[Isolating and culturing rat marrow mesenchymal stem cells and studying their phenotypical and functional properties].
OBJECTIVE
To establish a method for isolating and cultivating mesenchymal stem cells (MSCs) from SD rat bone marrow and to study their phenotypical and functional properties.


METHODS
MSCs from rat bone marrow were separated and purified by gradient centrifugation and adherence to the culture plastic; then the cells were expanded by subculture successively. The growth curves were drawn, and the morphology was observed. In an attempt to analyze immuno- and adhesive-phenotype and differentiation properties, the MSCs were evaluated with cytochemical and immunocytochemical methods.


RESULTS
MSCs belonged to the mononuclear cells of marrow, they could be isolated and purified by gradient centrifugation and adherence to the culture plastic; their living behavior was quite stable in Dulbecco's Modified Eagle's Medium with low glucose (L-DMEM) containing 100 ml/L newborn bovine serum; the growth curves of passage 1, 3 and 5 were much similar, exhibiting a 2.2-fold increase in cell number after each passage. The cells were noted to have a large expansive potential and a typical fibroblast-like morphology, and they uniformly expressed CD44, CD54, fibronectin and collagen I. When incubated in medium supplemented with dexamethasone, beta-glycerophosphate and ascorbic acid, the MSCs underwent differentiation into osteoblasts, showing positive stain of alkaline phosphatase activity and mineralized nodules.


CONCLUSIONS
The cells obtained in this experiment possess phenotypical and functional properties of mesenchymal stem cells, and the method for isolating and culturing rat marrow-derived MSCs has been established.
Zhang
Bensi
B
Department of Anatomy, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Wang
Fan
F
Deng
Li
L
Dun
Aishe
A
Dong
Lihua
L
Li
Jian
J
Yang
Huijun
H
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002469:Cell Separation / Q000379:methods*; D018960:Hyaluronan Receptors / Q000378:metabolism; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D010641:Phenotype; D051381:Rats; D017207:Rats, Sprague-Dawley
D018960:Hyaluronan Receptors; D018799:Intercellular Adhesion Molecule-1