Fig. 1

Optical microscopy and proliferation analysis of Huh7 spheres in DMEM(+)GF, DMEM(+)FBS and DMEM(−)FBS medium on days 3, 7, 15, and 20. Hepatocellular carcinoma cell lines, including a Huh7 and b HepG2, could form the spheres in DMEM(+)GF and DMEM(+)FBS medium. Scale bar 100 µm, scale bar 100 µm. Huh7 and HepG2 cells were cultured in three media types (DMEM(+)GF, DMEM(+)FBS and DMEM(−)FBS media) and were observed over time. Cells were not adhered to culture plates but grew in suspension and formed spheres. Cells in DMEM(+)GF and DMEM(+)FBS media formed spheres that were similar in size, which increased after day 15. Cells in the DMEM(−)FBS medium died and were excluded from further analyses. The proliferation of sphere-forming cells in DMEM(+)GF, DMEM(+)FBS and DMEM(−)FBS was compared over time. Cells were analyzed on days 3, 7, 15, and 20 using a CCK-8 kit. c The Huh7 sphere-forming cells showed a cell proliferation. d The HepG2 sphere-forming cells showed a cell proliferation. After 3 days in culture, the proliferation of sphere-forming cells in DMEM(+)GF medium was increased. After 7 days in culture, the proliferation of DMEM(+)GF and DMEM(+)FBS media cultured cells became similar. b The HepG2 sphere-forming cells showed a cell proliferation. Cells in DMEM(+)FBS media were increased for 15 days and in DMEM(+)GF media decreased from 7 days