Abstract
Inflammatory bowel disease (IBD) is an idiopathic gastrointestinal disease with drastically increasing incidence rates. Due to its multifactorial etiology, a precise investigation of the pathogenesis is extremely difficult. Although reductionist cell culture models and more complex disease models in animals have clarified the understanding of individual disease mechanisms and contributing factors of IBD in the past, it remains challenging to bridge research and clinical practice. Conventional 2D cell culture models cannot replicate complex host–microbiota interactions and stable long‐term microbial culture. Further, extrapolating data from animal models to patients remains challenging due to genetic and environmental diversity leading to differences in immune responses. Human intestine organ‐on‐chip (OoC) models have emerged as an alternative in vitro model approach to investigate IBD. OoC models not only recapitulate the human intestinal microenvironment more accurately than 2D cultures yet may also be advantageous for the identification of important disease‐driving factors and pharmacological interventions targets due to the possibility of emulating different complexities. The predispositions and biological hallmarks of IBD focusing on host–microbiota interactions at the intestinal mucosal barrier are elucidated here. Additionally, the potential of OoCs to explore microbiota‐related therapies and personalized medicine for IBD treatment is discussed.
Keywords: host–microbiota interactions, IBD treatment, inflammatory bowel disease, intestine‐on‐chip
The challenges of studying inflammatory bowel disease (IBD), highlighting limitations of traditional 2D cell cultures and animal models are discussed here. It introduces human intestine organ‐on‐chip (OoC) models as a promising alternative, capable of more accurately mimicking the intestinal microenvironment. The potential of OoCs in identifying disease mechanisms and developing personalized therapies for IBD is also explored here.
1. Introduction
Inflammatory bowel disease (IBD) is a chronic gastrointestinal disease that comprises two main entities, namely ulcerative colitis (UC) and Crohn's disease (CD). UC is confined to the colon and rectum, whereas CD can affect any region of the digestive system. The global prevalence of IBD has witnessed a dramatic rise, affecting ≈6–8 million people across diverse geographic regions.[ 1 , 2 ] The highest prevalence rates of about 0.5% of the population have been reported in Western industrialized countries. A forecast model has predicted that up to 1% of the Western population could be affected by IBD by 2030.[ 3 ] Epidemiological studies further revealed drastic increases in cases even in traditionally low‐prevalence areas, highlighting the dynamic nature of IBD distribution.[ 4 ]
Both diseases can present with overlapping symptoms such as abdominal pain and diarrhea and systemic manifestations like weight loss, fever, and fatigue. However, the development of intestinal fistulae and abscesses is more commonly observed in CD patients.[ 5 ] Extraintestinal manifestations occur in roughly one‐third of IBD patients and most frequently affect not only joints, skin, or eyes but also the liver, lungs, and pancreas.[ 6 ] Sometimes, these manifestations are diagnosed even before the onset of intestinal symptoms.
