With the rising interest in the effects of orally ingested engineered nanomaterials (ENMs), much effort is undertaken to develop and advance intestinal in vitro models. The cytotoxic, proinflammatory, and DNA damaging properties of polyvinylpyrrolidone‐capped silver (Ag‐PVP) and titanium dioxide (TiO2, P25) ENM in four in vitro models of increasing complexity—from proliferating Caco‐2 and HT29‐MTX‐E12 monocultures to long‐term transwell triple cultures including THP‐1 macrophages to reproduce the human intestine in healthy versus inflamed‐like state—are studied. Results are compared against in vivo effects of the same ENM through intestinal tissue analysis from 28‐day oral exposure studies in mice. Adverse responses are only observed in monocultures and suggest toxic potential for both ENM, typically showing stronger effects for Ag‐PVP than for TiO2. By contrast, no adverse effects are observed in either the transwell cultures or the analyzed murine tissues. The data provide further support that monoculture models represent a cost and time efficient tool for early‐phase hazard assessment. However, the observed similarities in morphology and ENM effects in murine intestinal tissue and the in vitro triple culture model suggest that advanced multifacetted research questions concerning oral ENM exposure are more adequately addressed by the more complex and time intensive models.

Would an engineered nanomaterial (ENM) still have the same identity once it reaches a secondary target tissue after a journey through several physiological compartments? Probably not. Does it matter? ENM pre‐treatments may enhance the physiological relevance of in vitro testing via controlled transformation of the ENM identity. The implications of material transformation upon reactivity, cytotoxicity, inflammatory, and genotoxic potential of Ag and SiO2 ENM on advanced gastro‐intestinal tract cell cultures and 3D liver spheroids are demonstrated. Pre‐treatments are recommended for certain ENM only.

The development and improvement of advanced intestinal in vitro models has received increasing attention in recent years. While the availability of relevant in vitro models is pivotal to advance the replacement and reduction of animal use in research, their robustness is a crucial determinant for intra- and interlaboratory reproducibility. We have developed a standard protocol to build a triple culture model combining two types of human intestinal epithelial cells (Caco-2, HT29-MTX-E12) and macrophages (THP-1), which was tested for transferability and reproducibility between three laboratories. The epithelial tissue barrier development and triple culture stability were investigated as well as the models’ responses to the non-steroidal anti-inflammatory drug diclofenac in terms of barrier integrity, cytotoxicity, and cytokine release. The results of two partner laboratories were compared to previously established benchmark results and quality criteria. For the epithelial co-cultures, the results were overall highly comparable between the laboratories. The addition of THP-1 cells resulted in increased variability and reduced reproducibility. While good correlation was achieved in several endpoints, others showed substantial response differences between the laboratories. Some variations may be addressed with training or demonstrations, whereas others might be related to fundamental differences in the cell lines introduced during routine cell culture and maintenance. Our results underline the importance of interlaboratory transfer studies using standardised experimental procedures, including defined quality criteria and benchmarks, as well as of training when newly establishing complex in vitro models in laboratories.

The increasing use of engineered nanomaterials (ENM) in food has fueled the development of intestinal in vitro models for toxicity testing. However, ENM effects on intestinal mucus have barely been addressed, although its crucial role for intestinal health is evident. We investigated the effects of ENM on mucin expression and aimed to evaluate the suitability of four in vitro models of increasing complexity compared to a mouse model exposed through feed pellets. We assessed the gene expression of the mucins MUC1, MUC2, MUC5AC, MUC13 and MUC20 and the chemokine interleukin-8 in pre-confluent and confluent HT29-MTX-E12 cells, in stable and inflamed triple cultures of Caco-2, HT29-MTX-E12 and THP-1 cells, and in the ileum of mice following exposure to TiO2, Ag, CeO2 or SiO2. All ENM had shared and specific effects. CeO2 downregulated MUC1 in confluent E12 cells and in mice. Ag induced downregulation of Muc2 in mice. Overall, the in vivo data were consistent with the findings in the stable triple cultures and the confluent HT29-MTX-E12 cells but not in pre-confluent cells, indicating the higher relevance of advanced models for hazard assessment. The effects on MUC1 and MUC2 suggest that specific ENM may lead to an elevated susceptibility towards intestinal infections and inflammations.

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