Study on Isolation and Cultivation of Rat Bone Marrow-derived Mesenchymal Stem Cells
Zhang Bensi Chen Hong Wang Song Deng Shishan Wang Fan (Department of Anatomy, School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China)
Objective To establish a method for isolation and cultivation of mesenchymal stem cells (MSCs) from rat bone marrow for offering an experimental foundation for their further differentiation and actual application.MethodsSacrifice the SD rats, separate and purify the marrow MSCs by density gradient centrifugation and by adhering to the culture plastic, select suitable culture medium and concentration of serum. After the in vitro subculture and expansion, observe the living characteristic of the cells, draw the growth curve ,measure the adhesive rate, study the morphology of the cells with phase contrast microscope, and examine the cell surface antigen and matrix protein by immunocytochemistry technique.Results The isolated MSCs belonged to the mononuclear cells of marrow, the living characteristic was quite stable in L-DMEM medium containing 100ml/L newborn bovine serum, the growh curves and adhesive rate of passage 1?3 and 5 were much similar, about 89% subcultured cells adhered the wall in 12hs, exhibiting a large expansive potential and high suitability, a population doubling time of 34h, a typical fibroblastlike morphology,and uniformly expressing CD44?CD54?fibronectin and collagen I.ConclusionThe marrow MSCs could be effectively isolated and purified by density gradient centrifugation and adhering to the culture plastic and expanded satisfactorily in L-DMEM with 10% newborn bovine serum.
【CateGory Index】： R329