Trust your gut: Establishing confidence in gastrointestinal models - An overview of the state of the science and contexts of use.

The webinar series and workshop titled Trust Your Gut: Establishing Confidence in Gastrointestinal Models - An Overview of the State of the Science and Contexts of Use was co-organized by NICEATM, NIEHS, FDA, EPA, CPSC, DoD, and the Johns Hopkins Center for Alternatives to Animal Testing (CAAT) and hosted at the National Institutes of Health in Bethesda, MD, USA on October 11-12, 2023. New approach methods (NAMs) for assessing issues of gastrointestinal tract (GIT)-related toxicity offer promise in addressing some of the limitations associated with animal-based assessments. GIT NAMs vary in complexity, from two-dimensional monolayer cell line-based systems to sophisticated 3-dimensional organoid systems derived from human primary cells. Despite advances in GIT NAMs, challenges remain in fully replicating the complex interactions and processes occurring within the human GIT. Presentations and discussions addressed regulatory needs, challenges, and innovations in incorporating NAMs into risk assessment frameworks; explored the state of the science in using NAMs for evaluating systemic toxicity, understanding absorption and pharmacokinetics, evaluating GIT toxicity, and assessing potential allergenicity; and discussed strengths, limitations, and data gaps of GIT NAMs as well as steps needed to establish confidence in these models for use in the regulatory setting.

Non-animal methods to assess whether chemicals may be toxic to the human digestive tract promise to complement or improve on animal-based methods. These approaches, which are based on human or animal cells and/or computer models, are faced with their own technical challenges and need to be shown to predict adverse effects in humans. Regulators are tasked with evaluating submitted data to best protect human health and the environment. A webinar series and workshop brought together scientists from academia, industry, military, and regulatory authorities from different countries to discuss how non-animal methods can be integrated into the risk assessment of drugs, food additives, dietary supplements, pesticides, and industrial chemicals for gastrointestinal toxicity.

Suspended hydrogel culture as a method to scale up intestinal organoids.

Primary tissue-derived epithelial organoids are a physiologically relevant in vitro intestinal model that have been implemented for both basic research and drug development applications. The existing method of culturing intestinal organoids in surface-attached native extracellular matrix (ECM) hydrogel domes is not readily amenable to large-scale culture and contributes to culture heterogeneity. We have developed a method of culturing intestinal organoids within suspended basement membrane extract (BME) hydrogels of various geometries, which streamlines the protocol, increases the scalability, enables kinetic sampling, and improves culture uniformity without specialized equipment or additional expertise. We demonstrate the compatibility of this method with multiple culture formats, and provide examples of suspended BME hydrogel organoids in downstream applications: implementation in a medium-throughput drug screen and generation of Transwell monolayers for barrier evaluation. The suspended BME hydrogel culture method will allow intestinal organoids, and potentially other organoid types, to be used more widely and at higher throughputs than previously possible.

Toward Inclusivity in Preclinical Drug Development: A Proposition to Start with Intestinal Organoids.

Representation of humans from diverse backgrounds in the drug development process is key to advancing health equity, and while clinical trial design has recently made strides toward greater inclusivity, preclinical drug development has struggled to make those same gains. One barrier to inclusion is the current lack of robust and established in vitro model systems that simultaneously capture the complexity of human tissues while representing patient diversity. Here, the use of primary human intestinal organoids as a mechanism to advance inclusive preclinical research is proposed. This in vitro model system not only recapitulates tissue functions and disease states, but also retains the genetic identity and epigenetic signatures of the donors from which they are derived. Thus, intestinal organoids are an ideal in vitro prototype for capturing human diversity. In this perspective, the authors call for an industry-wide effort to leverage intestinal organoids as a starting point to actively and intentionally incorporate diversity into preclinical drug programs.

Controlling the polarity of human gastrointestinal organoids to investigate epithelial biology and infectious diseases.

Human epithelial organoids-3D spheroids derived from adult tissue stem cells-enable investigation of epithelial physiology and disease and host interactions with microorganisms, viruses and bioactive molecules. One challenge in using organoids is the difficulty in accessing the apical, or luminal, surface of the epithelium, which is enclosed within the organoid interior. This protocol describes a method we previously developed to control human and mouse organoid polarity in suspension culture such that the apical surface faces outward to the medium (apical-out organoids). Our protocol establishes apical-out polarity rapidly (24-48 h), preserves epithelial integrity, maintains secretory and absorptive functions and allows regulation of differentiation. Here, we provide a detailed description of the organoid polarity reversal method, compatible characterization assays and an example of an application of the technology-specifically the impact of host-microbe interactions on epithelial function. Control of organoid polarity expands the possibilities of organoid use in gastrointestinal and respiratory health and disease research.

