Isolution And Culturing Rabbit Mesenchymal Sstem Cells In Vitro And Osteogensis

OBJECTIVE: To investigate the proliferative and multilineage potential cells of MSCs in vito,as seeding cells of tissue engineering,a kind of which were isolated from adult bone marrow,cultured and observed on biological properties.METHODS:The rabbit MSCs were aspirated from the iliac crests.Mononuclear cells were separated by centrifugation in Percoll solution(1 .073g/ml) followed by the method of Pitenger M.F. and Majumda MK.Then they were seeded in DMEM-LG supplemented with 15% fetal calf-serum at a concentration of 2× 10⁵cells/cm² in culture flask.Clutures were maintained at 37℃ in a humidified atmosphere chamber containing 5% CO₂.The medium was changed 48-72 hours,nonadherent cells were removed with changed in medium,and twice weekly therafter.When the culture reached nealy 80-90% of confluence,cells were trypsinized with EDTA,and passaged at 5 × 10³ cell/cm² to subculture.In order to investigare the biological charateristics of MSCs,RESULT:1 .Morphology and ultrastruture of MSCs:A samall percentage of isolated cells were adherent to the flasks and grow as typically fibroblastic or sprindle shape,which proliferated rapidly.Primary cultures reached 80-90% of confluence in about 7 to 10 days.Nonadherent or loosely adherent small round cells were present ound cells in primary cultures.2.Osteogensis Differentiation:When MSCs were grown in osteogensisinducing medium,the cells showed typical cuboidal shape.The trial groups stably expressed Alkaline Phosphatase while the controlled group remained at low level.CONCLUSION: 1 Jt is a simple,effective and pratical method to separate and obtain higher purity and activity of MSCs from the bone marrow of rabbits by gradeient centrifugation in percoll.2 -. MSCs that are in loe abundance can grow quickly when culture vitro.MSCs can differentiate to be osteogenic phenotype when cultured in a difined medium.MSCs can meet the demand of bone tissue engineering by Percol density gradient centrifugtion,through culturing and induced in vitro.This study can prove that after being induced MSCs can have the strong osteogenic activity and proper proliferative activity...