Experimental Study Of Isolation, Culture And Induction Of Rabbit Adipose-derived Stem Cells

Objective: The purpose of this research is to isolate,culture rabbit ADSCs, to observe biologic characters and multipotential differentiation phenotype of ADSCs,and to make preparation for deeper research of tissue engineering. Methods: The adipose tissue is fully Shredded, digested, centrifuged. The cell suspension placed in culture dishes is cultured in incubator, the primary cells are gained and carried out Subculture and observe the proliferation ability in vitro. After adipogenic induction, the cells are stained for oil red O. Select the third generation of ADSCs, by adding osteogenic induction medium 2 times per week, and the hole joining the control medium is a negative control, for ALP, Alizarin red, Von Kossa staining. Detect ALP, OPN, OCN, and ONN expression by immunofluorescence and RT-PCR. Results: When inoculation afte 24 hours, it showed that a little of large cell begins adherent, with large, flat monolayer. After 48h the majority of cells were adherent, they were spindle-shaped.After 5-7 days, the cells gradually fission, integration into the monolayer. After Subculture the cells were spindle-shaped passage, the nuclear was in center, the majority of them had a nucleolus which was round or oval. Cell growth curve showed the fast-proliferating cells from 3 to 7 day. After adipogenic induction, it confirmed that the formation of lipid droplets were in the cells by the oil red O staining. The ALP, Alizarin red, Von Kossa staining results are positive after Osteogenic induction. by immunofluorescence and RT-PCR detection, ALP, OPN, OCN, and ONN are expressed. Conclusions: We can isolate fibroblast-like cells from rabbit subcutaneous adipose tissue,culture and passage them in vitro by primary cell culture technique.Rabbit ADSCs can be induced to osteocytes and adipogenesis in given mediums.