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Updated: Jun 25, 2026
Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice
Published on: January 6, 2018
[Effect of osteoblast conditioned culture medium on differentiation of BMSCs]
Kunpeng Zhou1, Xiaohe Chen, Li Chang
1Division of Stem cell and Tissue Engineering, State key Laboratory of Biotherapy, West China Hospital of Sichuan University, Chengdu Sichuan, 610041, P.R. China.
Objective:
To study the effect of rat osteoblast conditioned culture medium on the BMSCs differentiation of allogeneic rat and to find a new approach to provide seed cells for bone tissue engineering.
Methods:
BMSCs and osteoblasts were harvested from 10 healthy one-week-old SD rats (male and female, weighing 20-30 g) by adherent method and enzyme digestion method respectively. Cell identification was conducted. Osteoblast conditioned culture medium was prepared by mixing supernatant of osteoblasts at passage 1-5 with complete medium (1:1). Then, BMSCs at passage 2 were co-cultured with osteoblast conditioned culture medium (inducement group) and complete medium (control group), respectively. The morphological changes of co-cultured BMSCs were observed by inverted phase contrast microscope, the growth condition of BMSCs was detected by MTT method, the expressions of ALP, Col I and osteocalcin (OCN) in the co-cultured BMSCs were tested by immunohistochemistry staining, and the expressions of Col I and OCN mRNA were detected by RT-PCR.
Results:
In the inducement group, BMSCs grew bigger, changing from long fusiform to flat and polygon with protuberance 7 days after co-culture; the presence of cell colony-like growth was observed 9 days after co-culture. Cell growth curve demonstrated that the counts of BMSCs was increased with time, there were more cells in the control group than that of the inducement group, and there was a significant difference in cell counts between the control and the inducement group 4-7 days after co-culture (P < 0.05). For the inducement group, ALP staining was positive 12 days after co-culture, the calcium nodules were appeared 18 days after co-culture, Col I and OCN were positive 21 days after co-culture, and the expressions of Col I and OCN mRNA were detected by RT-PCR 21 days after co-culture.
Conclusion:
Rat osteoblast conditioned culture medium can significantly induce the differentiation of allogeneic rats' BMSCs towards osteoblasts.
