Comparison of iPSC-derived human intestinal epithelial cells with Caco-2 cells and human in vivo data after exposure to Lactiplantibacillus plantarum WCFS1.

To investigate intestinal health and its potential disruptors in vitro, representative models are required. Human induced pluripotent stem cell (hiPSC)-derived intestinal epithelial cells (IECs) more closely resemble the in vivo intestinal tissue than conventional in vitro models like human colonic adenocarcinoma Caco-2 cells. However, the potential of IECs to study immune-related responses upon external stimuli has not been investigated in detail yet. The aim of the current study was to evaluate immune-related effects of IECs by challenging them with a pro-inflammatory cytokine cocktail. Subsequently, the effects of Lactiplantibacillus plantarum WCFS1 were investigated in unchallenged and challenged IECs. All exposures were compared to Caco-2 cells and in vivo data where possible. Upon the inflammatory challenge, IECs and Caco-2 cells induced a pro-inflammatory response which was strongest in IECs. Heat-killed L. plantarum exerted the strongest effect on immune parameters in the IEC model, while L. plantarum in the stationary growth phase had most pronounced effects on immune-related gene expression in Caco-2 cells. Unfortunately, comparison to in vivo transcriptomics data showed limited similarities, which could be explained by essential differences in the study setups. Altogether, hiPSC-derived IECs show a high potential as a model to study immune-related responses in the intestinal epithelium in vitro.

Transport of perfluoroalkyl substances across human induced pluripotent stem cell-derived intestinal epithelial cells in comparison with primary human intestinal epithelial cells and Caco-2 cells.

Humans can be exposed to per- and polyfluoroalkyl substances (PFASs) via many exposure routes, including diet, which may lead to several adverse health effects. So far, little is known about PFAS transport across the human intestinal barrier. In the current study, we aimed to assess the transport of 5 PFASs (PFOS, PFOA, PFNA, PFHxS and HFPO-DA) in a human induced pluripotent stem cell (hiPSC)-derived intestinal epithelial cell (IEC) model. This model was extensively characterized and compared with the widely applied human colonic adenocarcinoma cell line Caco-2 and a human primary IEC-based model, described to most closely resemble in vivo tissue. The hiPSC-derived IEC layers demonstrated polarized monolayers with tight junctions and a mucus layer. The monolayers consisted of enterocytes, stem cells, goblet cells, enteroendocrine cells, and Paneth cells that are also present in native tissue. Transcriptomics analysis revealed distinct differences in gene expression profiles, where the hiPSC-derived IECs showed the highest expression of intestinal tissue-specific genes relative to the primary IEC-based model and the Caco-2 cells clustered closer to the primary IEC-based model than the hiPSC-derived IECs. The order of PFAS transport was largely similar between the models and the apparent permeability (Papp) values of PFAS in apical to basolateral direction in the hiPSC-derived IEC model were in the following order: PFHxS > PFOA > HFPO-DA > PFNA > PFOS. In conclusion, the hiPSC-derived IEC model highly resembles human intestinal physiology and is therefore a promising novel in vitro model to study transport of chemicals across the intestinal barrier for risk assessment of chemicals.

Systematic comparison of transcriptomes of Caco-2 cells cultured under different cellular and physiological conditions.

There is a need for standardized in vitro models emulating the functionalities of the human intestinal tract to study human intestinal health without the use of laboratory animals. The Caco-2 cell line is a well-accepted and highly characterized intestinal barrier model, which has been intensively used to study intestinal (drug) transport, host-microbe interactions and chemical or drug toxicity. This cell line has been cultured in different in vitro models, ranging from simple static to complex dynamic microfluidic models. We aimed to investigate the effect of these different in vitro experimental variables on gene expression. To this end, we systematically collected and extracted data from studies in which transcriptome analyses were performed on Caco-2 cells grown on permeable membranes. A collection of 13 studies comprising 100 samples revealed a weak association of experimental variables with overall as well as individual gene expression. This can be explained by the large heterogeneity in cell culture practice, or the lack of adequate reporting thereof, as suggested by our systematic analysis of experimental parameters not included in the main analysis. Given the rapidly increasing use of in vitro cell culture models, including more advanced (micro) fluidic models, our analysis reinforces the need for improved, standardized reporting protocols. Additionally, our systematic analysis serves as a template for future comparative studies on in vitro transcriptome and other experimental data.