IBD is a multifactorial disease involving heterogeneous genetic, environmental, and immunological factors.[ 7 ] Genetic predisposition significantly influences the pathology of IBD, with over 240 susceptibility loci linked to genes controlling immune function and epithelial barrier integrity.[ 8 ] Studies conducted with families and identical twins have also provided substantial evidence of the heritability of IBD[ 9 , 10 ] and have suggested the role of specific genetic variations in disease susceptibility. Furthermore, environmental influences contribute to IBD pathogenesis, particularly factors such as smoking,[ 11 ] dietary habits,[ 12 ] intake of medication[ 13 , 14 ] or antibiotics,[ 15 ] and environmental exposure to chemicals.[ 16 ]
In recent years, the importance of microbiota composition and altered host–microbiota interactions has increasingly gained attention in IBD research.[ 17 ] In particular, IBD is characterized by a dysbiotic microbiome[ 18 , 19 ] involving the disruption of beneficial symbionts and an increased appearance of pathogens and pathobionts. This imbalance within the intestinal microecology can lead to increased microbial adhesion and invasion[ 20 , 21 ] resulting in inflammatory immune responses, further potentiating tissue damage and microbial composition changes. Drastic compositional alterations and reduction in microbial diversity can subsequently lead to a depletion of metabolic diversity.[ 22 ]
The lack of curative treatment for IBD emphasizes the critical need for innovative preclinical models to unravel new disease mechanisms and explore novel therapeutic intervention strategies. Due to the emerging awareness of microbial alterations and their link to IBD, preventive and curative microbiota‐based therapies are an innovative approach. Nevertheless, traditional in vitro systems and animal models are only partially suitable for the investigation of IBD and associated changes in host–microbiota interactions. Reductionistic in vitro models cannot represent the complex microenvironment and physiology of the human intestine and the underlying mechanical forces. Furthermore, long‐term coculture with microbiota remains challenging due to overgrowth or excessive waste‐product accumulation in the culture medium.[ 23 ] Animal models have provided seminal insights into systemic responses during IBD pathogenesis and progression by providing a holistic and systemic approach. Nevertheless, differences in microbiome composition[ 24 ] and immunological reactivity between mice and humans,[ 25 ] for example, limit clinical translation. The utilization of organ‐on‐chip (OoC) in vitro models recapitulating the human intestine under near‐physiological conditions is a promising approach to dissecting driving pathological factors and providing an additional platform to investigate in vivo‐like host–microbiota interactions and preventive treatment strategies.
This review aims to provide a contemporary summary of the current knowledge about host and microbiota interactions in IBD, and how state‐of‐the‐art in vitro models, particularly OoC models, have improved our understanding of disease mechanisms and pathophysiology. We further elaborate on the advantages, limitations, and innovative findings using intestine‐on‐chip (IoC) models, particularly for unraveling disease‐driving factors. In addition, we discuss recently uncovered treatment strategies using chip‐based approaches, including restoring intestinal microecology by integrating living microorganisms and microbiota‐associated nutrition and metabolites. Pointing the way ahead, we outline perspectives for OoC as a tool for personalized medicine by integrating patient‐derived cell material and the microbiome.
2. Host–Microbiota Interactions in the Homeostatic Intestine
The human intestine constitutes a highly complex and diverse host–microbiota interaction environment. The homeostasis of the host depends on a balance of these interactions, which can be disturbed by different conditions, especially inflammatory diseases.
To limit the direct contact between the luminal microbiota and the adjacent host tissue, the intestinal epithelial barrier is coated with a protective mucus layer secreted by goblet cells.[ 26 ] Cells of the intestinal epithelium are tightly connected by apical junctional complexes (AJCs),[ 27 , 28 ] including tight junctions and adherens junctions, which regulate cell–cell contacts and cell adhesion between neighboring cells. This creates a semi‐permeable barrier that allows specific molecules to pass through but prevents microbes to spontaneously translocate across and to stimulate the immune system of the host. In addition, Paneth cells are essential to limit the growth of enteric pathogens through the release of antimicrobial peptides (AMPs).[ 29 , 30 ] Moreover, intestinal epithelial cells are able to secrete bactericidal lectin,[ 31 ] which promotes host‐bacterial mutualism through the spatial separation of microbiota and the intestinal tissue.
The intestine also harbors specialized gut‐associated lymphoid tissue (GALT), which houses specialized lymphocyte populations and other antigen‐presenting cells (APCs) acting as key immune cells in the surveillance and responses to pathogens.[ 32 ] Particularly, specialized M‐cells lining the intestinal lumen facilitate the transfer of intestinal microbes to the innate and adaptive immune system in Peyer's patches.[ 33 ] The role of this intestinal immune system in mediating immune tolerance and homeostatic effector and regulator responses is further of particular importance. APCs capture and recognize microbe‐ or pathogen‐ associated molecular patterns (MAMPs or PAMPs) via pattern recognition receptors (PRRs)[ 34 ] to protect against microbial pathogens while conferring immune tolerance to the normal intestinal microbiota. Moreover, plasma cells in the lamina propria produce immunoglobulin A (IgA), which plays an important role by coating and protecting commensal microbiota,[ 35 ] neutralizing microbial toxins,[ 36 , 37 ] mediating immune exclusion and pathogen clearance,[ 38 ] thereby modulating the microbiota composition.
