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Simple Summary: This review aims to improve our understanding of gastrointestinal tract cancer and the side effects of cancer treatment by using advanced in vitro systems. Traditional models like cell cultures and animal studies provide valuable insights but have limitations in replicating the complexity of human disease and raise ethical concerns. By focusing on bioreactor-based in vitro systems, which can mimic the physical and chemical environment of the gastrointestinal tract, this study aims to provide more accurate models for studying cancer and its treatment side effects. These advancements could lead to better insights into disease mechanisms, potentially improving treatment strategies and benefiting the broader research community. Human diseases are multifaceted, starting with alterations at the cellular level, damaging organs and their functions, and disturbing interactions and immune responses. In vitro systems offer clarity and standardisation, which are crucial for effectively modelling disease. These models aim not to replicate every disease aspect but to dissect specific ones with precision. Controlled environments allow researchers to isolate key variables, eliminate confounding factors and elucidate disease mechanisms more clearly. Technological progress has rapidly advanced model systems. Initially, 2D cell culture models explored fundamental cell interactions. The transition to 3D cell cultures and organoids enabled more life-like tissue architecture and enhanced intercellular interactions. Advanced bioreactor-based devices now recreate the physicochemical environments of specific organs, simulating features like perfusion and the gastrointestinal tract's mucus layer, enhancing physiological relevance. These systems have been simplified and adapted for high-throughput research, marking significant progress. This review focuses on in vitro systems for modelling gastrointestinal tract cancer and the side effects of cancer treatment. While cell cultures and in vivo models are invaluable, our main emphasis is on bioreactor-based in vitro modelling systems that include the gut microbiome. [ABSTRACT FROM AUTHOR]

Exopolysaccharides (EPSs) producing lactic acid bacteria have been claimed to confer various health benefits to the host, including the ability to face oxidative and inflammatory-related stress. This study investigated the ability of food-borne Lactiplantibacillus (Lpb.) plantarum to improve the antioxidant activity of fermented milks by producing EPSs. Two Lpb. plantarum strains, selected as lower and higher EPSs producers, have been applied in lab-scale fermented milk production, in combination with conventional starters. Antioxidant activity was investigated in vitro using DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS (2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and FRAP (ferric reducing antioxidant power) assays while the ability to modulate reactive oxygen species (ROS) level was evaluated in an intestinal healthy model, subjected to both oxidative and inflammatory stress. Furthermore, to verify whether digestion affects functionality, fermented milks were evaluated before and after in vitro-simulated INFOGEST digestion. The results showed an improved antioxidant activity of fermented milk enriched with Lpb. plantarum LT100, the highest EPSs producer. Furthermore, the data showed a different ROS modulation with a protective anti-inflammatory effect of samples enriched with Lpb. plantarum strains. Our data suggest the use of selected EPS-producing strains of Lpb. plantarum as a natural strategy to enrich the functionality of fermented milks in terms of ROS modulation and inflammatory-related stress. [ABSTRACT FROM AUTHOR]

A novel bioreactor simulating human colonic conditions for in vitro cultivation of intestinal microbiota is presented. The PEristaltic mixed Tubular bioReactor (PETR) is modular designed and periodically kneaded to simulate intestinal peristalsis. The reactor is introduced, characterized from a bioprocess engineer's perspective and discussed in its ability to mimic colon conditions. PETR provides physiological temperature and appropriate anaerobic conditions, simulates intestinal peristalsis, and has a mean residence time of 32.8 ± 0.8 h comparable to the adult human colon. The single‐tube design enables a time‐constant and longitudinally progressive pH gradient from 5.5 to 7.0. Using a dialysis liquid containing high molecular weight polyethylene glycol, the integrated dialysis system efficiently absorbs short chain fatty acids (up to 60%) and water (on average 850 mL d−1). Cultivation of a typical gut bacterium (Bifidobacterium animalis) was performed to demonstrate the applicability for controlled microbiota cultivation. PETR is unique in combining simulation of the entire colon, peristaltic mixing, dialytic water and metabolite absorption, and a progressive pH gradient in a single‐tube design. PETR is a further step to precise replication of colonic conditions in vitro for reliable and reproducible microbiota research, such as studying the effect of food compounds, prebiotics or probiotics, or the development and treatment of infections with enteric pathogens, but also for further medical applications such as drug delivery studies or to study the effect of drugs on and their degradation by the microbiota. [ABSTRACT FROM AUTHOR]

