Thle 3

The HCC cell line Sk-Hep1 was purchased from the American Type Cell Culture Collection (ATCC), HuH-7 was purchased from RIKEN Resource Centre, Japan, SMMC-7721 was purchased from the Cell Bank of the Chinese Academy of Sciences Cell Bank of Type Culture Collection. The metastatic HCC cell lines MHCC-97 L, MHCC-97H and MHCC-LM3 were obtained from the Liver Cancer Institute, Zhongshan Hospital, Fudan University. The HCC cell line CSQT-2 derived from a portal vein tumor thrombus (PVTT) has been described elsewhere [45 (link)]. Human liver epithelial cell line THLE-3 was purchased from ATCC;. All cell lines except THLE-3 were cultivated in DMEM (Dulbecco’s modified Eagle medium) supplemented with 10% (v/v) fetal calf serum at 37°C in a humidified incubator under 5% CO₂ condition. THLE-3 was cultivated in BEGM (Lonza) with the addition of BEGM bullet kit according to the instruction on ACTT.

The following mammalian cell lines were purchased from ATCC through Cedarlane, Burlington, ON, Canada: MCF-7 (ATCC-HTB-22, human breast epithelial adenocarcinoma), HepG2 (ATCC-HB-8065, human hepatocellular carcinoma), HOS (ATCC-CRL-1543, Human Osteosarcoma), D-17 (ATCC-CCL-183, Canine osteosarcoma), DH-82 (ATCC-CRL-10389, canine histiocytosis), MCF10-A (ATCC-CRL-10317, human breast epithelial cell), AML-12 (ATCC-CRL-2254, mice liver epithelial cell), THLE-3 (ATCC-CRL-11233, human liver epithelial), and CnOb (cell applications-canine osteoblast). DH-82, HOS, and D-17 cells were cultured in EMEM (Sigma–Aldrich, Oakville, ON, Canada), MCF-7 and HepG2 in DMEM (Gibco), CnOb in CnOb basal medium supplemented with growth medium (cell applications), MCF-10A cultured in basal medium supplemented with growth medium, and THLE-3 in BEGM with supplements (Lonza Bioscience, Burlington, ON, Canada), as recommended. The cells were supplemented with 15% fetal bovine serum as required (Fisher Scientific, Ottawa, ON, Canada) and antibiotic (100 U/mL penicillin and 100 µg/mL streptomycin). The cells were maintained in a 5% CO₂ humidified incubator at 37°C (Fisher Scientific, Ottawa, ON, Canada). All experiments were performed after the second passage of the cells and repeated at least three times, independently.

Human liver cell line (THLE-3), cancer cell lines (SNU-475 and SNU-423), normal B lymphocyte (CCL-156) and normal monocyte/macrophage (CRL-9855) were purchased from American Type Culture Collection (ATCC, Manassas, Virginia, USA). SNU-475, SNU-423 and CCL-156 were cultured in Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 10% heat inactivated fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO) and 1% penicillin and streptomycin. THLE-3 was grown in Bronchial Epithelial Cell Growth Medium (BGEM) bulletkitTM (Lonza/Clonetics Corporation, Walksrsville, MD 21793) whereas, CRL-9855 was grown in Iscove’s Modified Dulbecco’s Medium (IMDM, ATCC 30-2005, Manassas, Virginia, USA) with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate and supplemented with 0.05 mM 2-mercaptoethanol, 0.1 mM hypoxanthine and 0.016 mM thymidine, 90%; fetal bovine serum, 10%. All cell lines were cultured in a humidified incubator with 5% CO₂ at 37 °C. All experiments were conducted on cell lines with passage number 1 to 10.

Human HCC cell lines SNU-423 (ATCC-CRL-2238) and SNU-182 (ATCC-CRL-2235), and human immortalized liver cell lines THLE-2 and THLE-3, were obtained from the American Type Cell Collection (ATCC, Manassas, VA, USA). HCC cells were grown in RPMI culture media (Lonza, Walkersville, MD, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Life Technologies, Waltham, MA, USA). THLE2 and THLE3 cells were cultured in a BEGM Bullet kit (Lonza), which contains BEBM basal medium and supplements. The final growth medium consists of BEBM supplemented with 10% FCS, bovine pituitary gland extract, hydrocortisone, epidermal growth factor (EGF), insulin, triiodothyronine, transferrin, and retinoic acid. THLE cells require a special coating medium that consists of the following: RPMI1640 without glutamine supplemented with 0.01 mg mL⁻¹ bovine serum albumin, (heat shock fraction, Sigma, Burlington, MA, USA), 0.03 mg mL⁻¹ type I collagen from bovine skin (Sigma), and 0.01 mg mL⁻¹ fibronectin from human plasma (Sigma). All cell lines were maintained independently in the recommended medium at 37 °C and 5% CO₂.