A novel membrane-on-chip guides morphogenesis for the reconstruction of the intestinal crypt-villus axis.

Reconstructing the microscale villous organisation and functionality of the small intestine is essential for developingin vitroplatforms tailored for absorption studies as well as for investigating intestinal morphogenesis in development and disease. However, the current fabrication techniques able to mimic the villus-crypt axis poses significant challenges in terms of reconstruction of the complex 3D microarchitecture. These challenges extend beyond mere structural intricacies to encompass the incorporation of diverse cell types and the management of intricate fluid dynamics within the system. Here, we introduce a novel microfluidic device calledIn-Crypts, which integrates a cell-instructive membrane aimed at inducing and guiding Caco-2 cells morphogenesis. Patterned topographical cues embossed onto the porous membrane induce the formation of a well-organized intestinal epithelium, characterized by proliferating crypt-like domains and differentiated villus-like regions. Notably, our cell-instructive porous membrane effectively sustains stem cells development, faithfully replicating the niche environment ofin vivointestinal crypts thus mirroring the cell biogeography observedin vivo. Moreover, by introducing dynamic fluid flow, we provide a faithful recapitulation of the native microenvironmental shear stress experienced by the intestinal epithelium. This stress plays a crucial role in influencing cell behaviour, differentiation, and overall functionality, thus offering a highly realistic model for studying intestinal physiology and pathology. The resulting intestinal epithelium exhibits significantly denser regions of mucus and microvilli, characteristic typically absent in static cultures, upregulating more than 1.5 of the amount expressed in the classical flattened configuration, enhanced epithelial cell differentiation and increased adsorptive surface area. Hence, the innovative design ofIn-Cryptsproves the critical role of employing a cell-instructive membrane in argument the physiological relevance of organs-on-chips. This aspect, among others, will contribute to a more comprehensive understanding of organism function, directly impacting drug discovery and development.

An iPSC-derived small intestine-on-chip with self-organizing epithelial, mesenchymal, and neural cells.

Human induced pluripotent stem cell (hiPSC)-derived intestinal organoids are valuable tools for researching developmental biology and personalized therapies, but their closed topology and relative immature state limit applications. Here, we use organ-on-chip technology to develop a hiPSC-derived intestinal barrier with apical and basolateral access in a more physiological in vitro microenvironment. To replicate growth factor gradients along the crypt-villus axis, we locally expose the cells to expansion and differentiation media. In these conditions, intestinal epithelial cells self-organize into villus-like folds with physiological barrier integrity, and myofibroblasts and neurons emerge and form a subepithelial tissue in the bottom channel. The growth factor gradients efficiently balance dividing and mature cell types and induce an intestinal epithelial composition, including absorptive and secretory lineages, resembling the composition of the human small intestine. This well-characterized hiPSC-derived intestine-on-chip system can facilitate personalized studies on physiological processes and therapy development in the human small intestine.

Engineering Cell Instructive Microenvironments for In Vitro Replication of Functional Barrier Organs.

Multicellular organisms exhibit synergistic effects among their components, giving rise to emergent properties crucial for their genesis and overall functionality and survival. Morphogenesis involves and relies upon intricate and biunivocal interactions among cells and their environment, that is, the extracellular matrix (ECM). Cells secrete their own ECM, which in turn, regulates their morphogenetic program by controlling time and space presentation of matricellular signals. The ECM, once considered passive, is now recognized as an informative space where both biochemical and biophysical signals are tightly orchestrated. Replicating this sophisticated and highly interconnected informative media in a synthetic scaffold for tissue engineering is unattainable with current technology and this limits the capability to engineer functional human organs in vitro and in vivo. This review explores current limitations to in vitro organ morphogenesis, emphasizing the interplay of gene regulatory networks, mechanical factors, and tissue microenvironment cues. In vitro efforts to replicate biological processes for barrier organs such as the lung and intestine, are examined. The importance of maintaining cells within their native microenvironmental context is highlighted to accurately replicate organ-specific properties. The review underscores the necessity for microphysiological systems that faithfully reproduce cell-native interactions, for advancing the understanding of developmental disorders and disease progression.

