C3867

An insert-based MAC-T cell culture system was established as previously described with slight modifications [50 (link)] to assess the paracellular permeability of MAC-T cell monolayer. MAC-T cells were seeded at 2.5 × 10⁴ cells per Transwell® insert (Corning® Transwell® #3470, 6.5 mm, 0.4 µm pore size) pre-coated with Type I collagen at 10 μg/cm² (C3867, Sigma-Aldrich) according to the manufacturer’s instruction and grown for 33 days in the same medium described above to ensure stable transepithelial electrical resistance (TEER) readings were yield according to our preliminary studies (data not shown). Medium was refreshed for both apical (AP) and basal compartments (BL) of the Transwell® inserts every other day [82 (link)]. On day 33, the cells were exposed to the increasing non-cytotoxic concentrations of either OTA (0, 2, 4.8 and 9.6 μmol/L), or CIT (0, 30, 60 and 80 μmol/L) in the AP compartment for 48 h. The TEER readings were measured before the addition of mycotoxins (TEER₀) and after 48 h mycotoxin exposure (TEER₄₈) using a Millicell ERS-2 Voltohmmeter (EMD Millipore Corporation) according to the manufacturer’s instruction. The change in TEER was expressed as TEER₄₈ to TEER₀ ratio, which was calculated according to the following formula [112 (link)]:

Before the cells were loaded, a 1 ml type I collagen solution (~4.26 mg/ml; C3867, SIGMA) was prepared by adding cell culture medium and 1 N sodium hydroxide for pH adjustment (details are reported in the Supplementary Table S1). Before each individual experiment, the pH of the collagen solution was measured to be around pH 7.4. The cells were then resuspended in solutions with different concentrations of collagen (0, 50, 500, and 1000 μg/ml). In 3D multicellular spheroid culture Fig. 1(c), cellular drops with a volume of 1 μl were dispensed onto the PDMS-HDA device (step 1) by a liquid handing machine (Versa 10 spotter, Aurora Instruments Ltd.) Fig. 1(b). Each drop had an average diameter of 1.4 mm. The device was flipped and placed in a 6-cm cell culture dish, which had been pre-filled with medium of 750 μl and sealed with parafilm to prevent evaporation of the cell-containing drops. The whole set was transferred immediately to a humidified incubator at 37 °C overnight to allow the formation of collagen fibrils, which contributed to the sedimentation and aggregation of the cells (Steps 2 and 3), promoting the generation of cellular spheroids (Step 4).

Hepatocytes were plated in a collagen-coated (Sigma-Aldrich C3867) seahorse XF96 cell culture microplates at 5000 cells/well in 80 µL in Buffer C and incubated at 37 °C for 6 h. After 6 h media was changed, and hepatocytes were cultured overnight before any experimental procedure. Mitochondrial measurements and FAO were performed using the Seahorse XF Cell Mito Stress Test (Agilent 103015-100) according to manufacturer’s instructions. Briefly, primary hepatocytes were plated as indicated above and the following morning regular media was replaced for Seahorse XF DMEM medium (Agilent 103575-100) supplemented with 10 mM glucose (Agilent 103577-100), 1 mM Sodium pyruvate (Agilent 103578-100) and 2 mM glutamine (Agilent 103579-100). Oxygen consumption Rate (OCR) was measured following the sequential addition of 40 µM of Etomoxir (MedChemTronica HY-502002; port A), 1.5 µM of oligomycin (port B), 0.3 µM of carbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone (FCCP)(port C) and 0.5 µM of Antimycin/Rotenone (Rot/AA) (port C). The oligomycin, FCCP and the Rot/AA were purchase from Agilent (103015-100). Oxygen consumption rate (OCR) was normalized to protein concentration using BCA Protein Assay (Thermo Scientific™ 23222). All parameters were represented as normalize OCR.