Optimisation of primary osteoblast cell culture from suckling mouse
10.16352/j.issn.1001-6325.2024.02.0159
- VernacularTitle:乳鼠原代成骨细胞的培养优化
- Author:
Zuoyu WANG
1
;
Yang ZHOU
;
Junwei YANG
Author Information
1. 南京医科大学第二附属医院 肾脏病中心,南京 210003
- Keywords:
suckling mouse;
osteoblasts;
primary culture
- From:
Basic & Clinical Medicine
2024;44(2):159-166
- CountryChina
- Language:Chinese
-
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.Meth-ods Primary osteoblasts were collected from skull of CD1 suckling mouse by interrupted enzyme digestion.The pu-rified osteoblasts were harvested by differential centrifugation.The incubation time,concentration of fetal bovine se-rum(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 ac-tivity 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 trans-generational 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 dexam-ethasone.The results showed that the expression of osteoblast maturation markers was higher under the culture con-ditions 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 osteo-blasts isolated from the skull of suckling CD1 mice cultured in induction medium containing 10%fetal bovine ser-um,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.