A modified method for in vitro isolation and cultivation of periosteal cells in rabbits

doi:10.3969/j.issn.2095-4344.2016.24.004

Abstract

Abstract:

BACKGROUND: Periosteum is considered as a source of seed cells for cell therapy due to its biological features.
OBJECTIVE: To seek the optimal way to isolate and culture rabbit periosteal cells and identify their biological features.
METHODS: Rabbit periosteum on facies medialis tibiae was taken out under aseptic conditions. Periosteal cells isolated through the digestion of type II collagenase with the explants culture method were cultured in DMEM/F12 complete medium. Cell ultrastructure was observed under an inverted microscope. Periosteal cell proliferation was determined by cell counting kit-8 assay. Cell surface antigens CD90 and CD105 were determined using flow cytometry. Osteogenic and lipogenic induction mediums were applied to induce periosteal cells to differentiate into osteocytes and adipocytes, respectively. After 2 weeks of induction, cells were harvested for alizarin red staining and oil red O staining to assay the calcium nodules and lipid droplet.
RESULTS AND CONCLUSION: The digestion of type II collagenase with the explants culture method shortened the period of primary cells culture and enhanced the survival rate, which caused higher purity and stronger reproductive activity of harvested periosteal cells. Primary cultured periosteal cells grew in form of spindle spiral or parallel. Alizarin red and Oil red O staining verified the multi-directional differentiation potentiality of periosteal cells. These findings suggest that the periosteal cells with high purity, strong reproductive activity, and multi-directional differentiation potentiality can be harvested in short time using digestion of type II collagenase with the explants culture method.

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

Key words: Periosteum, Cell Separation, Collagenases, Cell Differentiation, Tissue Engineering

CLC Number:

R318

Zhang Jun-wei1, Lu Hai-tao1, Yuan Feng2, Yang Yu-ming1. A modified method for in vitro isolation and cultivation of periosteal cells in rabbits[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(24): 3523-3528.

Figures/Tables 3

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