Engineered Matrices Enable the Culture of Human Patient-Derived Intestinal Organoids.

Human intestinal organoids from primary human tissues have the potential to revolutionize personalized medicine and preclinical gastrointestinal disease models. A tunable, fully defined, designer matrix, termed hyaluronan elastin-like protein (HELP) is reported, which enables the formation, differentiation, and passaging of adult primary tissue-derived, epithelial-only intestinal organoids. HELP enables the encapsulation of dissociated patient-derived cells, which then undergo proliferation and formation of enteroids, spherical structures with polarized internal lumens. After 12 rounds of passaging, enteroid growth in HELP materials is found to be statistically similar to that in animal-derived matrices. HELP materials also support the differentiation of human enteroids into mature intestinal cell subtypes. HELP matrices allow stiffness, stress relaxation rate, and integrin-ligand concentration to be independently and quantitatively specified, enabling fundamental studies of organoid-matrix interactions and potential patient-specific optimization. Organoid formation in HELP materials is most robust in gels with stiffer moduli (G' ≈ 1 kPa), slower stress relaxation rate (t1/2 ≈ 18 h), and higher integrin ligand concentration (0.5 × 10-3-1 × 10-3 m RGD peptide). This material provides a promising in vitro model for further understanding intestinal development and disease in humans and a reproducible, biodegradable, minimal matrix with no animal-derived products or synthetic polyethylene glycol for potential clinical translation.

Retinoic Acid and Lymphotoxin Signaling Promote Differentiation of Human Intestinal M Cells.

BACKGROUND & AIMS: Intestinal microfold (M) cells are a unique subset of intestinal epithelial cells in the Peyer's patches that regulate mucosal immunity, serving as portals for sampling and uptake of luminal antigens. The inability to efficiently develop human M cells in cell culture has impeded studies of the intestinal immune system. We aimed to identify signaling pathways required for differentiation of human M cells and establish a robust culture system using human ileum enteroids. METHODS: We analyzed transcriptome data from mouse Peyer's patches to identify cell populations in close proximity to M cells. We used the human enteroid system to determine which cytokines were required to induce M-cell differentiation. We performed transcriptome, immunofluorescence, scanning electron microscope, and transcytosis experiments to validate the development of phenotypic and functional human M cells. RESULTS: A combination of retinoic acid and lymphotoxin induced differentiation of glycoprotein 2-positive human M cells, which lack apical microvilli structure. Upregulated expression of innate immune-related genes within M cells correlated with a lack of viral antigens after rotavirus infection. Human M cells, developed in the enteroid system, internalized and transported enteric viruses, such as rotavirus and reovirus, across the intestinal epithelium barrier in the enteroids. CONCLUSIONS: We identified signaling pathways required for differentiation of intestinal M cells, and used this information to create a robust culture method to develop human M cells with capacity for internalization and transport of viruses. Studies of this model might increase our understanding of antigen presentation and the systemic entry of enteric pathogens in the human intestine.

Mucin glycans attenuate the virulence of Pseudomonas aeruginosa in infection.

A slimy, hydrated mucus gel lines all wet epithelia in the human body, including the eyes, lungs, and gastrointestinal and urogenital tracts. Mucus forms the first line of defence while housing trillions of microorganisms that constitute the microbiota1. Rarely do these microorganisms cause infections in healthy mucus1, suggesting that mechanisms exist in the mucus layer that regulate virulence. Using the bacterium Pseudomonas aeruginosa and a three-dimensional (3D) laboratory model of native mucus, we determined that exposure to mucus triggers downregulation of virulence genes that are involved in quorum sensing, siderophore biosynthesis and toxin secretion, and rapidly disintegrates biofilms-a hallmark of mucosal infections. This phenotypic switch is triggered by mucins, which are polymers that are densely grafted with O-linked glycans that form the 3D scaffold inside mucus. Here, we show that isolated mucins act at various scales, suppressing distinct virulence pathways, promoting a planktonic lifestyle, reducing cytotoxicity to human epithelia in vitro and attenuating infection in a porcine burn model. Other viscous polymer solutions lack the same effect, indicating that the regulatory function of mucin does not result from its polymeric structure alone. We identify that interactions with P. aeruginosa are mediated by mucin-associated glycans (mucin glycans). By isolating glycans from the mucin backbone, we assessed the collective activity of hundreds of complex structures in solution. Similar to their grafted counterparts, free mucin glycans potently regulate bacterial phenotypes even at relatively low concentrations. This regulatory function is likely dependent on glycan complexity, as monosaccharides do not attenuate virulence. Thus, mucin glycans are potent host signals that 'tame' microorganisms, rendering them less harmful to the host.

