Culturing in vitro and phenotype of human mesenchymal stem cells

Objective To isolate and culture the mesenchymal stem cell(MSCs) of human bone marrow in vitro and then study its biological characteristics.Methods To isolate,purify the MNCs from human bone marrow by density gradient centrifugation and then passage them in vitro by adherent culture; observe the cells′ gowth by inverted phase contrast microscope; observe the cell′s morphous and structure by Wright′s staining and transmission electron microscope; detect the cell membrane′s mark and reproductive cycle by flow cytometer; identify the cultured cells by immunocytochemistry and cytochemical staining methods.Results The primary cells and cultured passage cells mostly were fibroblast cells which had powerful capacity of growth and reproduction. The membrane mark showed that CD29 and CD105 are positive while the CD34 was negative; the cell cycle analysis showed that more than 90% cells were in the G_0/G_1 phase; the expression of CD106,fibronection were positive and the expression of PAS was strong positive, the expression of CD45,CD14,laminin,collagen protein Ⅱ and ALP were negative. All the above results shows that the cells are mesenchymal stem cells instead of hematopoietic cell.Conclusion Mesenchymal stem cells can be isolated and cultured by means of density gradient centrifugation and adherent culture, and the mesenchymal stem cells have special biological characteristics.