Exopolysaccharides (EPSs) producing lactic acid bacteria have been claimed to confer various health benefits to the host, including the ability to face oxidative and inflammatory-related stress. This study investigated the ability of food-borne Lactiplantibacillus (Lpb.) plantarum to improve the antioxidant activity of fermented milks by producing EPSs. Two Lpb. plantarum strains, selected as lower and higher EPSs producers, have been applied in lab-scale fermented milk production, in combination with conventional starters. Antioxidant activity was investigated in vitro using DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS (2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and FRAP (ferric reducing antioxidant power) assays while the ability to modulate reactive oxygen species (ROS) level was evaluated in an intestinal healthy model, subjected to both oxidative and inflammatory stress. Furthermore, to verify whether digestion affects functionality, fermented milks were evaluated before and after in vitro-simulated INFOGEST digestion. The results showed an improved antioxidant activity of fermented milk enriched with Lpb. plantarum LT100, the highest EPSs producer. Furthermore, the data showed a different ROS modulation with a protective anti-inflammatory effect of samples enriched with Lpb. plantarum strains. Our data suggest the use of selected EPS-producing strains of Lpb. plantarum as a natural strategy to enrich the functionality of fermented milks in terms of ROS modulation and inflammatory-related stress.

The intestine plays an important role in the absorption, metabolism, and transportation of xenobiotics including drugs and foods. Therefore, an in vitro intestinal model for predicting bioavailability of xenobiotics is important for the drug and food industries. Caco-2 is a human colon carcinoma cell line widely employed as a model to represent xenobiotic absorption, transportation, and metabolism in the human intestinal tract. However, Caco-2 has limitations as an intestinal model due to the low expression of metabolizing enzymes and the long time period required for cell differentiation. Hence, several modified Caco2 models have been developed to overcome these limitations and to establish Caco-2 models that correlate well with human intestine. These include xenobiotic-induced cytochrome P450 and transporter expression models, nuclear receptor gene-modified Caco-2 cells, and a novel Caco-2 culturing method that mimics the physiological characteristics of the human intestine and accelerates the growth rate of Caco-2

Oral administration is the preferred route for drug administration that leads to better therapy compliance. The intestine plays a key role in the absorption and metabolism of oral drugs, therefore, new intestinal models are being continuously proposed, which contribute to the study of intestinal physiology, drug screening, drug side effects, and drug–drug interactions. Advances in pharmaceutical processes have produced more drug formulations, causing challenges for intestinal models. To adapt to the rapid evolution of pharmaceuticals, more intestinal models have been created. However, because of the complexity of the intestine, few models can take all aspects of the intestine into account, and some functions must be sacrificed to investigate other areas. Therefore, investigators need to choose appropriate models according to the experimental stage and other requirements to obtain the desired results. To help researchers achieve this goal, this review summarised the advantages and disadvantages of current commonly used intestinal models and discusses possible future directions, providing a better understanding of intestinal models. [ABSTRACT FROM AUTHOR]

Considerable effort has been put forth to understand mechanisms by which the microbiota modulates and responds to inflammation. Here, we explored whether oxidation metabolites produced by the host during inflammation, sodium nitrate and trimethylamine oxide, impact the composition of a human stool bacterial population in a gut simulator. We then assessed whether an immune-competent in vitro intestinal model responded differently to spent medium from bacteria exposed to these cues compared to spent medium from a control bacterial population. The host-derived oxidation products were found to decrease levels of Bacteroidaceae and overall microbiota metabolic potential, while increasing levels of proinflammatory Enterobacteriaceae and lipopolysaccharide in bacterial cultures, reflecting shifts that occur in vivo in inflammation. Spent microbiota media induced elevated intracellular mucin levels and reduced intestinal monolayer integrity as reflected in transepithelial electrical resistance relative to fresh medium controls. However, multiplexed cytokine analysis revealed markedly different cytokine signatures from intestinal cultures exposed to spent medium with added oxidation products relative to spent control medium, while cytokine signatures of cultures exposed to fresh media were similar regardless of addition of host-derived cues. Further, the presence of immune cells in the intestinal model was required for this differentiation of cytokine signatures. This study indicates that simple in vitro immune-competent intestinal models can capture bacterial-mammalian cross-talk in response to host-derived oxidation products and supports utility of these systems for mechanistic studies of interactions between the gut microbiome and host in inflammation.

