Experimental study of primary culture and multi-directional differentiation of rabbit bone marrow derived mesenchymal stem cells in vitro

Objective To culture bone marrow derived MSCs (mesenchymal stem cells) in vitro,and then observe its biological characteristics and potentiality of multi-directional differentiation.Methods To isolate,purify and amplify the MSCs from rabbit bone marrow by density gradient centrifugation and adherent culture in vitro,observe the characteristics of MSCs morphology by inverted phase contrast microscope and transmission electron microscope ;survey and map MSCs growth curve ;detect cell membrane's marks and cell cycle by flow cytometer;MSCs were induced into osteogenetic cells in the osteogenesis supplement medium or adipose cells in adipogenesis supplement medium,and then they were verified.Results The primary cells and the passage cells were mostly fusiform in shape,to be similar to fibroblast cell.the growth curve showed the growth regularity of the passage cells was similar; the flow cytometer analysis showed that the membrane mark CD44 was positive,CD45 was negative; the cell cycle analysis showed that more than 87% cells were in G0/G1 phase; after MSCs were induced,cell alkaline phosphatase stain showed that alkaline phosphatase was positive in osteogenetic cell,oil red O stain showed that red lipid droplet existed in adipose cell.Conclusion MSCs can be isolated and cultured by the method of density gradient centrifugation and adherent culture in vitro,they have the better potentiality of proliferation and multi-directional differentiation.