Studies On Culturing Of Human Mesenchymal Stem Cells Under Serum-free Condition

Objective.To establish a method of culturing mesenchymal stem cells from bone marrow,and provide seed cells for tissue engineering of bone.Methods:The mononuclear cell fraction of mesenchymal stem cells was isolated,then MSCs were cultivated in serum-containing medium and serum-free medium,comparing the cells growth conditions in the two different medium.Identification of surface markers and cell cycle analysis of MSCs were performed with flow cytometry.Results:In serum-containing medium,adherent cells appeared 2～3 days after plating of mononuclear cells.The cells formed adherent heterogeneous cell populations after about 1 week in culture,and they consisted of round and spindle-like cells in 12～14 days.In serum-free medium,adherent cells appeared 4～5days after plating of mononuclear cells.The cells formed adherent heterogeneous cell populations after 8～10days in culture,and they consisted of round and spindle-like cells in 16～20 days.Though colonies in the serum-containing medium formed earlier than those in the serum-free medium,passage times and cell morphology were the same.By flow cytometry analysis,cells of two groups were negative for CD13,CD34 and CD45,but strongly positive for specific surface markers such asCD29,CD54 and CD105.The percentage of cells at G₀/G₁cell cycle was higher in serum-free group than that of serum-containing group with significant difference(P＜0.05).Conclusion:1.The self-preparation serum-free medium can be used to culture and amplify human bone marrow MSCs.2.The percentage of cells at G₀/G₁cell cycle was higher in serum-free group than that of serum-containing group with significant difference.Comparing to serum-containing medium,serum-free medium can preserve the stem cells much better.