Organ Model: Digestive System

Article Type: Review

Organ Models: Intestine (Colon, Duodenum)

Application: Toxicology

Abstract: 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.

In this session, our expert speakers highlight how they have used Organ-Chips to study viral and bacterial infection, including SARS-CoV-2, Nipah virus, and Mycobacterium tuberculosis. Watch to learn how you can gain deeper insights into infectious disease pathogenesis, infection-induced inflammatory response, and more. 

Talk 1: Human Lung Microfluidic Chip: Nipah Virus Disease Modeling and Antiviral Treatments in Maximum Containment

Dr. Bhosle’s research group at the National Institute of Allergy and Infectious Diseases (NIAID) leveraged human Small Airway Lung-Chips to model Nipah (NiV) virus disease modelling in maximum containment. They demonstrated the application of NiV-infected Small Airway Lung-Chips towards therapeutic evaluation of antiviral drugs. Further, successful recapitulation of neutrophil infiltration, critical immune responses, activation of endothelium leading to inflammation was achieved with human Lung-Chips. 

Talk 2: Early events in tuberculosis—harnessing microphysiological models to study humanity’s oldest foe

Tissue microenvironments profoundly influence infection and treatment outcomes; but their roles can be difficult to dissect in a mechanistic manner. This is particularly so for tuberculosis, whose early stages of infection in alveoli are difficult to study in any animal model and not recapitulated in typical in vitro models of infection. In this talk, Dr. Thacker will describe how microphysiological models such as Organ-Chips are powerful tools to fill this gap and provide new insights into how obligate pathogens, such as Mycobacterium tuberculosis, adapt to specific tissue niches and what consequences this may have for treatment outcomes.

For this research, Dr. Thacker and team received the 2024 SwissTB Award.

Talk 3: Application of Airway-on-Chip Models to study Bacterial Lung Infection

Many lung diseases, both acute and chronic, are associated with bacterial infection damaging the integrity of the epithelial barrier. Dr. Ryan’s recent collaborative research has developed Airway-on-chip models, which offer a microscale platform that mimics many features of human lung physiology. This platform facilitates the investigation of bacterial lung infections by replicating key features of the airway environment, including directional air flow and stretch. These models enable real-time monitoring of bacterial behavior and host responses, advancing our understanding of infection dynamics and the development of targeted therapeutic strategies. 

Organ Model: Intestine (Caco2)

Application: ADME-Tox

Abstract: Oral delivery is considered the most patient preferred route of drug administration, however, the drug must be sufficiently soluble and permeable to successfully formulate an oral formulation. There have been advancements in the development of more predictive solubility and dissolution tools, but the tools that has been developed for permeability assays have not been validated as extensively as the gold-standard Caco-2 Transwell assay. Here, we evaluated Caco-2 intestinal permeability assay in Transwells and a commercially available microfluidic Chip using 19 representative Biopharmaceutics Classification System (BCS) Class I-IV compounds. For each selected compound, we performed a comprehensive viability test, quantified its apparent permeability (Papp), and established an in vitro in vivo correlation (IVIVC) to the human fraction absorbed (fa) in both culture conditions. Permeability differences were observed across the models as demonstrated by antipyrine (Transwell Papp: 38.5 ± 6.1 ◊ 10-8 cm/s vs Chip Papp: 32.9 ± 11.3 ◊ 10-8 cm/s) and nadolol (Transwell Papp: 0.6 ± 0.1 ◊ 10-7 cm/s vs Chip Papp: 3 ± 1.2 ◊ 10-7 cm/s). The in vitro in vivo correlation (IVIVC; Papp vs. fa) of the Transwell model (r2 = 0.59-0.83) was similar to the Chip model (r2 = 0.41-0.79), highlighting similar levels of predictivity. Comparing to historical data, our Chip Papp data was more closely aligned to native tissues assessed in Ussing chambers. This is the first study to comprehensively validate a commercial Gut-on-a-Chip model as a predictive tool for assessing oral absorption to further reduce our reliance on animal models.

In this webinar from Drug Discovery Day 2024, Carly Strelez, PhD, discusses her work using Organ-on-a-Chip technology to better understand colorectal cancer progression and transform approaches to personalized medicine.

Speaker:

Organ Model: Intestine (Caco2)

Application: ADME-Tox

Abstract: 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.

