Biological features and ultrastructure of rabbit mesenchymal stem cells in vitro / 解放军医学杂志

ABSTRACT

Objective To isolate and culture the rabbit mesenchymal stem cells,and explore the biological features in vitro.Methods Two milliliter of bone marrow was extracted from the tibia of juvenile rabbit under anesthesia and sterile conditions,and mesenchymal stem cells were obtained by density gradient centrifugation and inoculated to 100ml culture flask.The morphological changes,adherence rate and ultrastructure of the stem cells were observed.The surface markers and growth cycle of the mesenchymal stem cells were detected by flow cytometry,and biological features were observed by inducing the cells differentiate into osteoblasts.Results The adherence of mesenchymal stem cells started 2 hours after culturing,basically finished 20 hours later,and more than 90% of the cells were fused at the 15th day.Observed with transmission electron microscopy,the cultured cells showed typical ultrastructure of stem cells numbers of microvillus mainly distributed on one side of the cells,irregular nuclei with indentation,and lots of cell organs.Analysis for the cell surface markers indicated positive expression of CD44 and CD90 and negative expression of CD45 in mesenchymal stem cells.Cell growth cycle assay showed that most mesenchymal stem cells(about 97%) stayed in stage G1 and G2.Three weeks after osteoblastic induction,the mesenchymal stem cells gradually produced small calcium salt crystals,the cells were positive in alkaline phosphatase staining,and Von Kossa staining showed many calcium salts in the cell clone as black reaction deposits.Conclusions The rabbit bone marrow mesenchymal stem cells isolated by density gradient centrifugation are of high purity and stable growth rate,and the cells' characters can be still maintained after serial subcultivation.Since easy to operate on isolation and proliferation,the mesenchymal stem cells could be used as the tissue engineering seed cells in repairing of intervertebral disc degeneration.