Culture of rabbit osteoblasts digested by collagenase in DMEM containing fetal bovine serum
- VernacularTitle:含胎牛血清胶原酶消化法培养兔成骨细胞
- Author:
Chao CHEN
;
Guanghui LI
;
Anmin CHEN
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2006;10(41):223-225,封3
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: The skill to culture osteoblasts primarily has been well developed, however, variousdigestive enzymes can affect membrane protein of osteoblasts when they are used on primary tissue separately.OBJECTIVE: To avoid the damage from enzyme to cell as best possible,digest cranial osseous tissue with collagenase in DMEM containing fetal bovine serum and carry out in vitro culture of osteoblasts, then observe the effect of collagenase in DMEM containing fetal bovine serum on the digestion of osteoblasts.DESIGN: Control experiment.SETTING: Department of Orthopaedic Surgery, Tongji Hospital Affiliated to Huazhong University of Science and Technology.MATERIALS: This experiment was carried out in the Department of Orthopaedic Surgery, Tongji Hospital Affiliated to Huazhong University of Science and Technology from March to December 2004. Two Japanese white rabbits with big ears that were pregnant for 28 days were used in the experiment.METHODS: Type I collagenase with the same batch number was prepared into 0.1% collagenase with fetal bovine serum and 0.1% collagenase without fetal bovine serum by using culture medium containing 0.15 volume fraction of fetal bovine serum and DMEM solution respectively, serving as the enzyme digestive juice A and B for experimental group and control group respectively. Abdominal delivery was performed to take ont the fetal rabbit which was at embryonic 28 days under aseptic condition.Then, craniums of fetal rabbits were dissected, rinsed and chipped into pieces. 5 mL solution A and 5 mL solution B were added into the experimental group and control group at 37℃, respectively. Culture solution containing 0.15 volume fraction of fetal bovine serum was gradiently diluted into 0,1,2 and 4 folds in each group, and continued to digest and culture osteoblasts. The cellular survival rate was measured with trypan blue staining, and the osteoblast and its purity were identified with alkaline phosphatase(ALP) staining. Cellular growth was observed under the microscope.MAIN OUTCOME MEASURES: ①Cellular survival rate of two groups was detected with trypan blue staining. ② Cellular purity of two groups was detected with ALP staining in the cells. ③Cellular growth was distinguished by observing cellular morphology under the microscope.RESULTS: ① Results of trypan blue staining: Through cell counting, the rate of cells, which refused to be stained, of the experimental group reached 98%, and the cellular survival rate was high; that of control group reached 95%, and the cellular survival rate was also very high. ② Results of ALP staining in the cells: The area of ALP staining positive of experimental group was not less than 95%, and cellular purity was very high; but that of control group was not more than 75%, and cellular purity was very low. ③ Observation results under the microscope: Cells of experimental group were well stacked, convex and stereoscopic, they grew prosperously and had enough nutrition; while those of control group were not significantly in comparison with experimental group.CONCLUSION: Collagenase containing fetal bovine serum is used to digest the osseous tissue of cranium of fetal rabbits, and the cultured osteoblasts have typical properties of osteoblasts with simple component and high survival rate.
