ABSTRACT
Objective To establish a method of culturing mesenchymal stem cells from umbilical cord blood. Methods The mononuclear cell fraction of umbilical cord blood was isolated with the aid of a Ficoll/Hypaque gradient, then MSC were cultivated in serum containing medium or serum-free medium. The serum containing medium (SC) consisted of LG-DMEM supplemented with 10% fetal calf serum and 10-6M hydrocortisone, with a final cell concentration of 1?106 cells/ml. The serum-free medium (SF) was supplemented with 1?10 -6M hydrocortisone and 40ng/ml fibronectin with a final cell concentration of 1?106 cells/ml. After a week, 50% of the media were renewed. Before reaching the monolayer phase cells were trypsinised and re-cultured under similar conditions for the another 3 weeks. Cells were counted at the beginning and the end of the culturing period. Phenotypes were identifiad by PAS, NAE, ALP staining. Identification of surface markers and cell cycle analysis of MSCs were performed with flow cytometry. Results Adherent cells appeared 24h after plating of mononuclear cells. The cells formed adherent heterogeneous cell populations after 4-7 days in culture, and they consisted of round and spindle-like cells. In the primary passage of culture, the cells proliferated slowly and became confluent in 14-20 days. When subcultured, the heterogeneous cell populations became homogeneous assuming flat and fibroblast-like shape. Though colonies in the medium containing serum formed earlier than those in the serum-free medium, passage times and cell morphology were the same. The total cell numbers in SF group were lower than those of SC group. The percentage of cells at G0-G1 cell cycle was higher in SF group than that of SC group with significant difference. By flow cytometry analysis, cells of two groups were negative for CD34, CD13 and CD45, but strongly positive for specific surface markers such as CD166, CD29, CD105 and CD54. Conclusion Serum-free medium was superior to medium containing serum for culturing MSCs.