D glucose and l glutamine

The Human Caucasian Hepatocellular Carcinoma derived epithelial cell line, HepG2 (ECACC 85011430 and ATCC HB-8065) monolayers were cultured in Dubecco’s Modified Eagle Medium (DMEM) with 4.5g/L d-Glucose and L-Glutamine (GIBCO, Paisley, UK) supplemented with 10% Foetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin antibiotic (GIBCO, Paisley, UK). HepG2 cells were sub-cultured every 3–5 days, once 80% confluency was reached, they were trypsinised (0.05% trypsin/EDTA solution; GIBCO, Paisley, UK) and a cell stock of 2.0 × 10⁵ cells/mL was prepared. To form the 3D spheroid structure, HepG2 cells were cultured in 96-well plates using the hanging drop method developed by Llewellyn et al. and described by Conway et al. [28 (link), 29 ]. Extensive information regarding the establishment, culture, characterisation, exposure protocol, harvest and application of the 3D HepG2 spheroid model can be found in the published protocol by Llewellyn et al., 2020. In short, 20 μL of the cell suspension (4000 HepG2 cells per 20 μL hanging drop) was pipetted onto the inverted side of a 96-well tissue culture plate, before gently inverting the lid and placing back onto the 96-well plate filled with 100µl of PBS [28 (link)]. The plate was then placed in the incubator at 37 °C with 5% CO₂ for 3 days before agarose transfer.