Under homeostatic conditions, the intestinal microbiota maintains a mutualistic relationship with the host. The microbiota participates in the fermentation of nutrients into short‐chain fatty acids (SCFAs),[ 39 , 40 ] the conversion of bile acids (BAs),[ 41 ] and the de novo synthesis of vitamins,[ 42 ] lipids,[ 43 ] and amino acids.[ 44 ] These products are essential for maintaining the integrity and immunological functionality of the host[ 45 ] and their secretion is highly dependent on the microbiota composition. Furthermore, the microbiota itself can limit excessive pathogen abundance by producing antimicrobial substances[ 46 , 47 ] and via nutrient competition,[ 48 ] which is important for the development of intestinal immune maturation and reactivity.[ 49 ]
Collectively, a controlled interplay between the microbiota, specialized intestinal cell populations, and mucosal barriers is required for intact homeostasis, while dysfunction of any of these elements can have serious consequences for the human health.
3. Intestinal Dysbiosis in IBD
3.1. Changes in Microbiota Composition and Diversity
IBD has been closely associated with dysbiosis of the intestinal microbiota. The microbial composition in stool or mucosal samples from IBD patients was associated with decreased alpha diversity and shifts in the phyla populations compared to samples from healthy controls.[ 50 , 51 , 52 , 53 , 54 ] For example, studies reported a higher amount of Proteobacteria (especially Proteus, Pasteurella, Neisseria, Rhodococcus, and Escherichia coli) and lower levels of the phyla Firmicutes (especially Eubacterium, Faecalibacterium, and Ruminococcus spp.) and Bacteroides.[ 55 ] Changes in specific families or species have also been suggested as IBD biomarkers.[ 56 ] For example, Clostridium and Veronococcus spp. tend to increase their abundance while the class Clostridia, Akkermansia muciniphila, Enterococcus faecalis, and Faecalibacterium prausnitzii decrease.[ 57 , 58 , 59 ] Particularly, the reduction of F. prausnitzii has been associated with an increased risk of relapse after surgery in the case of ileal CD.[ 60 ] In addition, it was demonstrated that postsurgical CD patients exhibit reductions in members of an identified taxonomical cluster, involving Ruminococcaceae , Oscillospira, Ruminococcus, Bilophila, Parabacteroides, and Clostridiales , while Enterobacteriaceae, were significantly increased.[ 61 ] These compositional alterations can result in decreased SCFA production and dysregulated oxygen metabolism,[ 62 ] allowing facultative aerobic bacteria such as Enterobacteriaceae to proliferate and potentially cause a disease relapse. Especially the decrease in beneficial members of the microbiota, such as F. prausnitzii,[ 59 ] Bifidobacterium longum,[ 63 ] or Clostridium clusters IV and XIVa,[ 18 ] can also contribute to increased inflammation during active IBD since, in homeostatic conditions, they facilitate intact barrier functionality,[ 64 , 65 ] regulatory T cell differentiation,[ 66 ] and maintenance of an anti‐inflammatory milieu.[ 67 ]
Moreover, mucolytic bacteria such as Ruminococcus gnavas and Romonococcus torques are more frequently present in both CD and UC,[ 58 ] which can lead to alterations in mucosal permeability and an increased abundance of mucosa‐associated bacteria leading to inflammatory reactions in IBD.