From animal models to gut-on-chip: the challenging journey to capture inter-individual variability in chronic digestive disorders.

Chronic digestive disorders are of increasing incidence worldwide with expensive treatments and no available cure. Available therapeutic schemes mainly rely on symptom relief, with large degrees of variability in patients' response to such treatments, underlining the need for new therapeutic strategies. There are strong indications that the gut microbiota's contribution seems to be a key modulator of disease activity and patients' treatment responses. Hence, efforts have been devoted to understanding host-microbe interactions and the mechanisms underpinning such variability. Animal models, being the gold standard, provide valuable mechanistic insights into host-microbe interactions. However, they are not exempt from limitations prompting the development of alternative methods. Emerging microfluidic technologies and gut-on-chip models were shown to mirror the main features of gut physiology and disease state, reflect microbiota modification, and include functional readouts for studying host responses. In this commentary, we discuss the relevance of animal models in understanding host-microbe interactions and how gut-on-chip technology holds promises for addressing patient variability in responses to chronic digestive disease treatment.

Addressing the ADME Challenges of Compound Loss in a PDMS-Based Gut-on-Chip Microphysiological System.

Microphysiological systems (MPSs) are promising in vitro technologies for physiologically relevant predictions of the human absorption, distribution, metabolism, and excretion (ADME) properties of drug candidates. However, polydimethylsiloxane (PDMS), a common material used in MPSs, can both adsorb and absorb small molecules, thereby compromising experimental results. This study aimed to evaluate the feasibility of using the PDMS-based Emulate gut-on-chip to determine the first-pass intestinal drug clearance. In cell-free PDMS organ-chips, we assessed the loss of 17 drugs, among which testosterone was selected as a model compound for further study based on its substantial ad- and absorptions to organ chips and its extensive first-pass intestinal metabolism with well-characterized metabolites. A gut-on-chip model consisting of epithelial Caco-2 cells and primary human umbilical vein endothelial cells (HUVECs) was established. The barrier integrity of the model was tested with reference compounds and inhibition of drug efflux. Concentration-time profiles of testosterone were measured in cell-free organ chips and in gut-on-chip models. A method to deduce the metabolic clearance was provided. Our results demonstrate that metabolic clearance can be determined with PDMS-based MPSs despite substantial compound loss to the chip. Overall, this study offers a practical protocol to experimentally assess ADME properties in PDMS-based MPSs.

Chips, guts, and gas: unraveling volatile microbial mysteries in real time!

Exploring the gastrointestinal role of hydrogen sulfide (H2S) is difficult because of its volatility and the absence of a precisely controllable model system for manipulating the gut environment. Hayes et al. address this issue by engineering Escherichia coli to titrate H2S levels in a gas-impermeable gut-on-chip device.

Recent Advances in Cell-Based In Vitro Models to Recreate Human Intestinal Inflammation.

Inflammatory bowel disease causes a major burden to patients and healthcare systems, raising the need to develop effective therapies. Technological advances in cell culture, allied with ethical issues, have propelled in vitro models as essential tools to study disease aetiology, its progression, and possible therapies. Several cell-based in vitro models of intestinal inflammation have been used, varying in their complexity and methodology to induce inflammation. Immortalized cell lines are extensively used due to their long-term survival, in contrast to primary cultures that are short-lived but patient-specific. Recently, organoids and organ-chips have demonstrated great potential by being physiologically more relevant. This review aims to shed light on the intricate nature of intestinal inflammation and cover recent works that report cell-based in vitro models of human intestinal inflammation, encompassing diverse approaches and outcomes.

Bioinspired in vitro intestinal mucus model for 3D-dynamic culture of bacteria.