Organ Model: Liver, Intestine (Caco2)

Application: Organ transplantation

How Organ-Chips Were Used: Drugs that induce reversible slowing of metabolic and physiological processes would have great value for organ preservation, especially for organs with high susceptibility to hypoxia-reperfusion injury, such as the heart. Here, Gut- and Liver-Chips were used to evaluate the metabolic suppression capability of the test compound. The Chips showed a decrease in the tissue’s total ATP production in presence of SNC80, which is accompanied by a global slowing of metabolism.

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Organ Model: Intestine (Duodenum)

Application: Metabolic Disease

Abstract:

Background

Sulfadoxine-pyrimethamine (SP) antimalarial therapy has been suggested to potentially increase the birth weight of infants in pregnant women in sub-Saharan Africa, independently of malarial infection. Here, we utilized female intestinal organoid-derived cells cultured within microfluidic Organ Chips to investigate whether SP could directly impact intestinal function and thereby improve the absorption of essential fats and nutrients crucial for fetal growth.

Methods

Using a human organ-on-a-chip model, we replicated the adult female intestine with patient organoid-derived duodenal epithelial cells interfaced with human intestinal endothelial cells. Nutrient-deficient (ND) medium was perfused to simulate malnutrition, resulting in the appearance of enteric dysfunction indicators such as villus blunting, reduced mucus production, impaired nutrient absorption, and increased inflammatory cytokine secretion. SP was administered to these chips in the presence or absence of human peripheral blood mononuclear cells (PBMCs).

Findings

Our findings revealed that SP treatment effectively reversed multiple intestinal absorptive abnormalities observed in malnourished female Intestine Chips, as validated by transcriptomic and proteomic analyses. SP also reduced the production of inflammatory cytokines and suppressed the recruitment of PBMCs in ND chips.

Interpretation

Our results indicate that SP could potentially increase birth weights by preventing enteric dysfunction and suppressing intestinal inflammation. This underscores the potential of SP as a targeted intervention to improve maternal absorption, subsequently contributing to healthier fetal growth. While SP treatment shows promise in addressing malabsorption issues that can influence infant birth weight, we did not model pregnancy in our chips, and thus its usefulness for treatment of malnourished pregnant women requires further investigation through clinical trials.

Organ Model: Intestine (Duodenum)

Application: ADME-Tox

Abstract: Microphysiological systems (MPS) incorporating human intestinal organoids have shown the potential to faithfully model intestinal biology with the promise to accelerate development of oral prodrugs. We hypothesized that an MPS model incorporating flow, shear stress, and vasculature could provide more reliable measures of prodrug bioconversion and permeability. Following construction of jejunal and duodenal organoid MPS derived from 3 donors, we determined the area under the concentration-time (AUC) curve for the active drug in the vascular channel and characterized the enzymology of prodrug bioconversion. Fosamprenavir underwent phosphatase mediated hydrolysis to amprenavir while dabigatran etexilate (DABE) exhibited proper CES2- and, as anticipated, not CES1-mediated de-esterification, followed by permeation of amprenavir to the vascular channel. When experiments were conducted in the presence of bio-converting enzyme inhibitors (orthovanadate for alkaline phosphatase; bis(p-nitrophenyl)phosphate for carboxylesterase), the AUC of the active drug decreased accordingly in the vascular channel. In addition to functional analysis, the MPS was characterized through imaging and proteomic analysis. Imaging revealed proper expression and localization of epithelial, endothelial, tight junction and catalytic enzyme markers. Global proteomic analysis was used to analyze the MPS model and 3 comparator sources: an organoid-based transwell model (which was also evaluated for function), Matrigel embedded organoids and finally jejunal and duodenal cadaver tissues collected from 3 donors. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) of global proteomic data demonstrated that all organoid-based models exhibited strong similarity and were distinct from tissues. Intestinal organoids in the MPS model exhibited strong similarity to human tissue for key epithelial markers via HCA. Quantitative proteomic analysis showed higher expression of key prodrug converting and drug metabolizing enzymes in MPS-derived organoids compared to tissues, organoids in Matrigel, and organoids on transwells. When comparing organoids from MPS and transwells, expression of intestinal alkaline phosphatase (ALPI), carboxylesterase (CES)2, cytochrome P450 3A4 (CYP3A4) and sucrase isomaltase (SI) was 2.97-, 1.2-, 11.3-, and 27.7-fold higher for duodenum and 7.7-, 4.6-, 18.1-, and 112.2-fold higher for jejunum organoids in MPS, respectively. The MPS approach can provide a more physiological system than enzymes, organoids, and organoids on transwells for pharmacokinetic analysis of prodrugs that account for 10% of all commercial medicines.