The intestine plays an important role in the absorption, metabolism, and transportation of xenobiotics including drugs and foods. Therefore, an in vitro intestinal model for predicting bioavailability of xenobiotics is important for the drug and food industries. Caco-2 is a human colon carcinoma cell line widely employed as a model to represent xenobiotic absorption, transportation, and metabolism in the human intestinal tract. However, Caco-2 has limitations as an intestinal model due to the low expression of metabolizing enzymes and the long time period required for cell differentiation. Hence, several modified Caco-2 models have been developed to overcome these limitations and to establish Caco-2 models that correlate well with human intestine. These include xenobiotic-induced cytochrome P450 and transporter expression models, nuclear receptor gene-modified Caco-2 cells, and a novel Caco-2 culturing method that mimics the physiological characteristics of the human intestine and accelerates the growth rate of Caco-2. [ABSTRACT FROM AUTHOR]

Intestinal dysfunction is frequently driven by abnormalities of specific genes, microbiota, or microenvironmental factors, which usually differ across individuals, as do intestinal physiology and pathology. Therefore, it's necessary to develop personalized therapeutic strategies, which are currently limited by the lack of a simulated intestine model. The mature human intestinal mucosa is covered by a single layer of columnar epithelial cells that are derived from intestinal stem cells (ISCs). The complexity of the organ dramatically increases the difficulty of faithfully mimicking in vivo microenvironments. However, a simulated intestine model will serve as an indispensable foundation for personalized drug screening. In this article, we review the advantages and disadvantages of conventional 2-dimensional models, intestinal organoid models, and current microfluidic intestine-on-a-chip (IOAC) models. The main technological strategies are summarized, and an advanced microfluidic primary IOAC model is proposed for personalized intestinal medicine. In this model, primary ISCs and the microbiome are isolated from individuals and co-cultured in a multi-channel microfluidic chip to establish a microengineered intestine device. The device can faithfully simulate in vivo fluidic flow, peristalsis-like motions, host-microbe crosstalk, and multi-cell type interactions. Moreover, the ISCs can be genetically edited before seeding, and monitoring sensors and post-analysis abilities can also be incorporated into the device to achieve high-throughput and rapid pharmaceutical studies. We also discuss the potential future applications and challenges of the microfluidic platform. The development of cell biology, biomaterials, and tissue engineering will drive the advancement of the simulated intestine, making a significant contribution to personalized medicine in the future. [ABSTRACT FROM AUTHOR]

Glucosinolates are plant secondary metabolites found in cruciferous vegetables (Brassicaceae) that are valued for their potential health benefits. Frequently consumed representatives of these vegetables, for example, are white or red cabbage, which are typically boiled before consumption. Recently, 3-alk(en)yl-4-hydroxythiazolidine-2-thiones were identified as a class of thermal glucosinolate degradation products that are formed during the boiling of cabbage. Since these newly discovered compounds are frequently consumed, this raises questions about their potential uptake and their possible bioactive functions. Therefore, 3-allyl-4-hydroxythiazolidine-2-thione (allyl HTT) and 4-hydroxy-3-(4-(methylsulfinyl) butyl)thiazolidine-2-thione (4-MSOB HTT) as degradation products of the respective glucosinolates sinigrin and glucoraphanin were investigated. After consumption of boiled red cabbage broth, recoveries of consumed amounts of the degradation products in urine collected for 24 h were 18 ± 5% for allyl HTT and 21 ± 4% for 4-MSOB HTT (mean ± SD, n = 3). To investigate the stability of the degradation products during uptake and to elucidate the uptake mechanism, both an in vitro stomach and an in vitro intestinal model were applied. The results indicate that the uptake of allyl HTT and 4-MSOB HTT occurs by passive diffusion. Both compounds show no acute cell toxicity, no antioxidant potential, and no change in NAD(P)H dehydrogenase quinone 1 (NQO1) activity up to 100 μM. However, inhibition of glycogen synthase kinases-3 (GSK-3) in the range of 20% for allyl HTT for the isoform GSK-3β and 29% for 4-MSOB HTT for the isoform GSK-3α at a concentration of 100 μM was found. Neither health-promoting nor toxic effects of 3-alk(en)yl-4-hydroxythiazolidine-2-thiones were found in the four tested assays carried out in this study, which contrasts with the properties of other glucosinolate degradation products, such as isothiocyanates.

Supplemental dietary amino acids (AAs) need to be provided in a form that prevents their degradation along the gastrointestinal tract to guarantee their high bioavailability and bioactivity. In this study, methionine (Met) protected via organo-clay intercalation (natural carriers) has been developed as a sustainable alternative to polymeric coating. Specifically, two different bentonite-zeolite-based mineral clays were tested, Adsorbene (ADS) and BioKi (BIO). Briefly, 1 g of the carrier (ADS or BIO) was contacted with 50 mL of an aqueous solution at a pH of 3.0, 5.8, and 8.9. Solid-liquid separation was conducted. The released Met in the liquid phase was analysed by Chemical Oxygen Demand, while residual Met in the solid phase was analysed by Fourier Transform Infra-Red (FT-IR) spectroscopy. The effect of Met-ADS complex on cell viability was tested on IPEC-J2 cells incubated 3 h with Met-ADS 2.5 mM. Jejunum segments obtained by entire male pigs (Swiss Large White, body weight 100 ± 5 kg) were used as ex vivo models to compare the absorption of 2.5 mM Met released by ADS with 2.5 mM free Met and its influence on epithelial integrity in perfusion Ussing chambers. The carriers released a very low amount of Met and Met-BIO interaction was stronger than Met-ADS. The maximum release of Met was at pH 3, with 3% and 6% of Met release from Met-BIO and Met-ADS, respectively. Cell viability experiments revealed that Met-ADS did not alter cell metabolic activity. No differences in Met absorption and intestinal epithelial integrity were observed ex vivo between free Met and Met-ADS. This study provided new insights into the release of Met from natural clays such as ADS and BIO, the safety of its use in the porcine intestine and the ability of ADS-released Met to absorb to the same extent as the free Met in porcine jejunum. [ABSTRACT FROM AUTHOR]