The intestinal mucus is a biological barrier that supports the intestinal microbiota growth and filters molecules. To perform these functions, mucus possesses optimized microstructure and viscoelastic properties and it is steadily replenished thus flowing along the gut. The available in vitro intestinal mucus models are useful tools in investigating the microbiota-human cells interaction, and are used as matrices for bacterial culture or as static component of microfluidic devices like gut-on-chips. The aim of this work is to engineer an in vitro mucus models (I-Bac3Gel) addressing in a single system physiological viscoelastic properties (i.e., 2-200 Pa), 3D structure and suitability for dynamic bacterial culture. Homogeneously crosslinked alginate hydrogels are optimized in composition to obtain target viscoelastic and microstructural properties. Then, rheological tests are exploited to assess a priori the hydrogels capability to withstand the flow dynamic condition. We experimentally assess the suitability of I-Bac3Gels in the evolving field of microfluidics by applying a dynamic flow to a bacterial-loaded mucus model and by monitoring E. coli growth and survival. The engineered models represent a step forward in the modelling of the mucus, since they can answer to different urgent needs such as a 3D structure, bioinspired properties and compatibility with dynamic system.

Colon Cancer: From Epidemiology to Prevention.

Colorectal cancer (CRC) is one of the most prevalent cancers affecting humans, with a complex genetic and environmental aetiology. Unlike cancers with known environmental, heritable, or sex-linked causes, sporadic CRC is hard to foresee and has no molecular biomarkers of risk in clinical use. One in twenty CRC cases presents with an established heritable component. The remaining cases are sporadic and associated with partially obscure genetic, epigenetic, regenerative, microbiological, dietary, and lifestyle factors. To tackle this complexity, we should improve the practice of colonoscopy, which is recommended uniformly beyond a certain age, to include an assessment of biomarkers indicative of individual CRC risk. Ideally, such biomarkers will be causal to the disease and potentially modifiable upon dietary or therapeutic interventions. Multi-omics analysis, including transcriptional, epigenetic as well as metagenomic, and metabolomic profiles, are urgently required to provide data for risk analyses. The aim of this article is to provide a perspective on the multifactorial derailment of homeostasis leading to the initiation of CRC, which may be explored via multi-omics and Gut-on-Chip analysis to identify much-needed predictive biomarkers.

Implementing organ-on-chip in a next-generation risk assessment of chemicals: a review.

Organ-on-chip (OoC) technology is full of engineering and biological challenges, but it has the potential to revolutionize the Next-Generation Risk Assessment of novel ingredients for consumer products and chemicals. A successful incorporation of OoC technology into the Next-Generation Risk Assessment toolbox depends on the robustness of the microfluidic devices and the organ tissue models used. Recent advances in standardized device manufacturing, organ tissue cultivation and growth protocols offer the ability to bridge the gaps towards the implementation of organ-on-chip technology. Next-Generation Risk Assessment is an exposure-led and hypothesis-driven tiered approach to risk assessment using detailed human exposure information and the application of appropriate new (non-animal) toxicological testing approaches. Organ-on-chip presents a promising in vitro approach by combining human cell culturing with dynamic microfluidics to improve physiological emulation. Here, we critically review commercial organ-on-chip devices, as well as recent tissue culture model studies of the skin, intestinal barrier and liver as the main metabolic organ to be used on-chip for Next-Generation Risk Assessment. Finally, microfluidically linked tissue combinations such as skin-liver and intestine-liver in organ-on-chip devices are reviewed as they form a relevant aspect for advancing toxicokinetic and toxicodynamic studies. We point to recent achievements and challenges to overcome, to advance non-animal, human-relevant safety studies.

Gut-on-chip for ecological and causal human gut microbiome research.

There is a growing interest to understand if and how the gut microbiome is causally linked to the pathogenesis and/or progression of diseases. While in vitro cell line models are commonly used for studying specific aspects of the host-microbe interaction, gnotobiotic murine models are considered the preferred platform for studying causality in microbiome research. Nevertheless, findings from animal studies provide limited opportunity for delineating various areas of interest to the human gut microbiome research. Gut-on-chips are biomimetics recapitulating intestinal physiology which enable investigation of bidirectional effects of the host and microbiome. We posit that they could advance causal and ecological gut microbiome research in three major areas: (i) diet-microbiome and drug-microbiome interaction; (ii) microbiome-targeted therapeutics pharmacoecology; and (iii) mechanistic studies of gut microbiome and microbiome-targeted intervention in extraintestinal pathologies.

