Non essential amino acids solution

NIH3T3 cells were obtained from the RIKEN Cell Bank. Cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/ml of penicillin, and 100 μg/ml of streptomycin at 37°C. Sodium ascorbate or its stabilized form, AA2G, was supplied as required at concentrations of 250 μM; however, the culture medium included a sufficient amount of ascorbate to facilitate collagen biosynthesis in many cases. Cells were seeded at a density of 1.5 × 10⁵ on 35-mm dishes and cultured for 24 h. Cells were treated with 1 μg of a plasmid mixed with 6 μl of polyethylenimine transfection reagent (Polyscience) following the manufacturer’s instructions. Medium was changed 24 h after transfection and subjected to analysis. To detect secreted collagen, cells were cultured on fibronectin-coated dishes. The HSC clones, CFSC-2G and -5H cells, were cultured in DMEM supplemented with 10% fetal bovine serum, 1× non-essential amino acids solution (FUJIFILM Wako Pure Chemical), 100 units/ml of penicillin, and 100 μg/ml of streptomycin at 37°C. The transfection procedure was identical to that for NIH3T3 cells. Cells were stimulated with 1 ng/ml of TGF-β for 3 h as needed.

HepG2 cells, HUVECs, and MSCs were independently cultured for 24 h. HepG2 cells were cultured in cell culture dishes (VTC‐D150; AS ONE Corporation, Osaka, Japan) with low glucose Dulbecco's modified Eagle's medium (DMEM; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) containing 10% FBS (Thermo Fisher Scientific Inc., Waltham, MA, USA) and 1% penicillin/streptomycin solution (FUJIFILM Wako Pure Chemical). HUVECs (PromoCell GmbH, Heidelberg, Germany) were cultured in gelatin‐coated cell culture dishes (FUJIFILM Wako Pure Chemical Corporation) containing endothelial cell growth medium 2 (PromoCell) supplemented with 1% penicillin/streptomycin solution (×100). MSCs (SCRC‐4000; ATCC, Manassas, VA, USA) were cultured in gelatin‐coated cell culture dishes containing high‐glucose DMEM (FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% FBS, 1% nonessential amino acids solution (×100) (FUJIFILM Wako Pure Chemical), and 1% penicillin/streptomycin solution (×100). All cells were cultured at 37 °C in a 5% CO₂ atmosphere. The cells were dissociated with TrypLE Express (Thermo Fisher Scientific) 1 day after plating and mixed at the same ratio as the tubular liver tissue (3 : 3 : 0.5) (Fig. 1C) for extraction of RNA.

HepG2 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum (FBS; Thermo Fisher Scientific Inc., Waltham, MA, USA) and 1% penicillin-streptomycin solution (×100; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). HUVECs (PromoCell GmbH, Heidelberg, Germany) were cultured in endothelial cell growth medium 2 (EGM-2; PromoCell) supplemented with growth factors included in the medium kit and 1% penicillin-streptomycin solution (×100). Immortalised MSCs (SCRC-4000, ATCC, Manassas, Virginia, USA) were grown in DMEM supplemented with 10% FBS, 1% non-essential amino acids solution (×100) (FUJIFILM Wako Pure Chemical), and 1% penicillin-streptomycin solution (×100). All cells were maintained at 37 °C in a 5% CO₂ atmosphere.