The small intestine is the primary site of drug absorption following oral administration, making paramount the proper monitoring of the absorption process. In vitro tools to predict intestinal absorption are particularly important in preclinical drug development since they are less laborious and cost-intensive and raise less ethical considerations compared to in vivo studies. The Caco-2 model is considered the gold standard of in vitro intestinal models regarding the prediction of absorption of orally delivered compounds. However, this model presents several drawbacks, such as the expression of tighter tight junctions, not being suitable to perform permeability of paracellular compounds. Besides, cells are representative of only one intestinal cell type, without considering the role of non-absorptive cells on the absorption pathway of drugs. In the present study, we developed a new three-dimensional (3D) intestinal model that aims to bridge the gap between in vitro tools and animal studies. Our 3D model comprises a collagen layer with human intestinal fibroblasts (HIFs) embedded, mimicking the intestinal lamina propria and providing 3D support for the epithelium, composed of Caco-2 cells and mucus-producing HT29-MTX cells, creating a model that can better resemble, both in terms of composition and regarding the outcomes of drug permeability when testing paracellular compounds, the human small intestine. The optimization of the collagen layer with HIFs was performed, testing different collagen concentrations and HIF seeding densities in order to avoid collagen contraction before day 14, maintaining HIF metabolically active inside the collagen disks during time in culture. HIF morphology and extracellular matrix (ECM) deposition were assessed, confirming that fibroblasts presented a normal and healthy elongated shape and secreted fibronectin and laminin, remodeling the collagen matrix. Regarding the epithelial layer, transepithelial electrical resistance (TEER) values decreased when cells were in the 3D configuration, comparing with the 2D analogs (Caco-2 and coculture of Caco-2+HT29-MTX models), becoming more similar with in vivo values. The permeability assay with fluorescein isothiocyanate (FITC)–Dextran 4 kDa showed that absorption in the 3D models is significantly higher than that in the 2D models, confirming the importance of using a more biorelevant model when testing the paracellular permeability of compounds.

Microphysiometry has proved to be a useful tool for monitoring the energy metabolism of living cells and their interactions with other cells. The technique has mainly been used for monitoring two-dimensional (2D) monolayers of cells. Recently, our group showed that it is also possible to monitor the extracellular acidification rate and transepithelial electrical resistance (TEER) of 3D skin constructs in an automated assay maintaining an air–liquid interface (ALI) with a BioChip extended by 3D-printed encapsulation. In this work, we present an optimized multichannel intestine-on-a-chip for monitoring the TEER of the commercially available 3D small intestinal tissue model (EpiIntestinalTM from MatTek). Experiments are performed for 1 day, during which a 60 min cycle is repeated periodically. Each cycle consists of three parts: (1) maintain ALI; (2) application of the measurement medium or test substance; and (3) the rinse cycle. A cytotoxic and barrier-disrupting benchmark chemical (0.2% sodium dodecyl sulfate) was applied after 8 h of initial equilibration. This caused time-dependent reduction of the TEER, which could not be observed with typical cytotoxicity measurement methods. This work represents a proof-of-principle of multichannel time-resolved TEER monitoring of a 3D intestine model using an automated ALI. Reconstructed human tissue combined with the Intelligent Mobile Lab for In vitro Diagnostic technology represents a promising research tool for use in toxicology, cellular metabolism studies, and drug absorption research.

In order to find dietary conditions favouring endogenous equol biosynthesis, a pooled faecal homogenate from equol-producing women was used to inoculate the TIM-2 artificial model of the human proximal colon. The model was fuelled with control diets not supplemented (C) or supplemented (C-ISO) with isoflavones, and two isoflavone-containing diets rich in carbohydrate (CH-ISO) or protein (PR-ISO). Compared to the C-ISO control, the CH-ISO diet doubled the production of equol, while with the PR-ISO diet the production of equol in cultures decreased sharply. The CH-ISO diet was also associated with enhanced butyrate production. The numbers of most bacterial populations analysed did not significantly change along cultures with any of the diets. Surprisingly, counts for a gene involved in equol production (tdr) were reduced in all cultures, reflecting a reduction in the number of equol-producing bacteria. In conclusion, under the TIM-2 culture conditions established, the CH-ISO diet favoured the synthesis of equol.

