained from sucleling mouse skull bone by interrupted enzyme digestion for proliferation and transgenerational expansion.The expression of osteoblast maturation markers was parallel to the time of induction culture and the concentration of FBS. Mature osteoblasts were obtained by culturing the cells with 10% FBS for 14 days. The differentiation of primary osteoblasts was induced by different concentrations of β-glycerophosphate and dexamethasone. The results showed that the expression of osteoblast maturation markers was higher under the culture conditions of 10 mmol/L β-glycerophosphate and 5 nmol/L dexamethasone(P＜0.01), and the staining of alkaline phosphatase and alizarin red was obvious,and the osteogenic activity was better too. Conclusions Primary osteoblasts isolated from the skull of suckling CD1 mice cultured in induction medium containing 10% fetal bovine serum, 10 mmol/L β-glycerophosphate sodium and 5 nmol/L dexamethasone for 14 days show good osteogenic activity and are suitable for in vitro experimental studies.

Keywords

• suckling mouse|osteoblasts|primary culture