Cell culture and chip loading
Cell culture. The human hepatoma cell line HepG2/C3A (CRL-10741) was purchased from the American Type Culture Collection (ATCC). Cells were expanded in DMEM low glucose supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, MEM non-essential amino acid solution (100x), 10% FBS, and incubated in a humidified 37 °C atmosphere containing 5% CO2.
Chip loading. HepG2 cells were detached at subconfluence, and loaded into chips at a density of 1.0×106 cells/mLvia the central cell loading channel as previously described (Gori et al., 2016). Thereafter, cells were cultured on chip, in 3D fashion, under microfluidic perfusion obtained by applying a height differential to the culture medium contained in the inlet/outlet reservoirs. The sinusoid-like microarchitecture of the chip allowed the continuous diffusion of nutrients and the removal of metabolic waste products, with negligible shear stress thanks to the microchannel barrier. After chip loading, cells were cultured overnight (o.n.) in standard culture medium; the next day, medium was discarded and replaced with fresh steatosis medium containing FFAs and polyphenols according to the different treatments as described in the following.