Abstract
BackgroundCancer 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.MethodsHuh7 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.ResultsCells 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.ConclusionTherefore, growth factor-free medium is an adaptable, efficient, and cost-effective tool for in vitro cultivation of CSCs.
Highlights
Cancer stem cells (CSCs) constitute 1–2 % of cancer tissue and are a major cause of tumor metastasis and recurrence
Sphere formation in Huh7 and HepG2 cells Sphere formation was evaluated in the three types of media, Dulbecco’s Modified Eagle Medium (DMEM)(+)GF, DMEM(+)fetal bovine serum (FBS) and DMEM(−) FBS
Sphere formation began in DMEM(+)GF and DMEM(+)FBS cultured cells on day 5
Summary
Cancer stem cells (CSCs) constitute 1–2 % of cancer tissue and are a major cause of tumor metastasis and recurrence. Studies have been conducted to elucidate the role of CSCs in liver cancer to reduce the high recurrence rate, but the findings are controversial regarding accurate stem cell markers and cell separation methods. These studies show that the methods to efficiently culture CSCs from cancer cells are lacking, and more effective culture conditions are required. Sphere-forming culture was first used by Reynold and Weiss to isolate stem cells and has been widely used since [7] In this technique, cells are attached to an extracellular matrix (ECM) that limits the growth, proliferation, and differentiation of the cells. When cells obtained from sphere-forming culture methods were transplanted into mice, cancer formed more readily than when using an equal number of cells grown in a monolayer [13]
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