In vitro models and ex vivo systems used in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic, relapsing gastrointestinal condition. Ulcerative colitis and Crohn's disease are types of inflammatory bowel disease. Over many decades, the disease has been a topic of study, with experts still trying to figure out its cause and pathology. Researchers have established many in vivo animal models, in vitro cell lines, and ex vivo systems to understand its cause ultimately and adequately identify a therapy. However, in vivo animal models cannot be regarded as good models for studying IBD since they cannot completely simulate the disease. Furthermore, because species differences are a crucial subject of concern, in vitro cell lines and ex vivo systems can be employed to recreate the condition properly. In vitro models serve as the starting point for biological and medical research. Ex vivo and in vitro models for replicating gut physiology have been developed. This review aims to present a clear understanding of several in vitro and ex vivo models of IBD and provide insights into their benefits and limits and their value in understanding intestinal physiology.

Intestinal Stem Cell-on-Chip to Study Human Host-Microbiota Interaction.

The gut is a tubular organ responsible for nutrient absorption and harbors our intestinal microbiome. This organ is composed of a multitude of specialized cell types arranged in complex barrier-forming crypts and villi covered by a mucosal layer controlling nutrient passage and protecting from invading pathogens. The development and self-renewal of the intestinal epithelium are guided by niche signals controlling the differentiation of specific cell types along the crypt-villus axis in the epithelium. The emergence of microphysiological systems, or organ-on-chips, has paved the way to study the intestinal epithelium within a dynamic and controlled environment. In this review, we describe the use of organ-on-chip technology to control and guide these differentiation processes in vitro. We further discuss current applications and forthcoming strategies to investigate the mechanical processes of intestinal stem cell differentiation, tissue formation, and the interaction of the intestine with the microbiota in the context of gastrointestinal diseases.

The Progress of Intestinal Epithelial Models from Cell Lines to Gut-On-Chip.

Over the past years, several preclinical in vitro and ex vivo models have been developed that helped to understand some of the critical aspects of intestinal functions in health and disease such as inflammatory bowel disease (IBD). However, the translation to the human in vivo situation remains problematic. The main reason for this is that these approaches fail to fully reflect the multifactorial and complex in vivo environment (e.g., including microbiota, nutrition, and immune response) in the gut system. Although conventional models such as cell lines, Ussing chamber, and the everted sac are still used, increasingly more sophisticated intestinal models have been developed over the past years including organoids, InTESTine™ and microfluidic gut-on-chip. In this review, we gathered the most recent insights on the setup, advantages, limitations, and future perspectives of most frequently used in vitro and ex vivo models to study intestinal physiology and functions in health and disease.

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.

Gut-on-chip: Recreating human intestine in vitro.

The human gut is important for food digestion and absorption, as well as a venue for a large number of microorganisms that coexist with the host. Although numerous in vitro models have been proposed to study intestinal pathology or interactions between intestinal microbes and host, they are far from recapitulating the real intestinal microenvironment in vivo. To assist researchers in further understanding gut physiology, the intestinal microbiome, and disease processes, a novel technology primarily based on microfluidics and cell biology, called "gut-on-chip," was developed to simulate the structure, function, and microenvironment of the human gut. In this review, we first introduce various types of gut-on-chip systems, then highlight their applications in drug pharmacokinetics, host-gut microbiota crosstalk, and nutrition metabolism. Finally, we discuss challenges in this field and prospects for better understanding interactions between intestinal flora and human hosts, and then provide guidance for clinical treatment of related diseases.

A Membrane-free Liver-Gut-on-Chip Platform for the Assessment on Dysregulated Mechanisms of Cholesterol and Bile Acid Metabolism Induced by PM2.5.