Further, an increased appearance of pathobiont species, which are microorganisms native to the host that become pathogenic under specific circumstances, like adherent‐invasive Escherichia coli (AIEC),[ 68 ] Clostridium innocuum,[ 69 ] Candida albicans,[ 70 , 71 ] or Bacteroides vulgatus,[ 72 , 73 ] correlated with IBD. This was further supported by elevated levels of microbial factors that can be used as virulence mechanisms.[ 74 ] These pathobionts increasingly adhere to the intestinal epithelium, affect the intestinal permeability, and induce inflammation and tissue damage at the intestinal epithelial barrier.[ 75 ] Additionally, elevations in the number of sulfate‐reducing bacteria, such as Desulfovibrio,[ 76 , 77 ] have been implicated to increase hydrogen‐sulfate concentrations, which result in damage of intestinal epithelial cells and inflammation.
In comparison to the bacteriome, the involvement of the virome and mycobiome in IBD has been less explored for a long time due to lower sequencing resolution. By using novel cutting‐edge technologies such as deep metagenomics, it was possible to link IBD for example to an expansion of mucosal bacteriophages,[ 78 ] especially Caudovirales, accompanied by decreased diversity. Changes in the abundance and diversity of the mycobiome are also associated with IBD.[ 79 ] For example, Malassezia [ 80 ] and Candida [ 79 ] species are frequently enriched in IBD patients. Besides changes in the mycobiota, CD patients present anti‐Saccharomyces cerevisiae antibodies (ASCAs) in their blood,[ 81 , 82 ] often before any clinical diagnosis. These antibodies are not specific for S. cerevisiae but recognize fungal cell wall mannans, thus recognizing other fungi. Even though these ASCAs are a prognostic biomarker for CD,[ 83 ] how these antibodies are generated and their role in the disease remains unknown.[ 84 ] In addition, a study with UC patients revealed that candidalysin, a toxin secreted by C. albicans, can fuel host inflammation and release of interleukin (IL)‐1β.[ 85 ] However, only certain C. albicans isolates could induce inflammation, demonstrating that strain variability is responsible for different outcomes in IBD patients. This suggests that strain functionality could be more important for disease than taxonomy. Other species, such as Debaryomyces hansenii (formerly Candida famata), have also been reported to be involved in poor mucosal healing and inflammation.[ 86 ]
The role of microbial dysbiosis as a cause or a consequence of IBD remains not fully resolved. Changes in the abundance of specific bacteria (Ruminococcus torques, Blautia, Colidextribacter, Oscillospiraceae, and Roseburia) were identified to be predictive of CD onset.[ 87 ] However, mechanistic insights have not been provided yet. Combined data from clinical studies and preclinical in vivo and in vitro models revealed that microbial composition changes can increase inflammation in the intestine.[ 88 ] Simultaneously, intestinal inflammation can disrupt the microbial homeostasis in the intestine, with detrimental effects on the health of the host. Therefore, it seems likely that during IBD a positive feedback loop exists in which intestinal inflammation leads to microbiota compositional changes and a favorable niche for enteric pathobionts[ 89 ] and pathogens,[ 90 ] which in turn results in intestinal tissue damage and inflammation. While the exact causes are unknown, multiple disease‐driving factors that participate and exacerbate this feedback loop have already been unraveled.
Even though there is a consensus that microbial dysbiosis is a key feature of IBD, the characterized microbiome shifts highly depend on the stage and severity of the disease, the sample origin (from different areas of the mucosa or the stool), and particularly on the individual patient. To shed more light on this topic, metagenomic approaches are being implemented to study functional changes in the microbiome. Herein, the metabolic potential of the microbiome has been implicated and connected to IBD.
3.2. Alterations in the Intestinal Metabolite Pool
Changes in the microbiota composition go hand in hand with alterations in metabolic functionality and activity, thus changing the microbiome‐derived metabolite pool in the intestine.[ 22 , 91 ] Two groups of metabolites are of particular importance in the context of IBD: SCFAs and BAs.
