Hepg2

Human Caucasian hepatocyte carcinoma (HepG2) (Sigma-Aldrich, cat# 85011430) was used for the cytotoxicity experiments. Targeted fractions were assessed for 50% of cytotoxicity (CC₅₀) at concentrations from 200 to 1 μM in triplicate. Cell viability was performed using the MTT assay as described by Crusco et al. (2019 (link)).

HepG2 (HB-8065, human hepatocellular carcinoma cells) and MCF-7 (HTB-22, oestrogen receptor-positive human breast adenocarcinoma cells), were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The HepG2 and MCF-7 cells were routinely maintained and incubated at 37 °C with 5% CO2 in RPMI-1640 medium (Sigma-Aldrich, Munich, Germany), supplemented with 10% sterile-filtered fetal bovine serum (FBS) (Sigma-Aldrich, Germany) and 1% antibiotic (penicillin-streptomycin) (Sigma-Aldrich, Germany).

The hepatoblastoma cell line HepG2 and HCC cell line Huh7 were obtained from RIKEN (Tokyo, Japan). HCC cell lines HLE and HLF were purchased from Japanese Collection of Research Biosources (Osaka, Japan). HepG2, HLE, and HLF were maintained in DMEM (Sigma-Aldrich, Tokyo, Japan) medium and Huh7 was maintained in PRMI1640 (Sigma-Aldrich, Japan) medium supplemented with 10% (HLF only 5%) fetal calf serum (Invitrogen, Carlsbad, CA, USA) and 50 mg/mL streptomycin (Invitrogen) at 37 °C under 5% CO₂ in air.

Caki-2 cells were cultured in McCoy’s 5A (modified) medium supplemented with 10% FCS, Calu-3 cells were cultured in MEM with 10% FCS and 1x non-essential amino acids and both obtained from ATCC (Manassas, VA, USA). HEK293T cells were cultured in DMEM supplemented with GlutaMAX, 10% heat-inactivated FCS. Caco-2 cells were from Sigma Aldrich (Schnelldorf, Germany) and were cultured in EMEM medium supplemented with 10% FCS, L-glutamine and non-essential amino acids (M7145, Sigma Aldrich, Schnelldorf, Germany). HepG2 was obtained from Sigma Aldrich (Schnelldorf, Germany) and cultured in DMEM supplemented with GlutaMAX and 10% heat-inactivated FCS. All media contained 1% penicillin/streptomycin and the cells were cultured at 37 °C in a 5% CO₂ atmosphere. Silvestrol was dissolved in DMSO and further diluted in media (c_stock = 6 mM, maximal DMSO concentration during experiments 0.1% v/v). EGTA, famotidine and carbamazepine were from Sigma Aldrich (Schnellendorf, Germany). Lucifer Yellow (sc-215269) was obtained from Santa Cruz. Celecoxib was synthesized by WITEGA Laboratorien Berlin-Adlershof GmbH (Berlin, Germany). Forskolin and ionomycin were obtained from Sigma Aldrich (Schnelldorf, Germany). Silvestrol was provided by the Sarawak Biodiversity Centre (Kuching, North-Borneo, Malaysia; purity > 99%) and zotatifin was bought from MedChemExpress (USA, purity: 98%).

Human hepatocellular carcinoma human cell line (HepG2, 85011430, Sigma‐Aldrich, UK) was cultured in Dulbecco's modified Eagle's medium/Ham's F12 (DMEM/F12, Sigma‐Aldrich, UK) supplemented with 2 mM 1‐glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin and 10% (v/v) FCS (Serum Laboratories International UK) in a humidified atmosphere containing 5% CO₂ in air at 37°C.

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.