Development of reconstructed intestinal micronucleus cytome (RICyt) assay in 3D human gut model for genotoxicity assessment of orally ingested substances

Hui Kheng Lim · Christopher Owen Hughes · Michelle Jing Sin Lim · Jia’En Jasmine Li · Moumita Rakshit · Calvin Yeo · Kern Rei Chng · Angela Li · Joanne Sheot Harn Chan · Kee Woei Ng · David Ian Leavesley · Benjamin Paul Chapman Smith

The micronucleus (MN) assay is widely used as part of a battery of tests applied to evaluate the genotoxic potential of chemicals, including new food additives and novel food ingredients. Micronucleus assays typically utilise homogenous in vitro cell lines which poorly recapitulate the physiology, biochemistry and genomic events in the gut, the site of first contact for ingested materials. Here we have adapted and validated the MN endpoint assay protocol for use with complex 3D reconstructed intestinal microtissues; we have named this new protocol the reconstructed intestine micronucleus cytome (RICyt) assay. Our data suggest the commercial 3D microtissues replicate the physiological, biochemical and genomic responses of native human small intestine to exogenous compounds. Tissues were shown to maintain log-phase proliferation throughout the period of exposure and expressed low background MN. Analysis using the RICyt assay protocol revealed the presence of diverse cell types and nuclear anomalies (cytome) in addition to MN, indicating evidence for comprehensive DNA damage and mode(s) of cell death reported by the assay. The assay correctly identified and discriminated direct-acting clastogen, aneugen and clastogen requiring exogenous metabolic activation, and a non-genotoxic chemical. We are confident that the genotoxic response in the 3D microtissues more closely resembles the native tissues due to the inherent tissue architecture, surface area, barrier effects and tissue matrix interactions. This proof-of-concept study highlights the RICyt MN cytome assay in 3D reconstructed intestinal microtissues is a promising tool for applications in predictive toxicology.


SMI-200-FT, EpiIntestinal, genotoxicity, DNA damage, Micronuclei, direct-acting clastogen, aneugen, exogenous metabolic activation, chemically induced , Reconstructed intestine micronucleus cytome (RICyt) assay, TK6, mitomycin C, vinblastine sulphate, benzo(a)pyrene, Phenformin HCl, DMSO, tissue dissociation, Cytokinesis‑block, TEER, (FITC)-dextran 20 kDa, Alamar blue, Scanning electron microscopy (SEM), villi, microvilli, f-actin, brush border, nuclear buds, mononucleated cells, binucleated cells, condensed chromatin, karyorrhectic, pyknotic, karyolytic cells, caspase-3, apoptosis

Materials Tested

mitomycin C, vinblastine sulphate, benzo(a)pyrene, Phenformin HCl, DMSO

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