Sphere-forming cultivation of liver cancer cell line in culture media without growth factors

Purpose: Cancer stem cells have the characteristics similar to the normal stem cell but are cancer cells. Sphere forming culture has been adapted as a culture method for cancer stem cells. In this study, we validated to optimal culture condition for sphere formation of hepatocellular carcinoma cell line using three different types of culture media. Methods: Huh7 cells were plated in different culture media allowed for sphere formation. M1, Dulbecco’s Modified Eagle’s medium (DMEM)-high glucose with fetal bovine serum (FBS); M2, DMEM-high glucose without FBS; and M3, DMEM-F12 (Gibco, Grand Island, NY, USA) with B27, epidermal growth factor, basic fibroblast growth factor (bFGF, Invitrogen, Seoul, Korea). Sphere formation was observed by light microscope. Proliferation of the sphere-forming cells was evaluated by Cell counting kit-8 (CCK-8) analysis. Results: Cells in M1 and M3 media were formed sphere. Sphere-forming cells in M1 and M3 were bigger than M2 cells and had similar morphology. Sphere-forming cells in M3 media showed higher level of cell proliferation than M1 and M2 cells on 15 days, while sphere-forming cell in M1 media were exhibited higher level than others on post 20 days. Proliferation of M2 cells did not improve. Conclusion: Results showed that sphere-forming cells in M1 and M3 media had similar character. So, it is assumed that a growth factor-free medium is adaptable or efficient tool for in vitro cultivation of cancer stem cells, because the tool shall be reduced expense for experiments without the adding of growth factors.