Cellulose nanocrystals (CNCs) are a next-generation cellulose product with many unique properties including applications in the food industry as a food additive, food coating, and in food-contact packaging material. While CNC is anticipated to be safe due to its similarity to the many forms of cellulose currently used as food additives, special consideration is given to it as it is the first manufactured form of cellulose that is nanoscale in both length and width. A proactive approach to safety has been adopted by manufacturers to demonstrate CNC safety toward responsible commercialization. As part of the safety demonstration, in vivo and in vitro testing strategies were commissioned side-by-side with conventional cellulose, which has been safely used in food for decades. Testing included a 90-day rodent feeding study as well as additional physical, chemical, and biological studies in vitro that follow European Food Safety Authority (EFSA) guidance to demonstrate the safe use of novel food ingredients. The strategy includes assessment of neat materials side-by-side with simulated digestion, mimicking conditions that occur along the gastrointestinal tract as well as intracellularly. An intestinal co-culture model examined any potential toxicological effects from exposure to either pristine or digested forms of CNC including cytotoxicity, metabolic activity, membrane permeability, oxidative stress, and proinflammatory responses. None of the studies demonstrated any toxicity via oral or simulated oral exposure. These studies demonstrate that CNC produced by InnoTech Alberta is similarly safe by ingestion as conventional cellulose with a no-observed-adverse-effect level of 2085.3 (males) and 2682.8 (females) mg/kg/day. [ABSTRACT FROM AUTHOR]

Fibrillated cellulose is a next-generation material in development for a variety of applications, including use in food and food-contact materials. An alternative testing strategy including simulated digestion was developed to compare the physical, chemical, and biological characteristics of seven different types of fibrillated cellulose, following European Food Safety Authority guidance. Fibrillated forms were compared to a conventional form of cellulose which has been used in food for over 85 years and has Generally Recognized as safe regulatory status in the USA. The physical and chemical characterization of fibrillated celluloses demonstrate that these materials are similar physically and chemically, which composed of the same fundamental molecular structure and exhibit similar morphology, size, size distribution, surface charge, and low levels of impurities. Simulated gastrointestinal and lysosomal digestions demonstrate that these physical and chemical similarities remain following exposure to conditions that mimic the gastrointestinal tract or intracellular lysosomes. A toxicological investigation with an advanced intestinal co-culture model found that exposure to each of the fibrillated and conventional forms of cellulose, in either the pristine or digested form at 0.4% by weight, showed no adverse toxicological effects including cytotoxicity, barrier integrity, oxidative stress, or inflammation. The results demonstrate the physical, chemical, and biological similarities of these materials and provide substantive evidence to support their grouping and ability to read-across data as part of a food safety demonstration. [ABSTRACT FROM AUTHOR]

Consumption of probiotic bacteria can result in a transient colonization of the human gut and thereby in potential interactions with the commensal microbiota. In this study, we used novel PolyFermS continuous fermentation models to investigate interactions of the candidate probiotic strain Lactobacillus paracasei CNCM I-1518 (L. paracasei) with colonic microbiota from healthy elderly subjects using 16S rRNA gene amplicon sequencing and metatranscriptomics, or with microbiota in vitro-colonized with Clostridioides difficile (C. difficile NCTC 13307 and C. difficile DSM 1296)—an enteropathogen prevalent in the elderly population. Small changes in microbiota composition were detected upon daily addition of L. paracasei, including increased abundances of closely related genera Lactobacillus and Enterococcus, and of the butyrate producer Faecalibacterium. Microbiota gene expression was also modulated by L. paracasei with distinct response of the Faecalibacterium transcriptome and an increase in carbohydrate utilization. However, no inhibitory effect of L. paracasei was observed on C. difficile colonization in the intestinal models under the tested conditions. Our data suggest that, in the in vitro experimental conditions tested and independent of the host, L. paracasei has modulatory effects on both the composition and function of elderly gut microbiota without affecting C. difficile growth and toxin production.

