[Isolation and purification of BMScs of GFP transgenic mouse using the method of adhering to cuture plastic in different time].
OBJECTIVE
To adopt the method of adhering to culture plastic in different time for cultivating and purifying BMSCs of green fluorescent protein (GFP) transgenic mice.


METHODS
Bone marrow cells isolated from GFP transgenic mice are directly planted in culture flask and an exchange of the total volume medium is made at different time. Then the cells adhering to culture plastic are differently counted according to the cell types and are examined by immunohistochemistry using the antibodies of CD44, CD45 and CD54 in three days. Moreover, the cells after the exchange of the total volume medium in 4 hours, 8 hours and 24 hours are selected and successively subcultured down to the fifth passage. Then the result of amplification is calculated and the cells are examined by immunohistochemistry using the antibodies of CD44, CD45 and CD54.


RESULTS
With the extending of the time for the first exchange of medium, the density of cells adhering to culture plastic increased accordingly, but the BMSCs proportion decreased. The cells after first exchange of medium in 4 hours had high BMSCs proportion but low BMSCs density, and the cells in 24 hours had high BMSCs density and low BMSCs proportion. However, the cells in 8-10 hours had high BMSCs density and also high BMSCs proportion. The subcultured BMSCs could stably express GFP.


CONCLUSIONS
The method of adhering to culture plastic in different time for cultivating and purifying BMSCs of GFP transgenic mice is effective. It is suitable to make the first exchange of total volume medium in 8-10 hours. The subcultured cell has the capacity for amplification and will probably be a seed cell for the research of tissue engineering and gene therapy.
Li
Fu-Qiang
FQ
Department of Anatomy, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Zhou
Hong-Ying
HY
Yang
Hui-Lun
HL
Xiang
Tao
T
Mei
Yan
Y
Hu
Huo-Zhen
HZ
Wang
Ting-Hua
TH
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D051379:Mice; D008822:Mice, Transgenic
D049452:Green Fluorescent Proteins