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Background  Cancer stem cells (CSCs) constitute 1–2 % of cancer tissue and are a major cause of tumor metastasis and recurrence. The culture environment is important in maintaining CSCs in vitro. Sphere formation is one method of culturing CSCs. In this study, we identified and validated optimal culture conditions for sphere formation in hepatocellular carcinoma cells.    Methods  Huh7 and HepG2 cells were plated in three different media types and were allowed to form spheres. To confirm the pluripotency of sphere cells, the expression of stem cell markers was evaluated. EpCAM, Connexin 32, and Connexin 43 expression was evaluated using reverse transcription-polymerase chain reaction (RT-PCR). The expression of E-cadherin, β-catenin, CD90, and CD133 was evaluated using immunocytochemistry. Flow cytometry was performed to confirm the presence of stem cell markers CD133 and Connexin 32.    Results  Cells maintained in medium containing growth factors ((DMEM(+))GF) showed greater cell proliferation than cells in media with fetal bovine serum (FBS) (DMEM(+)FBS) or without FBS (DMEM(−)FBS). Cells cultured in DMEM(+)FBS medium exhibited greater proliferation after 20 days in culture than cells cultured under the other two conditions. Cells cultured in DMEM(−)FBS medium did not proliferate; therefore, this condition was removed from further analysis. RT-PCR and flow cytometry showed that sphere-forming cells cultured in DMEM(+)GF and DMEM(+)FBS media had similar expression of stem cell markers.    Conclusion  Therefore, growth factor-free medium is an adaptable, efficient, and cost-effective tool for in vitro cultivation of CSCs.

Ideal sphere-forming culture conditions to maintain pluripotency in a hepatocellular carcinoma cell lines