Differential gene expression in iPSC-derived human intestinal epithelial cell layers following exposure to two concentrations of butyrate, propionate and acetate.

Intestinal epithelial cells and the intestinal microbiota are in a mutualistic relationship that is dependent on communication. This communication is multifaceted, but one aspect is communication through compounds produced by the microbiota such as the short-chain fatty acids (SCFAs) butyrate, propionate and acetate. Studying the effects of SCFAs and especially butyrate in intestinal epithelial cell lines like Caco-2 cells has been proven problematic. In contrast to the in vivo intestinal epithelium, Caco-2 cells do not use butyrate as an energy source, leading to a build-up of butyrate. Therefore, we used human induced pluripotent stem cell derived intestinal epithelial cells, grown as a cell layer, to study the effects of butyrate, propionate and acetate on whole genome gene expression in the cells. For this, cells were exposed to concentrations of 1 and 10 mM of the individual short-chain fatty acids for 24 h. Unique gene expression profiles were observed for each of the SCFAs in a concentration-dependent manner. Evaluation on both an individual gene level and pathway level showed that butyrate induced the biggest effects followed by propionate and then acetate. Several known effects of SCFAs on intestinal cells were confirmed, such as effects on metabolism and immune responses. The changes in metabolic pathways in the intestinal epithelial cell layers in this study demonstrate that there is a switch in energy homeostasis, this is likely associated with the use of SCFAs as an energy source by the induced pluripotent stem cell derived intestinal epithelial cells similar to in vivo intestinal tissues where butyrate is an important energy source.

Responses of increasingly complex intestinal epithelium in vitro models to bacterial toll-like receptor agonists.

The intestine fulfills roles in the uptake of nutrients and water regulation and acts as a gatekeeper for the intestinal microbiome. For the latter, the intestinal gut barrier system is able to respond to a broad range of bacterial antigens, generally through Toll-like receptor (TLR) signaling pathways. To test the capacity of various in vitro intestinal models, we studied IL-8 secretion, as a marker of pro-inflammatory response through the TLR pathway, in a Caco-2 monoculture, Caco-2/HT29-MTX di-culture, Caco-2/HT29-MTX/HMVEC-d tri-culture and in a HT29-p monoculture in response to exposure to various TLR agonists. Twenty-one-day-old differentiated cells in Transwells were exposed to Pam3CSK4 (TLR1/2), lipopolysaccharide (TLR4), single-stranded RNA (TLR7/8), Poly(i:C) (TLR3) and flagellin (TLR5) for 24 h. In all systems IL-8 secretion was increased in response to flagellin exposure, with HT29-p cells also responding to Poly(I:C) exposure. All other agonists did not induce an IL-8 response in the tested in vitro models, indicating that the specific TLRs are either not present or not functional in these models. This highlights the need for careful selection of in vitro models when studying intestinal immune responses and the need for improved in vitro models that better recapitulate intestinal immune responses.

Comparison of gene expression and biotransformation activity of HepaRG cells under static and dynamic culture conditions.

