Huh7 cells

Human hepatocellular carcinoma Huh‐7 cells were purchased from the Riken Cell Bank (Ibaraki, Japan) and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C in a 5% CO₂ humidified atmosphere.^[37] For the levitation experiments, Huh‐7 cells were harvested by trypsinization (or 0.5 mM EDTA at pH 7.4 in PBS) and then suspended in DMEM containing 10% FBS at a concentration of 8  × 10³ cells/µL (Huh‐7 cell suspension).

The human HCC cell line SMMC-7721 and the immortalized hepatocyte cell line L-02 were purchased from the Cell Bank of the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). MHCC-97 L, MHCC-97H and MHCC-LM3 cells were provided by the Liver Cancer Institute of Zhongshan Hospital, Fudan University (Shanghai, China). PLC/PRF/5, SK-Hep1 and human embryonic kidney (HEK)-293 T cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Huh-7 cells were obtained from the Riken Cell Bank (Tsukuba, Japan). Li-7 cells were purchased from Shanghai Sixin Biological Technology. HCC-LY5 and HCC-LY10 cell lines were established in our laboratory. All the above cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, New York, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) along with penicillin (100 U/mL, Sigma-Aldrich) and streptomycin (100 μg/mL, Sigma-Aldrich) at 37 °C in a humidified incubator containing 5% CO₂. All the cells were authenticated and characterized by their respective suppliers.

Normal human fetal hepatocytes (Hc cells) were obtained from DS Pharma Biomedical Co. (Osaka, Japan) with a certification of informed consent for research and were cultured in CS-C medium (DS Pharma Biomedical Co.). Human hepatocellular carcinoma cells (HuH-7 cells) were purchased from RIKEN CELL BANK (Ibaraki, Japan) and incubated in Dulbecco’s modified Eagle’s medium (DMEM) (FUJIFILM Wako Chemicals, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; CAPRICORN, Hessen, Germany) and penicillin–streptomycin solution (FUJIFILM Wako Chemicals). In addition, Hc and HuH-7 cells were mixed-inoculated in CS-C medium (DC Pharma Biomedical Co.) such that the percentage of HuH-7 cells to total cells was 0, 25, 50, 75, and 100%. These cells were cultured in a 5% CO₂ humidified incubator at 37 °C.

The human HCC cell lines SMMC-7721 was used in our study and were supplied by the Cell Bank of the Institute of Biochemistry and Cell Biology, China Academy of Sciences (Shanghai, China) in 2007. The cell lines MHCC-97L and MHCC-LM3 were kindly provided by the Liver Cancer Institute of Zhongshan Hospital, Fudan University (Shanghai, China) in 2007, and human HCC-LY5 and HCC-LY10 were established in our laboratory in 2011, which was authenticated by ourselves. 293T was purchased from the American Type Culture Collection (Manassas, VA, USA), and Huh7 cells were obtained from the Riken Cell Bank (Tsukuba, Japan) in 2010. The cell nutrient solution for all of the above cells was Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS) (HyClone), which was heat-inactivated at 56 °C for 30 min; 100 IU/mL penicillin G; and 100 μg/mL streptomycin (Sigma-Aldrich). All cell lines were incubated at 37 °C in a humidified atmosphere with 5% CO₂. All cell lines used in this study were thawed fresh every two months and used within 20 passages. These cell lines were mycoplasma-free and authenticated by their examination of morphology and growth profile.

The human HCC cell lines, HepG2 (well differentiated, low metastatic potential), Hep3B (well differentiated, low metastatic potential), and Human liver adenocarc -inoma Endothelial cell line, Sk-Hep1 were obtained from the American Type Culture Collection (Manassas, VA, USA). Huh7 cells (well differentiated, low metastatic potential) were obtained from the Riken Cell Bank (Ibaraki, Japan). Bel-7402 cells (moderate differentiated, low metastatic potential) and the normal liver cell line LO2 were obtained from the Committee of Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) in 5% CO₂ at 37°C.

Hep3B, PLC/PRF/5, and 293T cells were all obtained from the American Type Culture Collection (Manassas, VA, USA). Huh7 cells were obtained from the Riken Cell Bank (Tsukuba, Japan). SMMC-7721 cells were provided by the Cell Bank of the Institute of Biochemistry and Cell Biology, China Academy of Sciences (Shanghai, China). MHCC-LM3 cells were provided by the Liver Cancer Institute of Zhongshan Hospital, Fudan University (Shanghai, China). HCC-LY5 cells were established from a human primary HCC tissue in our lab.