The intestine is a visceral organ that integrates absorption, metabolism, and immunity, which is vulnerable to external stimulus. Researchers in the fields such as food science, immunology, and pharmacology have committed to developing appropriate in vitro intestinal cell models to study the intestinal absorption and metabolism mechanisms of various nutrients and drugs, or pathogenesis of intestinal diseases. In the past three decades, the intestinal cell models have undergone a significant transformation from conventional two-dimensional cultures to three-dimensional (3D) systems, and the achievements of 3D cell culture have been greatly contributed by the fabrication of different scaffolds. In this review, we first introduce the developing trend of existing intestinal models. Then, four types of scaffolds, including Transwell, hydrogel, tubular scaffolds, and intestine-on-a-chip, are discussed for their 3D structure, composition, advantages, and limitations in the establishment of intestinal cell models. Excitingly, some of the in vitro intestinal cell models based on these scaffolds could successfully mimic the 3D structure, microenvironment, mechanical peristalsis, fluid system, signaling gradients, or other important aspects of the original human intestine. Furthermore, we discuss the potential applications of the intestinal cell models in drug screening, disease modeling, and even regenerative repair of intestinal tissues. This review presents an overview of state-of-the-art scaffold-based cell models within the context of intestines, and highlights their major advances and applications contributing to a better knowledge of intestinal diseases. Impact statement The intestine tract is crucial in the absorption and metabolism of nutrients and drugs, as well as immune responses against external pathogens or antigens in a complex microenvironment. The appropriate experimental cell model in vitro is needed for in-depth studies of intestines, due to the limitation of animal models in dynamic control and real-time assessment of key intestinal physiological and pathological processes, as well as the "R" principles in laboratory animal experiments. Three-dimensional (3D) scaffold-based cell cultivation has become a developing tendency because of the superior cell proliferation and differentiation and more physiologically relevant environment supported by the customized 3D scaffolds. In this review, we summarize four types of up-to-date 3D cell culture scaffolds fabricated by various materials and techniques for a better recapitulation of some essential physiological and functional characteristics of original intestines compared to conventional cell models. These emerging 3D intestinal models have shown promising results in not only evaluating the pharmacokinetic characteristics, security, and effectiveness of drugs, but also studying the pathological mechanisms of intestinal diseases at cellular and molecular levels. Importantly, the weakness of the representative 3D models for intestines is also discussed.

Supplemental dietary amino acids (AAs) need to be provided in a form that prevents their degradation along the gastrointestinal tract to guarantee their high bioavailability and bioactivity. In this study, methionine (Met) protected via organo-clay intercalation (natural carriers) has been developed as a sustainable alternative to polymeric coating. Specifically, two different bentonite-zeolite-based mineral clays were tested, Adsorbene (ADS) and BioKi (BIO). Briefly, 1 g of the carrier (ADS or BIO) was contacted with 50 mL of an aqueous solution at a pH of 3.0, 5.8, and 8.9. Solid-liquid separation was conducted. The released Met in the liquid phase was analysed by Chemical Oxygen Demand, while residual Met in the solid phase was analysed by Fourier Transform Infra-Red (FT-IR) spectroscopy. The effect of Met-ADS complex on cell viability was tested on IPEC-J2 cells incubated 3 h with Met-ADS 2.5 mM. Jejunum segments obtained by entire male pigs (Swiss Large White, body weight 100 ± 5 kg) were used as ex vivo models to compare the absorption of 2.5 mM Met released by ADS with 2.5 mM free Met and its influence on epithelial integrity in perfusion Ussing chambers. The carriers released a very low amount of Met and Met-BIO interaction was stronger than Met-ADS. The maximum release of Met was at pH 3, with 3% and 6% of Met release from Met-BIO and Met-ADS, respectively. Cell viability experiments revealed that Met-ADS did not alter cell metabolic activity. No differences in Met absorption and intestinal epithelial integrity were observed ex vivo between free Met and Met-ADS. This study provided new insights into the release of Met from natural clays such as ADS and BIO, the safety of its use in the porcine intestine and the ability of ADS-released Met to absorb to the same extent as the free Met in porcine jejunum.

An intestinal epithelium model able to produce mucus was developed to provide an environment suitable for testing the therapeutic activity of gut bacteriophages. We show that Enterococcus faecalis adheres more effectively in the presence of mucus, can invade the intestinal epithelia and is able to translocate after damaging tight junctions. Furthermore, Enterococcus phage vB_EfaM_A2 (a member of Herelleviridae that possesses virion associated immunoglobin domains) was found to translocate through the epithelium in the presence and absence of its host bacteria. Phage A2 protected eukaryotic cells by reducing mortality and maintaining the structure of the cell layer structure. We suggest the mammalian cell model utilized within this study as an adaptable in vitro model that can be employed to enable a better understanding of phage–bacteria interactions and the protective impact of phage therapy relating to the intestinal epithelium.