Flow conditions have been shown to be important in improving longevity and functionality of primary hepatocytes, but the impact of flow on HepaRG cells is largely unknown. We studied the expression of genes encoding CYP enzymes and transporter proteins and CYP1 and CYP3A4 activity during 8 weeks of culture in HepaRG cells cultured under static conditions (conventional 24-/96-well plate culture with common bicarbonate/CO2 buffering) and under flow conditions in an organ-on-chip (OOC) device. Since the OOC-device is a closed system, bicarbonate/CO2 buffering was not possible, requiring application of another buffering agent, such as HEPES. In order to disentangle the effects of HEPES from the effects of flow, we also applied HEPES-supplemented medium in static cultures and studied gene expression and CYP activity. We found that cells cultured under flow conditions in the OOC-device, as well as cells cultured under static conditions with HEPES-supplemented medium, showed more stable gene expression levels. Furthermore, only cells cultured in the OOC-device showed relatively high baseline CYP1 activity, and their gene expression levels of selected CYPs and transporters were most similar to gene expression levels in human primary hepatocytes. However, there was a decrease in baseline CYP3A4 activity under flow conditions compared to HepaRG cells cultured under static conditions. Altogether, the present study shows that HepaRG cells cultured in the OOC-device were more stable than in static cultures, being a promising in vitro model to study hepatoxicity of chemicals upon chronic exposure.

A versatile, compartmentalised gut-on-a-chip system for pharmacological and toxicological analyses.

A novel, integrated, in vitro gastrointestinal (GI) system is presented to study oral bioavailability parameters of small molecules. Three compartments were combined into one hyphenated, flow-through set-up. In the first compartment, a compound was exposed dynamically to enzymatic digestion in three consecutive microreactors, mimicking the processes of the mouth, stomach, and intestine. The resulting solution (chyme) continued to the second compartment, a flow-through barrier model of the intestinal epithelium allowing absorption of the compound and metabolites thereof. The composition of the effluents from the barrier model were analysed either offline by electrospray-ionisation-mass spectrometry (ESI-MS), or online in the final compartment using chip-based ESI-MS. Two model drugs, omeprazole and verapamil, were used to test the integrated model. Omeprazole was shown to be broken down upon treatment with gastric acid, but reached the cell barrier unharmed when introduced to the system in a manner emulating an enteric-coated formulation. In contrast, verapamil was unaffected by digestion. Finally, a reduced uptake of verapamil was observed when verapamil was introduced to the system dissolved in apple juice, a simple food matrix. It is envisaged that this integrated, compartmentalised GI system has potential for enabling future research in the fields of pharmacology, toxicology, and nutrition.

Transcriptome comparisons of in vitro intestinal epithelia grown under static and microfluidic gut-on-chip conditions with in vivo human epithelia.

Gut-on-chip devices enable exposure of cells to a continuous flow of culture medium, inducing shear stresses and could thus better recapitulate the in vivo human intestinal environment in an in vitro epithelial model compared to static culture methods. We aimed to study if dynamic culture conditions affect the gene expression of Caco-2 cells cultured statically or dynamically in a gut-on-chip device and how these gene expression patterns compared to that of intestinal segments in vivo. For this we applied whole genome transcriptomics. Dynamic culture conditions led to a total of 5927 differentially expressed genes (3280 upregulated and 2647 downregulated genes) compared to static culture conditions. Gene set enrichment analysis revealed upregulated pathways associated with the immune system, signal transduction and cell growth and death, and downregulated pathways associated with drug metabolism, compound digestion and absorption under dynamic culture conditions. Comparison of the in vitro gene expression data with transcriptome profiles of human in vivo duodenum, jejunum, ileum and colon tissue samples showed similarities in gene expression profiles with intestinal segments. It is concluded that both the static and the dynamic gut-on-chip model are suitable to study human intestinal epithelial responses as an alternative for animal models.

An in vitro model for microbial fructoselysine degradation shows substantial interindividual differences in metabolic capacities of human fecal slurries.