Human Intestinal Enteroids Model MHC-II in the Gut Epithelium.

The role of intestinal epithelial cells (IECs) in mucosal tolerance and immunity remains poorly understood. We present a method for inducing MHC class II (MHC-II) in human enteroids, "mini-guts" derived from small intestinal crypt stem cells, and show that the intracellular MHC-II peptide-pathway is intact and functional in IECs. Our approach enables human enteroids to be used for novel in vitro studies into IEC MHC-II regulation and function during health and disease.

Controlling Epithelial Polarity: A Human Enteroid Model for Host-Pathogen Interactions.

Human enteroids-epithelial spheroids derived from primary gastrointestinal tissue-are a promising model to study pathogen-epithelial interactions. However, accessing the apical enteroid surface is challenging because it is enclosed within the spheroid. We developed a technique to reverse enteroid polarity such that the apical surface everts to face the media. Apical-out enteroids maintain proper polarity and barrier function, differentiate into the major intestinal epithelial cell (IEC) types, and exhibit polarized absorption of nutrients. We used this model to study host-pathogen interactions and identified distinct polarity-specific patterns of infection by invasive enteropathogens. Salmonella enterica serovar Typhimurium targets IEC apical surfaces for invasion via cytoskeletal rearrangements, and Listeria monocytogenes, which binds to basolateral receptors, invade apical surfaces at sites of cell extrusion. Despite different modes of entry, both pathogens exit the epithelium within apically extruding enteroid cells. This model will enable further examination of IECs in health and disease.

Mucus and Mucin Environments Reduce the Efficacy of Polymyxin and Fluoroquinolone Antibiotics against Pseudomonas aeruginosa.

Mucus, a biopolymer hydrogel that covers all wet epithelia of the body, is a potential site for infection by pathogenic bacteria. Mucus can bind small molecules and influence bacterial physiology, two factors that may affect the efficacy of antibiotics. In spite of this, the impact of mucus on antibiotic activity has not been thoroughly characterized. We examined the activity of polymyxin and fluoroquinolone antibiotics against the opportunistic pathogen Pseudomonas aeruginosa in native mucus and purified mucin biopolymer environments. We found that mucus reduces the effectiveness of polymyxins and fluoroquinolones against P. aeruginosa. Mucin biopolymers MUC5AC, MUC2, and MUC5B are primary contributors to this reduction. Our findings highlight that the biomaterial environmental context should be considered when evaluating antibiotics in vitro.

A Dual-Function Antibiotic-Transporter Conjugate Exhibits Superior Activity in Sterilizing MRSA Biofilms and Killing Persister Cells.

New strategies are urgently needed to target MRSA, a major global health problem and the leading cause of mortality from antibiotic-resistant infections in many countries. Here, we report a general approach to this problem exemplified by the design and synthesis of a vancomycin-d-octaarginine conjugate (V-r8) and investigation of its efficacy in addressing antibiotic-insensitive bacterial populations. V-r8 eradicated MRSA biofilm and persister cells in vitro, outperforming vancomycin by orders of magnitude. It also eliminated 97% of biofilm-associated MRSA in a murine wound infection model and displayed no acute dermal toxicity. This new dual-function conjugate displays enhanced cellular accumulation and membrane perturbation as compared to vancomycin. Based on its rapid and potent activity against biofilm and persister cells, V-r8 is a promising agent against clinical MRSA infections.

Mucins trigger dispersal of Pseudomonas aeruginosa biofilms.

Mucus is a biological gel that lines all wet epithelia in the body, including the mouth, lungs, and digestive tract, and has evolved to protect the body from pathogenic infection. However, microbial pathogenesis is often studied in mucus-free environments that lack the geometric constraints and microbial interactions in physiological three-dimensional mucus gels. We developed fluid-flow and static test systems based on purified mucin polymers, the major gel-forming constituents of the mucus barrier, to understand how the mucus barrier influences bacterial virulence, particularly the integrity of Pseudomonas aeruginosa biofilms, which can become resistant to immune clearance and antimicrobial agents. We found that mucins separate the cells in P. aeruginosa biofilms and disperse them into suspension. Other viscous polymer solutions did not match the biofilm disruption caused by mucins, suggesting that mucin-specific properties mediate the phenomenon. Cellular dispersion depended on functional flagella, indicating a role for swimming motility. Taken together, our observations support a model in which host mucins are key players in the regulation of microbial virulence. These mucins should be considered in studies of mucosal pathogenesis and during the development of novel strategies to treat biofilms.