Culture of rabbit’s articular chondrocytes using type Ⅱ collagenase enzyme digestion method

Abstract

Abstract:

BACKGROUND: At present, the separation and culture technique of chondrocytes has been mature, but the chondrocytes grow slowly which are prone to degenerate using the present technique. It is not conducive to the follow-up test.
OBJECTIVE: To investigate and improve the separation and culture method of articular chondrocytes of New Zealand rats at 4 weeks of age.
METHODS: New Zealand rats aged 4 weeks were selected to take cartilage tissues from the bilateral knees that were resected under aseptic condition. Chondrocytes were isolated by type Ⅱ collagenase enzyme digestion and mechanical isolation method. The cells were cultured and passaged, and then identified by morphologic observation, toluidine blue staining and type Ⅱ collagen enzyme immunohistochemical methods. Growth curve was pictured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method.
RESULTS AND CONCLUSION: Inverted microscope observation showed that the primary cultured chondrocytes adhered at 6 hours after cultivation. The monolayer formation occurred at 72 hours after cultivation, and the cells were ready to be passaged at 96 hours after cultivation. In the fourth generation, some cells represented a spindle-like appearance. In the fifth generation, most cells turned into irregular shape appearance, and cell proliferation capacity diminished. Toluidine blue staining showed that the nuclei of cultured chondrocytes were blue and cytoplasm was pale blue. Immunofluorescent staining showed that cultured chondrocytes had a positive expression of collagen type Ⅱ and the color was tawny. Proliferative rate of chondrocytes in the first to third generations had no differences (P < 0.05), while differences were found compared with the fourth generation in 4- 7 days (P < 0.05) and the fifth generation in 1-7 days (P < 0.05). The results indicate that type Ⅱ collagenase enzyme digestion and mechanical isolation method is successful for isolating, cultivating New Zealand rat articular chondrocytes in vitro, and the first to third generations can be the best choice for the experiments of knee osteoarthritis.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: chondrocytes, cell culture techniques, collagenases, cartilage, articular

CLC Number:

R318

Yan Hu, Su You-xin, Lin Xue-yi, Chen Bao-jun, Zhou Bi-hong, Zhang Qing. Culture of rabbit’s articular chondrocytes using type Ⅱ collagenase enzyme digestion method[J]. Chinese Journal of Tissue Engineering Research.

Figures/Tables 3

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