Fructoselysine is formed upon heating during processing of food products, and being a key intermediate in advanced glycation end product formation considered to be potentially hazardous to human health. Human gut microbes can degrade fructoselysine to yield the short chain fatty acid butyrate. However, quantitative information on these biochemical reactions is lacking, and interindividual differences therein are not well established. Anaerobic incubations with pooled and individual human fecal slurries were optimized and applied to derive quantitative kinetic information for these biochemical reactions. Of 16 individuals tested, 11 were fructoselysine metabolizers, with Vmax, Km and kcat-values varying up to 14.6-fold, 9.5-fold, and 4.4-fold, respectively. Following fructoselysine exposure, 10 of these 11 metabolizers produced significantly increased butyrate concentrations, varying up to 8.6-fold. Bacterial taxonomic profiling of the fecal samples revealed differential abundant taxa for these reactions (e.g. families Ruminococcaceae, Christenellaceae), and Ruminococcus_1 showed the strongest correlation with fructoselysine degradation and butyrate production (ρ ≥ 0.8). This study highlights substantial interindividual differences in gut microbial degradation of fructoselysine. The presented method allows for quantification of gut microbial degradation kinetics for foodborne xenobiotics, and interindividual differences therein, which can be used to refine prediction of internal exposure.

Comparative study of the transcriptomes of Caco-2 cells cultured under dynamic vs. static conditions following exposure to titanium dioxide and zinc oxide nanomaterials.

Due to the widespread application of food-relevant inorganic nanomaterials, the gastrointestinal tract is potentially exposed to these materials. Gut-on-chip in vitro systems are proposed for the investigation of compound toxicity as they better recapitulate the in vivo human intestinal environment than static models, due to the added shear stresses associated with the flow of the medium. We aimed to compare cellular responses of intestinal epithelial Caco-2 cells at the gene expression level upon TiO2 (E171) and ZnO (NM110) nanomaterial exposure when cultured under dynamic and conventionally applied static conditions. Whole-genome transcriptome analyses upon exposure of the cells to TiO2 and ZnO nanomaterials revealed differentially expressed genes and related biological processes that were culture condition specific. The total number of differentially expressed genes (p < 0.01) and affected pathways (p < 0.05 and FDR < 0.25) after nanomaterial exposure was higher under dynamic culture conditions than under static conditions for both nanomaterials. The observed increase in nanomaterial-induced responses in the gut-on-chip model indicates that shear stress might be a major factor in cell susceptibility. This is the first report on the application of a gut-on-chip system in which gene expression responses upon TiO2 and ZnO nanomaterial exposure are evaluated and compared to a static system. It extends current knowledge on nanomaterial toxicity assessment and the influence of a dynamic environment on cellular responses. Application of the gut-on-chip system resulted in higher sensitivity of the cells and might thus be an attractive system for use in the toxicological hazard characterization of nanomaterials.

Cytochrome P450 expression, induction and activity in human induced pluripotent stem cell-derived intestinal organoids and comparison with primary human intestinal epithelial cells and Caco-2 cells.

Human intestinal organoids (HIOs) are a promising in vitro model consisting of different intestinal cell types with a 3D microarchitecture resembling native tissue. In the current study, we aimed to assess the expression of the most common intestinal CYP enzymes in a human induced pluripotent stem cell (hiPSC)-derived HIO model, and the suitability of that model to study chemical-induced changes in CYP expression and activity. We compared this model with the commonly used human colonic adenocarcinoma cell line Caco-2 and with a human primary intestinal epithelial cell (IEC)-based model, closely resembling in vivo tissue. We optimized an existing protocol to differentiate hiPSCs into HIOs and demonstrated that obtained HIOs contain a polarized epithelium with tight junctions consisting of enterocytes, goblet cells, enteroendocrine cells and Paneth cells. We extensively characterized the gene expression of CYPs and activity of CYP3A4/5, indicating relatively high gene expression levels of the most important intestinal CYP enzymes in HIOs compared to the other models. Furthermore, we showed that CYP1A1 and CYP1B1 were induced by ß-naphtoflavone in all three models, whereas CYP3A4 was induced by phenobarbital and rifampicin in HIOs, in the IEC-based model (although not statistically significant), but not in Caco-2 cells. Interestingly, CYP2B6 expression was not induced in any of the models by the well-known liver CYP2B6 inducer phenobarbital. In conclusion, our study indicates that hiPSC-based HIOs are a useful in vitro intestinal model to study biotransformation of chemicals in the intestine.