Organ Model: Intestine (Caco2)

Application: Immune Cell Recruitment

Abstract: Over 2 million people in North America suffer from inflammatory bowel disease (IBD), a chronic and idiopathic inflammatory condition. While previous research has primarily focused on studying immune cells as a cause and therapeutic target for IBD, recent findings suggest that non-immune cells may also play a crucial role in mediating cytokine and chemokine signaling, and therefore IBD symptoms. In this study, we developed an organ-on-a-chip co-culture model of Caco2 epithelial and HUVEC endothelial cells and induced inflammation using pro-inflammatory cytokines TNF-α and IFN-γ. We tested different concentration ranges and delivery orientations (apical vs. basal) to develop a consistently inducible inflammatory response model. We then measured pro-inflammatory cytokines and chemokines IL-6, IL-8, and CXCL-10, as well as epithelial barrier integrity. Our results indicate that this model 1. induces IBD-like cytokine secretion in non-immune cells and 2. decreases barrier integrity, making it a feasible and reliable model to test the direct actions of potential anti-inflammatory therapeutics on epithelial and endothelial cells.

Featured session at Netherlands MPS Day, which took place on November 15, 2023.

Joram Mooiweer from the University Medical Center Groningen presented his team’s work on modeling celiac disease using advanced human-relevant systems, including intestinal organoids and Organ-Chips. Celiac disease involves a complex interplay of genetic predisposition, immune responses (especially from tissue-resident T cells), and interactions with the intestinal epithelium and microbiota. By taking advantage of patient-derived tissue and induced pluripotent stem cells (iPSCs), the group aims to reconstruct these intricate processes in vitro.

Mooiweer highlights two approaches to modeling celiac disease. First, using patient biopsies, they isolate both intraepithelial lymphocytes (IELs) and intestinal stem cells. Culturing organoids derived from these stem cells together with IELs in a carefully optimized co-culture medium, they can recreate aspects of the cytotoxic activity associated with celiac disease—something that can be further manipulated using bispecific antibodies. These co-cultures maintain viability of both the epithelial and immune cells, enabling exploration of celiac-related immune-mediated epithelial damage.

Second, the group also uses iPSCs from patients and controls to generate small intestine-like epithelial tissues that self-organize into complex, 3D folded architectures when seeded on chip. With the help of growth factor gradients, they achieve a cell-type composition and maturation state that closely mirrors the human small intestine, including various specialized epithelial cells and mesenchymal cell types. This system is genetically tractable and can be further refined to incorporate inflammatory cues, immune cells, and eventually patient-specific microbiota under physiologically relevant conditions.

Ultimately, these multifaceted in vitro platforms will enable mechanistic insights into how genetic variants influence celiac disease onset and progression. They also provide a foundation for modeling interactions between epithelial cells, immune cells, and environmental triggers, paving the way for more predictive and personalized therapeutic strategies.

Key learnings from this presentation include:

• Complex disease modeling: Celiac disease involves interactions among epithelial cells, immune cells (IELs), and environmental factors (gluten). Recreating these dynamics in vitro is essential to dissect underlying mechanisms.
• Patient-derived systems: Using biopsies, organoids, and expanded IEL populations, the team can simulate immune-driven epithelial damage in controlled culture environments.
• iPSC-derived intestinal tissue: iPSC-based models yield highly structured and diverse epithelial cell populations on a chip, reflecting small-intestine-like architecture and enabling genetic manipulation for studying disease-associated loci.
• Synergistic modeling strategies: Combining adult-stem-cell-derived organoids, iPSC-based tissues, immune cells, and future microbiota co-cultures provides a comprehensive platform for understanding celiac pathophysiology.
• Toward translational impact: These advanced human-relevant models aim to identify genetic factors and potential therapeutic targets, contributing to more tailored interventions in celiac disease and related intestinal disorders.