Host‐produced primary BAs (pBAs) are produced in the liver from cholesterol and metabolized by the intestinal microbiota via reactions of deconjugation, oxidation, and dehydroxylation to secondary bile acids (sBAs).[ 92 ] Due to changes in bacterial populations, patients with IBD show impaired bile acid conversion,[ 22 , 93 , 94 ] leading to a general increase in pBAs and conjugated BA and a decrease in sBAs. This can lead to detrimental effects since pBAs can increase the virulence of pathobionts, such as C. albicans,[ 95 ] AIEC,[ 96 ] and Clostridioides difficile,[ 97 ] while sBAs can limit the virulence of enteric pathogens and pathobionts.[ 98 , 99 ] Moreover, sBAs exert anti‐inflammatory effects on the mucosa and promote epithelial barrier function and regeneration.[ 93 ]
SCFAs, such as acetate, propionate, and butyrate, are primarily produced by Firmicutes and Bacteroides.[ 39 , 40 , 100 ] They provide an energy source for intestinal epithelial cells,[ 101 ] modulate the microbiota by preventing the overgrowth of certain pathogens,[ 102 ] and protect against inflammation and epithelial barrier disruption.[ 103 ] Patients with active IBD present a decrease in SCFAs,[ 104 , 105 ] which is connected to a decreased abundance of SCFA‐producing bacteria. SLC16A1‐mediated butyrate uptake was further shown to be impaired in colonic mucosal biopsies,[ 106 ] which depleted the positive effects of butyrate.
In addition to the changes in the metabolite pool caused by microbiota dysbiosis, alterations in the intestinal metabolic environment can also result from dysregulated host responses. For example, metabolomic studies with patients revealed alterations in the metabolite pool alterations due to a compromised intestinal barrier and subsequent nutrient and amino acid malabsorption.[ 107 , 108 ] Other studies reported alterations in primary energy metabolism, involving for example tricarboxylic acid cycle‐related metabolites.[ 109 ] However, which role these changes play in IBD is still not discovered.
Collectively, metabolome and microbiome alterations are closely associated with each other during IBD. Changes in the microbial and host metabolite pool are likely to have a detrimental influence on inflammation and microbial dysbiosis. Studies with patients have identified changes in metabolome and microbiome that differ between healthy and IBD patients, quiescent and active disease, and treated and untreated disease.[ 110 ] However, studies with patients tend to be descriptive and rarely establish causative links between the factors involved in IBD. Besides, prospective studies that have recorded the onset and development of the disease are scarce. Thus, it should be investigated whether beneficial bacterial members and their produced metabolites can exert preventive effects before the onset or progression of IBD and which members and molecules can trigger an IBD development or disease exacerbation.
3.3. Defects of Host Tolerance and Defense Mechanisms
Several mechanisms that modulate immune tolerance or defense mechanisms against microorganisms at the mucosal interface are dysregulated in IBD patients. A myriad of variants in NOD2, encoding an intracellular receptor involved in bacterial recognition and downstream immune stimulation, are strong risk factors in CD.[ 111 , 112 , 113 , 114 ] Gene polymorphisms in NOD1 have also been associated with IBD,[ 115 ] but to a lesser extent and only in very specific populations. Similarly, genetic association studies showed that specific mutations in CARD9, an associated signaling molecule downstream to NOD2, were a high‐risk factor for IBD.[ 116 , 117 , 118 ] ATGL161 variants have further been implicated in contributing to an impaired autophagy‐mediated clearance of intracellular bacteria, inflammation, and Paneth cell defects.[ 119 ]
Genetic defects in genes related to the development of Paneth cells and the secretion of AMPs have also been linked to IBD.[ 120 ] Dysregulation of AMP secretion could also result from inflammation‐induced mitochondrial dysfunction in intestinal epithelial cells, causing aberrant Paneth cell differentiation.[ 121 ]
Alterations of mucus layer thickness, glycosylation patterns, and mucin deficiency have further been implicated in IBD[ 122 ] and were associated with a higher adhesion and invasion of luminal microorganisms into the epithelium. In patients with UC, the mucus layer in the colon is diminished due to reduced goblet cell numbers and decreased mucin synthesis.[ 122 , 123 , 124 ] In contrast, CD patients display a thicker mucus layer with higher mucin secretion but with reduced mucus quality due to shorter oligosaccharide length.[ 122 , 125 ] In this context, the mucin increase might not reflect enhanced synthesis but rather decreased post‐translational modification and changes in the viscoelastic properties of the mucus.[ 122 ] Alterations in the intestinal mucus layer can contribute to increased adherence and invasion of luminal microbes,[ 20 , 21 ] resulting in subepithelial immune cell activation and inflammation.
