E171-induced Toxicity in Human iPSC-Derived Colon Organoids: Effects on Cell Viability, ROS Generation, DNA Damage, and Gene Expression Changes
E171-induced Toxicity in Human iPSC-Derived Colon Organoids: Effects on Cell Viability, ROS Generation, DNA Damage, and Gene Expression Changes
Following 24 hours of incubation to 10, 100, 250, and 1000 µg/mL of E171 or VC, the medium was removed from the plates, and 100 µL of ice-cold 1x PBS was added into each well to collect the organoids into a 1.5 mL Eppendorf tube. The organoids were centrifuged at 1100 rpm for 5 minutes at 4°C, and the supernatant containing the excess matrigel was removed. Next, a total volume of 700 µL of QIAzol Lysis reagent (Qiagen, Venlo, The Netherlands) was added to each tube. Complete homogenization of organoids in the lysis reagent was reached by vigorous pipetting and vortexing for 1 minute. Samples were immediately frozen at -80°C until further processing.  The iPSC-derived colon organoids (#SCC300, Merck, Rahway, USA) were cultured according to the manufacturer's protocol. In short, the iPSC-derived organoids were cultured in 3dGRO™ Expansion Medium (#SCM304, Merck. Rahway, USA) supplemented with 1x PenStrep (Gibco, New York, USA) and 10 µM ROCK inhibitor (Y-27632, AbMol Bioscience, Houston, USA).  The medium was replaced daily for the first two days with the medium containing ROCK inhibitor, to increase stem cell survival, proliferation, and spheroid formation. Afterward, the organoids were maintained in the medium without the ROCK inhibitor but with antibiotics. The medium was refreshed every other day. The organoids were passaged every 10-12 days to prevent over-confluency and maintain healthy growth. The passaging involved dissociating the organoids from the growth factor reduced matrigel (#CLS356231, Corning, New York, USA) through mechanical breaking up of the matrigel domes with ice-cold 1x PBS and a 3dGRO™ Organoid Dissociation Reagent (ODR, #SCM300, Merck, Rahway, USA) to remove the old matrigel, followed re-encapsulation in fresh matrigel. The split ratio at passaging was typically 1:2, adjusted based on the growth rate and density of the organoids. The iPSC-derived human colon organoids were cultured at 37°C and 5 % CO2. Cell passages 4-10 were used for the experiments. The organoids were exposed to E171 ranging from 0.1-1000 µg/mL in 3dGRO™ Expansion Medium (1:10 dilution) from stock solutions of 0.1, 1.0, 2.5, and 10 mg/mL of E171. Therefore, in addition, to the initial particle characterization, we prepared and analyzed 6 mL of stocks at 0.1, 1.0, 2.5, and 10 mg/mL of E171 following the same procedure with small deviations to the Nanogenotox dispersion protocol. Instead of probe sonication, the samples were vortexed and bath sonicated for 16 minutes at 37 kHz. All stock solutions were diluted at least 1:10 in the 3dGRO™ Human Colon Organoid Expansion Medium (Sigma-Aldrich) for subsequent experiments (25). Particle size, size distribution, HD, and zeta-potential were analyzed via spICP-MS, TEM, and DLS as described in section 2.1.  RNA isolation was performed using the miRNeasy Mini Kit (Qiagen, Venlo, The Netherlands), following the manufacturer's protocol for animal cells, which included a DNase treatment. Total RNA yield was measured on Qubit according to the manufacturer's protocol (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and RNA quality was assessed using RNA Pico-/Nanochips on a 2100 Bioanalyzer (Agilent Technologies, Leuven, Belgium). All samples with an RNA integrity number (RIN) > 7 and a total amount of RNA ≥ 100 ng were used for RNA sequencing. We prepared three technical and three biological (cell passage 6-8) replicates per concentration, totaling nine replicates per condition.  Samples containing purified RNA were prepared for sequencing using a NEX Poly(A) Beads 2.0 auto kit (Revvity, Groningen, The Netherlands) on a Zephyr G3 NGS workstation (CLS150362, Revvity, Groningen, The Netherlands).