New approach methodologies (NAMs) for human-relevant biokinetics predictions. Meeting the paradigm shift in toxicology towards an animal-free chemical risk assessment

For almost fifteen years, the availability and regulatory acceptance of new approach methodologies (NAMs) to assess the absorption, distribution, metabolism and excretion (ADME/biokinetics) in chemical risk evaluations are a bottleneck. To enhance the field, a team of 24 experts from science, industry, and regulatory bodies, including new generation toxicologists, met at the Lorentz Centre in Leiden, The Netherlands. A range of possibilities for the use of NAMs for biokinetics in risk evaluations were formulated (for example to define species differences and human variation or to perform quantitative in vitro-in vivo extrapolations). To increase the regulatory use and acceptance of NAMs for biokinetics for these ADME considerations within risk evaluations, the development of test guidelines (protocols) and of overarching guidance documents is considered a critical step. To this end, a need for an expert group on biokinetics within the Organisation of Economic Cooperation and Development (OECD) to supervise this process was formulated. The workshop discussions revealed that method development is still required, particularly to adequately capture transporter mediated processes as well as to obtain cell models that reflect the physiology and kinetic characteristics of relevant organs. Developments in the fields of stem cells, organoids and organ-on-a-chip models provide promising tools to meet these research needs in the future.

Microfluidic chip for culturing intestinal epithelial cell layers: Characterization and comparison of drug transport between dynamic and static models.

Dynamic flow in vitro models are currently widely explored for their applicability in drug development research. The application of gut-on-chip models in toxicology is lagging behind. Here we report the application of a gut-on-chip model for biokinetic studies and compare the observed biokinetics of reference compounds with those obtained using a conventional static in vitro model. Intestinal epithelial Caco-2 cells were cultured on a porous membrane assembled between two glass flow chambers for the dynamic model, or on a porous membrane in a Transwell model. Confocal microscopy, lucifer yellow translocation, and alkaline phosphatase activity evaluation revealed that cells cultured in the gut-on-chip model formed tight, differentiated, polarized monolayers like in the static cultures. In the dynamic gut-on-chip model the transport of the high permeability compounds antipyrine, ketoprofen and digoxin was lower (i.e. 4.2-, 2.7- and 1.9-fold respectively) compared to the transport in the static Transwell model. The transport of the low permeability compound, amoxicillin, was similar in both the dynamic and static in vitro model. The obtained transport values of the compounds are in line with the compound Biopharmaceuticals Classification System. It is concluded that the gut-on-chip provides an adequate model for transport studies of chemicals.

Combination of the BeWo b30 placental transport model and the embryonic stem cell test to assess the potential developmental toxicity of silver nanoparticles.

BACKGROUND: Silver nanoparticles (AgNPs) are used extensively in various consumer products because of their antimicrobial potential. This requires insight in their potential hazards and risks including adverse effects during pregnancy on the developing fetus. Using a combination of the BeWo b30 placental transport model and the mouse embryonic stem cell test (EST), we investigated the capability of pristine AgNPs with different surface chemistries and aged AgNPs (silver sulfide (Ag2S) NPs) to cross the placental barrier and induce developmental toxicity. The uptake/association and transport of AgNPs through the BeWo b30 was characterized using ICP-MS and single particle (sp)ICP-MS at different time points. The developmental toxicity of the AgNPs was investigated by characterizing their potential to inhibit the differentiation of mouse embryonic stem cells (mESCs) into beating cardiomyocytes. RESULTS: The AgNPs are able to cross the BeWo b30 cell layer to a level that was limited and dependent on their surface chemistry. In the EST, no in vitro developmental toxicity was observed as the effects on differentiation of the mESCs were only detected at cytotoxic concentrations. The aged AgNPs were significantly less cytotoxic, less bioavailable and did not induce developmental toxicity. CONCLUSIONS: Pristine AgNPs are capable to cross the placental barrier to an extent that is influenced by their surface chemistry and that this transport is likely low but not negligible. Next to that, the tested AgNPs have low intrinsic potencies for developmental toxicity. The combination of the BeWo b30 model with the EST is of added value in developmental toxicity screening and prioritization of AgNPs.