Fine particulate matter (PM2.5)-induced metabolic diseases have attracted a great deal of attention recently. However, the relevant metabolic mechanisms of PM2.5 in vivo have not yet been fully described due to the lack of reliable platforms. Herein, a membrane-free liver-gut-on-chip (L-GOC) platform was developed to investigate metabolism dysregulation induced by PM2.5. A multiple organ system with a liver-gut structure and two circulation paths (L-G and G-L circulation paths) was created, and then cells were exposed to PM2.5 on this platform. Secreted high-density lipoprotein (HDL) levels were detected, which demonstrates that this multiple organ system functioned with normal physiological metabolism at the organ level. Untargeted metabolomic analysis showed that there were 364 metabolites of LO2 cells dysregulated after exposure to PM2.5 at a concentration of 200 µg/mL. Moreover, cholesterol and bile acid metabolism were significantly dysregulated. Further immunofluorescence and ELISA assays confirmed that signal transduction pathways related to cholesterol metabolism (LCAT-CE, PON1-HDL, and SRB1-HDL metabolic pathways) and bile acid metabolism (CYP7A1-CA/CDCA/DCA metabolic pathways) were disturbed. These results indicate that PM2.5 primarily disturbed cholesterol metabolism of the liver and then disrupted bile acid metabolism of the liver (primary bile acid biosynthesis) and gut (secondary bile acid biosynthesis) via related metabolic pathways. These findings may partially explain the metabolic mechanisms of cells triggered by PM2.5 exposure.

A Theoretical and Experimental Study to Optimize Cell Differentiation in a Novel Intestinal Chip.

Microphysiological systems have potential as test systems in studying the intestinal barrier, in which shear stress is critical for the differentiation of Caco-2 cells into enterocytes. The most commonly used in vitro gut model for intestinal barrier studies is based on trans-well cultures. Albeit useful, these culture systems lack physiological shear stress which is believed to be critical for the differentiation of Caco-2 cells into enterocytes and to form tight monolayers. Conversely, organ-on-chip models have presented themselves as a promising alternative since it provides cells with the required shear stress. To this end, a novel biocompatible 3D-printed microfluidic device was developed. In this device, Caco-2 cells were seeded under physiologically-relevant unidirectional shear stress and compared to cells cultured under gravity-driven flow. Using numerical studies, the flow rate that corresponds to the required shear stress was calculated. Experimental tests were conducted to verify the effect of this on cell differentiation. The experiments clearly showed an enhancement of cell differentiation potential in a unidirectional physiologically-relevant pump-driven flow system (PDFS) as opposed to the simpler bidirectional gravity-driven flow system (GDFS). Additionally, computational modeling of an adapted design confirmed its ability to supply all cells with a more homogeneous shear stress, potentially further enhancing their differentiation. The shear stress in the adapted design can be well-approximated with analytic methods, thus allowing for efficient predictions for all parameter values in the system. The developed novel microfluidic device led to the formation of a tighter monolayer and enhanced functional properties of the differentiated Caco-2 cells, which presents a promising tool for preclinical in vitro testing of drugs in an animal-free platform.

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.

Bioengineered Human Organ-on-Chip Reveals Intestinal Microenvironment and Mechanical Forces Impacting Shigella Infection.

Intestinal epithelial cells are constantly exposed to pathogens and mechanical forces. However, the impact of mechanical forces on infections leading to diarrheal diseases remains largely unknown. Here, we addressed whether flow and peristalsis impact the infectivity of the human pathogen Shigella within a 3D colonic epithelium using Intestine-Chip technology. Strikingly, infection is significantly increased and minimal bacterial loads are sufficient to invade enterocytes from the apical side and trigger loss of barrier integrity, thereby shifting the paradigm about early stage Shigella invasion. Shigella quickly colonizes epithelial crypt-like invaginations and demonstrates the essential role of the microenvironment. Furthermore, by modulating the mechanical forces of the microenvironment, we find that peristalsis impacts Shigella invasion. Collectively, our results reveal that Shigella leverages the intestinal microenvironment by taking advantage of the microarchitecture and mechanical forces to efficiently invade the intestine. This approach will enable molecular and mechanistic interrogation of human-restricted enteric pathogens.