Robotic endoscopes with locomotion ability are among the most promising alternatives to traditional endoscopes; the locomotion ability is an important factor when evaluating the performance of the robot. This article describes the research on the characteristics of an expanding–extending robotic endoscope’s locomotion efficiency in real intestine and explores an approach to improve the locomotion ability in this environment. In the article, the robot’s locomotion efficiency was first calculated according to its gait in the gut, and the reasons for step losses were analyzed. Next, dynamical models of the robot and the intestine were built to calculate the step losses caused by failed anchoring and intestinal compression/extension. Based on the models and the calculation results, methods for reducing step losses were proposed. Finally, a series of ex vivo experiments were carried out, and the actual locomotion efficiency of the robot was analyzed on the basis of the theoretical models. In the experiment, on a level platform, the locomotion efficiency of the robot varied between 34.2% and 63.7%; the speed of the robot varied between 0.62 and 1.29 mm/s. The robot’s efficiency when climbing a sloping intestine was also tested and analyzed. The proposed theoretical models and experimental results provide a good reference for improving the design of robotic endoscopy. [ABSTRACT FROM AUTHOR]

Abstract: A new mechanism of ischemia/reperfusion (I/R) injury is discovered recently operating through innate autoimmunity. Studies of different animal I/R models showed that reperfusion of ischemic tissues elicits an acute inflammatory response involving complement system which is activated by autoreactive natural IgM. Whether similar mechanism operating in human is still unknown. We investigated this important question by testing if human natural IgM could induce I/R injury in an established murine intestinal model. RAG-1−/− mice (immunoglobulin deficient), which are protected from I/R injury, were reconstituted with purified normal human IgM and subjected in an intestinal injury model. Reconstituted RAG-1−/− mice that were underwent sham treatment did not show tissue injury in intestine. In contrast, reconstituted RAG-1−/− mice that underwent 40min intestinal ischemia and 3h reperfusion showed significant injury in the local tissues. In addition, immunohistochemistry showed that complement C4 were deposited in intestinal villi of I/R but not sham treated mice. Therefore, our study is the first report describing that human natural IgM is capable to induce I/R injury in the intestinal model, and further suggests that innate autoimmunity may operate under pathogenic conditions in human. [Copyright &y& Elsevier]

The study of epithelial barrier properties in the human body is of paramount interest to a range of disciplines, including disease modeling, drug transport studies, toxicology, developmental biology, and regenerative biology. Current day in vitro studies largely rely on growing epithelial cells in a static environment on membrane cell culture inserts. With the advancement of microfluidic and organ-on-a-chip techniques it became possible to culture 3D intestinal tubules directly against an extracellular matrix (ECM) under flow and without the need for artificial membranes. Here we describe detailed protocols for culturing epithelial tubules in a high-throughput format, assessing their permeability and marker expression. The platform harbors 40 independent microfluidic chips in a microtiter plate format. The resulting 40 epithelial tubules are analyzed in parallel using a high-content microscopy. Protocols described here allow for adoption and routine application of microfluidic techniques by nonspecialized end-users., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

An intestinal epithelium model able to produce mucus was developed to provide an environment suitable for testing the therapeutic activity of gut bacteriophages. We show that Enterococcus faecalis adheres more effectively in the presence of mucus, can invade the intestinal epithelia and is able to translocate after damaging tight junctions. Furthermore, Enterococcus phage vB_EfaM_A2 (a member of Herelleviridae that possesses virion associated immunoglobin domains) was found to translocate through the epithelium in the presence and absence of its host bacteria. Phage A2 protected eukaryotic cells by reducing mortality and maintaining the structure of the cell layer structure. We suggest the mammalian cell model utilized within this study as an adaptable in vitro model that can be employed to enable a better understanding of phage–bacteria interactions and the protective impact of phage therapy relating to the intestinal epithelium. [ABSTRACT FROM AUTHOR]

• This search aimed to find dietary conditions enhancing endogenous equol production. • Control and experimental diets with isoflavones were run in an artificial intestine. • The model was inoculated with a pooled faecal homogenate from equol-producing women. • A carbohydrate-rich diet was found to favour the synthesis of equol. In order to find dietary conditions favouring endogenous equol biosynthesis, a pooled faecal homogenate from equol-producing women was used to inoculate the TIM-2 artificial model of the human proximal colon. The model was fuelled with control diets not supplemented (C) or supplemented (C-ISO) with isoflavones, and two isoflavone-containing diets rich in carbohydrate (CH-ISO) or protein (PR-ISO). Compared to the C-ISO control, the CH-ISO diet doubled the production of equol, while with the PR-ISO diet the production of equol in cultures decreased sharply. The CH-ISO diet was also associated with enhanced butyrate production. The numbers of most bacterial populations analysed did not significantly change along cultures with any of the diets. Surprisingly, counts for a gene involved in equol production (tdr) were reduced in all cultures, reflecting a reduction in the number of equol-producing bacteria. In conclusion, under the TIM-2 culture conditions established, the CH-ISO diet favoured the synthesis of equol. [ABSTRACT FROM AUTHOR]