Current Insights into Monitoring, Bioaccumulation, and Potential Health Effects of Microplastics Present in the Food Chain.

Microplastics (MPs) are considered an emerging issue as environmental pollutants and a potential health threat. This review will focus on recently published data on concentrations in food, possible effects, and monitoring methods. Some data are available on concentrations in seafood (fish, bivalves, and shrimps), water, sugar, salt, and honey, but are lacking for other foods. Bottled water is a considerable source with numbers varying between 2600 and 6300 MPs per liter. Particle size distributions have revealed an abundance of particles smaller than 25 µm, which are considered to have the highest probability to pass the intestinal border and to enter the systemic circulation of mammals. Some studies with mice and zebrafish with short- or medium-term exposure (up to 42 days) have revealed diverse results with respect to both the type and extent of effects. Most notable modifications have been observed in gut microbiota, lipid metabolism, and oxidative stress. The principal elements of MP monitoring in food are sample preparation, detection, and identification. Identified data gaps include a lack of occurrence data in plant- and animal-derived food, a need for more data on possible effects of different types of microplastics, a lack of in silico models, a lack of harmonized monitoring methods, and a further development of quality assurance.

Impact of in vitro digestion on gastrointestinal fate and uptake of silver nanoparticles with different surface modifications.

Nanomaterials, especially silver nanoparticles (AgNPs), are used in a broad range of products owing to their antimicrobial potential. Oral ingestion is considered as a main exposure route to AgNPs. This study aimed to investigate the impact of the biochemical conditions within the human digestive tract on the intestinal fate of AgNPs across an intestinal in vitro model of differentiated Caco-2/HT29-MTX cells. The co-culture model was exposed to different concentrations (250-2500 µg/L) of pristine and in vitro digested (IVD) AgNPs and silver nitrate for 24 h. ICP-MS and spICP-MS measurements were performed for quantification of total Ag and AgNPs. The AgNPs size distribution, dissolution, and particle concentration (mass- and number-based) were characterized in the cell fraction and in the apical and basolateral compartments of the monolayer cultures. A significant fraction of the AgNPs dissolved (86-92% and 48-70%) during the digestion. Cellular exposure to increasing concentrations of pristine or IVD AgNPs resulted in a concentration dependent increase of total Ag and AgNPs content in the cellular fractions. The cellular concentrations were significantly lower following exposure to IVD AgNPs compared to the pristine AgNPs. Transport of silver as either total Ag or AgNPs was limited (<0.1%) following exposure to pristine and IVD AgNPs. We conclude that the surface chemistry of AgNPs and their digestion influence their dissolution properties, uptake/association with the Caco-2/HT29-MTX monolayer. This highlights the need to take in vitro digestion into account when studying nanoparticle toxicokinetics and toxicodynamics in cellular in vitro model systems.

Dynamic in vitro intestinal barrier model coupled to chip-based liquid chromatography mass spectrometry for oral bioavailability studies.

