title: Ideal sphere-forming culture conditions to maintain pluripotency in a hepatocellular carcinoma cell lines.
abstract: 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.
  
  
  CONCLUSIONS
  Therefore, growth factor-free medium is an adaptable, efficient, and cost-effective tool for in vitro cultivation of CSCs.
authors.affiliation: Department of Surgery, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 120-752 Korea ; Graduate Program of Nano Science and Technology, Graduate School of Yonsei University, Seoul, Korea.
authors.forename: Seon Ok
authors.initials: SO
authors.lastname: Min
authors.affiliation: Department of Surgery, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 120-752 Korea ; Graduate Program of Nano Science and Technology, Graduate School of Yonsei University, Seoul, Korea.
authors.forename: Sang Woo
authors.initials: SW
authors.lastname: Lee
authors.affiliation: Department of Surgery, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 120-752 Korea ; Graduate Program of Nano Science and Technology, Graduate School of Yonsei University, Seoul, Korea.
authors.forename: Seon Young
authors.initials: SY
authors.lastname: Bak
authors.affiliation: Department of Surgery, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 120-752 Korea ; Graduate Program of Nano Science and Technology, Graduate School of Yonsei University, Seoul, Korea ; Cell Therapy Center, Severance Hospital, Yonsei University College of Medicine, Seoul, Korea.
authors.forename: Kyung Sik
authors.initials: KS
authors.lastname: Kim
keywords: Bovine serum albumin; Hep G2 cells; Neoplastic stem cells; Pluripotent stem cells