Mhcc 97h cells

Human hepatocellular carcinoma SMMC-7721 and MHCC97Hcells were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China).Human hepatic stellate cell line LX-2 cells were purchased from Haibo Valley Biotechnology Co., Ltd. (Shanghai, China). According to the method of literature [5 (link)], SMMC-7721 andMHCC97H cells as well as LX-2 cells were maintained in high glucose DMEM cell culture medium containing 10% fetal bovine serum (FBS), 100 IU/mL penicillin G, and 100 μg/ml streptomycin. Cell cultures were incubated at 37 °C in an atmosphere of 5% CO₂.
Cells at a density of 1, 000 cells/well were seeded into ultralow attachment 6-well plates (Corning, USA) using the free-serum stem cell conditioned medium. After culturing for 5-6 days, three-dimensional clonal growth of non-adherent spheres was obtained, and the sphere diameter was ≥50.0 μm. Spheres were collected by centrifugation at 600 rpm (200×g), digested by accutase, dispersed into single cell suspensions, sphere cultivate again. The second-generation spheroids served as LCSLCs. Colonies were then scored under ten independent fields of view by light microscopy (Olympus, Japan). The sphere formation rate is calculated as follows (%): Average number of spheroid per well/total number of inoculated living cells (1000 cells) × 100%.

Cells (HUVECs, HepG2, and Hep3B) from American Type Culture Collection (ATCC) were cultured at 37°C in a humidified incubator with 5% CO₂ according to the ATCC protocols. Cells were cytogenetically tested and authenticated before being frozen. Each vial of frozen cells was thawed and maintained for 2 months (10 passages). MHCC97-H cells were purchased from Cell Bank of Chinese Academy of Sciences, Shanghai, China. Of note, HUVECs were cultured with M199, supplemented with 20% FBS, Endothelial cell growth supplement (30 µg/ml) and heparin (100 µg/ml), Human liver cancer cells HepG2, Hep3B, and MHCC97-H were grown in Eagle’s Minimum Essential Medium supplemented with 10% FBS and antibiotics. Deguelin and chemical reagents, including Tris, NaCl, SDS, and DMSO, for molecular biology and buffer preparation, were purchased from Sigma-Aldrich (St. Louis, MO, USA) Gefitinib and Linsitinib were purchased from Selleckchem (Houston, TX, USA).