ABSTRACT
Objective:To isolate SD rat adipose-derived stem cells(ASCs)by suspended explant culture method.Methods: The healthy rat inguinal fat pads were obtained.The SD rat ASCs were isolated by suspended explant culture method and explant adherent culture method.The growth status and morphology were observed.The growth curve and cell surface markers CD29,CD44,CD45 of the 3rd passage cells were analyzed respectively by CCK-8,immunocytochemistry;the 3rd passage cells were induced individually by adipogenic differentiation medium and osteogenic differentiation medium,and cells were examined by oil red O staining and alizarin red staining.Results: The SD rat ASCs obtained by the two methods exhibited a spindle-shaped appearance and could rapidly expand.The cell growth curves were typical of S type.Immunocytochemistry analysis revealed that the third passage of SD rat ASCs were positive for CD29,CD44,but were negative for CD45.SD rat ASCs were positive for oil red O staining at 14 days after adipogenic induction,and positive for alizarin red staining at 14 days after osteogenic induction.Conclusion: Isolation of SD rat ASCs by suspended explant culture method is successfully established.The method is simple.It provides a new method for the isolation of SD rat ASCs in vitro.
ABSTRACT
Objective To study the effect of cryopreservation on some biological properties of rabbit adipose -de-rived mesenchymal stem cells (rADMSCs).Methods rADMSCs culture was isolated by tissue explants adherent meth-od.Morphology of the primary cells was observed by inverted microscopy .Immunophenotypes of the rADMSCs were deter-mined using flow cytometry .The third passage cells were preserved in liquid nitrogen for 6 months, and then were thawed , and subcultured to passage 7.The growth curves of the cryopreserved cells were analyzed by MTT assay , and the cryopre-served cells were cultured in adipogenic and osteogenic medium , with non-cryopreserved rADMSCs as a control group .The adipogenic and osteogenic abilities of the rADMSCs were evaluated by oil red O staining , alizarin red staining and alkaline phosphatase activity assay , respectively.Results The rADMSCs cultured in vitro exhibited a spindle-shaped appearance and rapid growth expansion .Flow cytometry analysis revealed that the third passage rADMSCs were CD 44-and CD90-posi-tive, but negative for hematopoietic cells surface marker CD 45.The growth curves of cells in the experimental and control groups were “S” shaped, showing a non-significant difference between the two groups (P>0.05).The oil red O staining and alizarin red staining results were positive at 2 weeks after adipogenic and osteogenic induction .The ALP activities of the two groups were increased with osteogenic induction time , with a non-significant difference (P>0.05).Conclusions Cryopreservation does not affect the growth and differentiation pluripotency of rADMSCs significantly .