Hepg2

The human immortalized liver LO2, HepG2, HepG2.2.15 and NIHT3T cell lines were purchased from Nanjing KeyGEN Biotech Co., Ltd (Nanjing, China). The co-transfection with the plasmids of pCMV-X and pcDNA3-sur
[12 (link)] was performed in LO2 cells using Lipofectamine 2000 (Invitrogen, USA) as described
[12 (link)]. After 48 hours, the transfected cells were incubated in selection medium containing 500 μg/ml or 800 μg/ml G418 (Genview, USA). The transfection efficiencies were tested by PCR (one primer is from survivin and another is from the vector), western blotting, as indicated. Then, the engineered cell line was termed LO2-X-S. All the engineered cell lines were cultured in RPMI Medium 1640 (GIBCO, USA) containing 100 U/ml penicillin, 100 μg/ml streptomycin and 10% fetal calf serum (FCS). HepG2 cells, HepG2-X cells
[42 (link)] and HepG2.2.15 cells were cultured in Dulbecco’s Modified Eagle’s medium (DMEM, GIBCO) containing 100 U/ml penicillin, 100 μg/ml streptomycin and 10% FCS.

The human hepatocellular carcinoma cell line HepG2 (Sigma Aldrich Sweden AB) was used to check the biocompatibility of the dLM-G-PEG bioink during the gelation and printing process. HepG2 cells were cultured according to the manufacturer’s protocol, with Eagle’s minimal essential medium (MEM, 11095080), 10% fetal bovine serum (FBS, A3160502), and penicillin-streptomycin (10,000 U mL⁻¹, 15140122). Cells were maintained in culture and passaged every 2–3 days at about 80–90% confluency. Once at proper confluency, cells were used for encapsulation. Briefly, HepG2 cells were dissociated from the culture plate with trypsin-EDTA (0.25% solution, Sigma-Aldrich AB) and centrifuged at 300× g for 4–5 min. The collected HepG2 cells were resuspended in 10% FBS. To sustain the same osmotic pressure in dLM-G-PEG, 10× concentrated MEM (21430020, Gibco) was added (1/10th volume) and the collected HepG2 cells in 10% FBS, were mixed thoroughly with the acellular bioink. HepG2 cells were used in the dLM-G-PEG at a concentration of 4 to 5 × 10⁶ cells per ml. The prepared bioink with HepG2 cells was crosslinked for 1 h at 37 °C and loaded into a sterilized syringe for bioprinting while maintaining the temperature. Cell culture reagents for HepG2 cell culture and maintenance were obtained from Thermofisher Scientific.

The HCC cell lines HepG2 and Hep3B were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Bel-7402 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Xuhui, Shanghai, China). HepG2/ADM cells were kindly provided by Prof. Kwok-Pui Fung (The Chinese University of Hong Kong, Hong Kong, China). The HepG2, Hep3B, HepG2/ADM and Bel-7402 cells were maintained in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% (v/v) foetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 1% (v/v) penicillin-streptomycin (PS; 10,000 U/ml, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C in a 5% CO₂ atmosphere.