In oral bioavailability studies, evaluation of the absorption and transport of drugs and food components across the intestinal barrier is crucial. Advances in the field of organ-on-a-chip technology have resulted in a dynamic gut-on-a-chip model that better mimics the in vivo microenvironment of the intestine. Despite a few recent integration attempts, ensuring a biologically relevant microenvironment while coupling with a fully online detection system still represents a major challenge. Herein, we designed an online technique to measure drug permeability and analyse unknown product formation across an intestinal epithelial layer of Caco-2 and HT29-MTX cells cultured on a flow-through Transwell system, while ensuring the quality and relevance of the biological model. Chip-based ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was coupled to the dynamic Transwell system via a series of switching valves, thus allowing alternating measurements of the apical and basolateral sides of the in vitro model. Two trap columns were integrated for online sample pre-treatment and compatibility enhancement. Temporal analysis of the intestinal permeability was successfully demonstrated using verapamil as a model drug and ergotamine epimers as a model for natural toxins present in foods. Evidence was obtained that our newly developed dynamic system provided reliable results versus classical static in vitro models, and moreover, for the first time, epimer-specific transport is shown for ergotamine. Finally, initial experiments with the drug granisetron suggest that metabolic activity can be studied as well, thus highlighting the versatility of the bio-integrated online analysis system developed. Graphical abstract.

Impact of nanoparticle surface functionalization on the protein corona and cellular adhesion, uptake and transport.

BACKGROUND: Upon ingestion, nanoparticles can interact with the intestinal epithelial barrier potentially resulting in systemic uptake of nanoparticles. Nanoparticle properties have been described to influence the protein corona formation and subsequent cellular adhesion, uptake and transport. Here, we aimed to study the effects of nanoparticle size and surface chemistry on the protein corona formation and subsequent cellular adhesion, uptake and transport. Caco-2 intestinal cells, were exposed to negatively charged polystyrene nanoparticles (PSNPs) (50 and 200 nm), functionalized with sulfone or carboxyl groups, at nine nominal concentrations (15-250 µg/ml) for 10 up to 120 min. The protein coronas were analysed by LC-MS/MS. RESULTS: Subtle differences in the protein composition of the two PSNPs with different surface chemistry were noted. High-content imaging analysis demonstrated that sulfone PSNPs were associated with the cells to a significantly higher extent than the other PSNPs. The apparent cellular adhesion and uptake of 200 nm PSNPs was not significantly increased compared to 50 nm PSNPs with the same surface charge and chemistry. Surface chemistry outweighs the impact of size on the observed PSNP cellular associations. Also transport of the sulfone PSNPs through the monolayer of cells was significantly higher than that of carboxyl PSNPs. CONCLUSIONS: The results suggest that the composition of the protein corona and the PSNP surface chemistry influences cellular adhesion, uptake and monolayer transport, which might be predictive of the intestinal transport potency of NPs.

Implementation of a dynamic intestinal gut-on-a-chip barrier model for transport studies of lipophilic dioxin congeners.

Novel microfluidic technologies allow the manufacture of in vitro organ-on-a-chip systems that hold great promise to adequately recapitulate the biophysical and functional complexity of organs found in vivo. In this study, a gut-on-a-chip model was developed aiming to study the potential cellular association and transport of food contaminants. Intestinal epithelial cells (Caco-2) were cultured on a porous polyester membrane that was tightly clamped between two glass slides to form two separate flow chambers. Glass syringes, polytetrafluoroethylene tubing and glass microfluidic chips were selected to minimize surface adsorption of the studied compounds (i.e. highly lipophilic dioxins), during the transport studies. Confocal microscopy studies revealed that, upon culturing under constant flow for 7 days, Caco-2 cells formed complete and polarized monolayers as observed after culturing for 21 days under static conditions in Transwells. We exposed Caco-2 monolayers in the chip and Transwell to a mixture of 17 dioxin congeners (7 polychlorinated dibenzo-p-dioxins and 10 polychlorinated dibenzofurans) for 24 h. Gas chromatography-high resolution mass spectrometry was used to assess the cellular association and transport of individual dioxin congeners across the Caco-2 cell monolayers. After 24 h, the amount of transported dioxin mixture was similar in both the dynamic gut-on-a-chip model and the static Transwell model. The transport of individual congeners corresponded with their number of chlorine atoms and substitution patterns as revealed by quantitative structure-property relationship modelling. These results show that the gut-on-a-chip model can be used, as well as the traditional static Transwell system, to study the cellular association and transport of lipophilic compounds like dioxins.