The Caco-2 cell monolayer has been extensively used for the high-throughput assessing of nutrient absorption, screening of drug permeability, and studying the intestinal physiological process in vitro . The most used Caco-2 cell model is the Transwell model with polycarbonate microporous membranes. However, Caco-2 cells in the classical Transwell model need 21 days to gain an intact and mature monolayer. Electrospun nanofiber scaffolds mimicking the natural extracellular matrix could improve cell adhesion, proliferation, and expression, whereas there are no reports that intestinal cells were cultured on the electrospun nanofiber scaffolds. Here, electrospun polylactic acid (PLA) nanofiber scaffolds were chosen as the ideal scaffolds for Caco-2 cell monolayers to construct a modified Transwell. Cell morphology and polarity were studied. Monolayer barrier properties were assessed by measuring transepithelial electrical resistance (TEER) and the leakage of phenol red. As found, intact Caco-2 cell monolayers were formed on the PLA nanofiber scaffolds after 4 days of culture. After 4 days, the TEER increased to 422 Ω·cm 2 and the apparent permeability coefficients of phenol red decreased to 1.0 ± 0.1 × 10 -6 cm/s, suggesting that Caco-2 cell monolayers developed a formidable barrier to small molecules on the surface of PLA nanofiber scaffolds. Microvilli and tight junctions were clearly visible after day 3. Besides, Caco-2 cell monolayers on the surface of PLA nanofiber scaffolds presented higher differentiation properties than on the surface of the polycarbonate microporous membrane in traditional Transwell including higher alkaline phosphatase activity and higher P-gp activity. Results of quercetin absorption and probiotics adhesion demonstrated that Caco-2 cell monolayers formed on the surface of PLA nanofiber scaffolds also had better physiological function and prediction function in vitro . Overall, the present study indicated that the Transwell with the structurally and functionally biomimetic electrospun PLA nanofiber scaffold could be potentially developed as a promising in vitro intestinal model.

Cellulose nanocrystals (CNCs) are a next-generation cellulose product with many unique properties including applications in the food industry as a food additive, food coating, and in food-contact packaging material. While CNC is anticipated to be safe due to its similarity to the many forms of cellulose currently used as food additives, special consideration is given to it as it is the first manufactured form of cellulose that is nanoscale in both length and width. A proactive approach to safety has been adopted by manufacturers to demonstrate CNC safety toward responsible commercialization. As part of the safety demonstration, in vivo and in vitro testing strategies were commissioned side-by-side with conventional cellulose, which has been safely used in food for decades. Testing included a 90-day rodent feeding study as well as additional physical, chemical, and biological studies in vitro that follow European Food Safety Authority (EFSA) guidance to demonstrate the safe use of novel food ingredients. The strategy includes assessment of neat materials side-by-side with simulated digestion, mimicking conditions that occur along the gastrointestinal tract as well as intracellularly. An intestinal co-culture model examined any potential toxicological effects from exposure to either pristine or digested forms of CNC including cytotoxicity, metabolic activity, membrane permeability, oxidative stress, and proinflammatory responses. None of the studies demonstrated any toxicity via oral or simulated oral exposure. These studies demonstrate that CNC produced by InnoTech Alberta is similarly safe by ingestion as conventional cellulose with a no-observed-adverse-effect level of 2085.3 (males) and 2682.8 (females) mg/kg/day., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Fibrillated cellulose is a next-generation material in development for a variety of applications, including use in food and food-contact materials. An alternative testing strategy including simulated digestion was developed to compare the physical, chemical, and biological characteristics of seven different types of fibrillated cellulose, following European Food Safety Authority guidance. Fibrillated forms were compared to a conventional form of cellulose which has been used in food for over 85 years and has Generally Recognized as safe regulatory status in the USA. The physical and chemical characterization of fibrillated celluloses demonstrate that these materials are similar physically and chemically, which composed of the same fundamental molecular structure and exhibit similar morphology, size, size distribution, surface charge, and low levels of impurities. Simulated gastrointestinal and lysosomal digestions demonstrate that these physical and chemical similarities remain following exposure to conditions that mimic the gastrointestinal tract or intracellular lysosomes. A toxicological investigation with an advanced intestinal co-culture model found that exposure to each of the fibrillated and conventional forms of cellulose, in either the pristine or digested form at 0.4% by weight, showed no adverse toxicological effects including cytotoxicity, barrier integrity, oxidative stress, or inflammation. The results demonstrate the physical, chemical, and biological similarities of these materials and provide substantive evidence to support their grouping and ability to read-across data as part of a food safety demonstration., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)