Snu449

Human liver cancer cells; Huh7 and SNU449 were obtained from the Korean Cell Line Bank (Seoul, Korea) and maintained in Roswell Park MEMorial Institute medium (RPMI 1640; Welgene, Daegu, Korea). The human immortalized hepatocyte Fa2N-4 cell line were purchased from Xenotech (Lenexa, KS, USA), and maintained in serum-containing plating mediun (Xenotech) at first. After cell attachment in the plate, the medium was replaced with supporting culture medium (Xenotech). Human hepatic stellate cells (HSCs), LX2, was purchased from Merck Millipore (Darmstadt, Germany), and maintained Dulbecco`s modified Eagle`s medium (DMEM; Welgene) containing 2% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% penicillin–streptomycin (P/S; Gibco). One of HCC cell lines, HepG2 and WI38 human fibroblast cell line were obtained from ATCC (Manassas, VA, USA). These cell lines were maintained in minimum essential media (MEM; Welgene) supplemented with 10% FBS and 1% P/S. the Human umbilical vein endothelial cells (HUVECs) was purchased from PromoCells (Heidelberg, Germarny), and cultured in endothelial basal medium with supplementary reagents from PromoCells (Heidelberg, Germarny). All cells were maintained at 37 °C in a humidified incubator with 5% CO₂.

Cell lines, SNU449, SNU475, Huh7 and Hep3B were purchased from the Korean Cell Line Bank (Korea). The cells were cultured at 37°C under 5% CO₂ in DMEM supplemented with 10% FBS, 100 units/ml of penicillin and 100 μg/ml streptomycin. THLE-2 cell line was purchased from the American Type Culture Collection (USA). THLE-2 cells originated from human primary normal liver cells were plated on culture plates pre-coated with a solution containing 0.01 mg/mL fibronectin, 0.03 mg/mL bovine collagen type I and 0.01 mg/mL bovine serum albumin dissolved in Bronchial Epithelium Basal Medium (BEBM, Lonza). THLE-2 cells were cultured at 37°C under 5% CO₂ in BEBM supplemented with BEGM SingleQuots (Lonza).

The human HCC cell line, SNU449, was acquired from the Korean Cell Line Bank (Seoul, South Korea). Immortalized normal hepatocytes (MIHA) were provided by Dr. Roy-Chowdhury (Albert Einstein College of Medicine, Bronx, NY, USA). MIHA cells were cultured in Dulbecco’s modified Eagle’s medium (GenDEPOT, Barker, TX, USA), while SNU449 cells were cultured in RPMI1640 (GenDEPOT). Both mediums were supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Waltham, MA, USA) and 100 U·mL⁻¹ penicillin-streptomycin (GenDEPOT). Cells were grown in a humidified incubator at 37 °C and 5% CO₂.

The HCC cell lines ;SNU449, SNU475, SNU398, SNU898, Huh7, HepG2, Hep3B and PLC/PRF/5 were purchased from the Korean Cell Line Bank. Huh6 cells were kindly provided by Dr. Ralf Bartenschlager (University of Heidelberg, Germany). All HCC cells were maintained in RPMI (Welgene, Korea) or Dulbecco’s Modified Eagle Medium (DMEM; Welgene, Korea) containing 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin solution (p/s; Gibco, Grand Island, NY, USA). Fa2N-4, a human immortalized hepatocyte cell line, was obtained from Xenotech (Lenexa, KS, USA) and first cultured in serum-containing plating medium (K4000; Xenotech). Miha. Cells were kindly provided by Dr. Kim. K. M. from ASAN medical center. To produce tumor spheroids, HCC cells seeded at a density of 6 × 10³ cells/well in 96-well round-bottom ULA microplates (Corning Life Sciences, Corning, NY, USA). All cells were maintained in a 5% CO₂ humidified incubator at 37 °C.

Human HCC cell lines, Hep3B, SNU449, and Huh7 cells were obtained from the Korean Cell line Bank (Seoul, Republic of Korea). Hep3B and SNU449 cells were grown in Roswell Park Memorial Institute-1640 medium (Welgene, Daegu, Republic of Korea) supplemented with 10% fetal bovine serum (FBS) (Welgene) and 1% penicillin streptomycin (PS) (Welgene). Huh7 cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% FBS and 1% PS. For hypoxic incubation, cells were incubated in a hypoxic incubator (New Brunswick Scientific, Edison, NJ, USA) with a humidified environment consisting of 1% O₂, 5% CO₂, and 94% N₂.