Several studies have indicated that increased epithelial permeability could precede the development of inflammation. In particular, relatives of CD patients were demonstrated to exhibit increased intestinal permeability without clinical symptoms,[ 126 , 127 ] which might be a risk factor for the onset of the disease. Abnormal intestinal permeability in CD is attributable to disruption and redistribution in different AJCs[ 128 , 129 ] including zonula occludens‐1, occludin, claudin‐3, ‐4, ‐5, ‐8, and junctional adhesion molecule (JAM)‐A. In contrast, decreased expression of occludin, claudin‐1, ‐4 and JAM‐A with a concomitant increase in claudin‐2 was observed in UC patients.[ 129 ] In both disease types, an increased expression of the “pore‐forming” claudin‐2 was shown,[ 130 , 131 ] which leads to tight junction strand breaks and decreased barrier function. Increased proinflammatory cytokines such as IL‐13, interferon (IFN)‐γ, tumor necrosis factor‐alpha (TNF‐α), and IL‐1β were identified to induce decreased intestinal barrier integrity in IBD.[ 132 ]
In addition, programmed cell death pathways such as anoikis, apoptosis, necroptosis, and pyroptosis have been implicated to play a role in the disease.[ 133 ] For example, Paneth cell death was induced by TNF‐α and associated with increased receptor‐interacting protein 3‐mediated necroptosis in CD patients,[ 134 ] which could explain the reduction of antimicrobial defense in the disease. So far, it has not been clarified, whether elevated cell death signaling is a cause or a consequence of IBD. However, it contributes to increased barrier permeability and aggravates inflammation by releasing endogenous immunogenic molecules, termed damage‐associated molecular patterns (DAMPs), from damaged cells.
3.4. Dysregulation of Mucosal Immunity
During IBD, the PAMPs and MAMPs of translocated microbiota and DAMPs released upon cellular injury elicit inflammatory responses. This includes activating innate immune cells like macrophages, dendritic cells, neutrophils, and lymphoid cells via PRRs.[ 34 ] Activation of PRRs leads to activation of immune transcription factors such as NF‐κB, AP‐1, IRF3, and IRF7, which promotes APC maturation and cytokine responses.[ 135 , 136 ] This results in increased antigen presentation and effector T cell activation.[ 135 , 137 ]
Increased proinflammatory macrophages and the recruitment of inflammatory monocytes into the inflamed lamina propria.[ 138 ] In addition, dendritic cell accumulation was shown to be increased in IBD,[ 139 , 140 ] particularly in GALT regions such as Peyer's patches and lymph follicles. Moreover, dendritic cells display an abnormal phenotype and increased Toll‐like receptor (TLR) 2/4 expression, activation markers, and elevated proinflammatory cytokine release perturbating the disease severity.[ 141 ]
Further, dysregulated neutrophil activity is associated with IBD.[ 142 ] An increased abundance of neutrophils and neutrophil extracellular trap‐associated proteins has been found in biopsies of UC patients.[ 143 ] Accumulation of hyperactive neutrophils and induction of increased reactive oxygen species, inflammation, morphological alterations such as abscess formation, and the attraction of further immune cells were implicated in UC pathophysiology.[ 144 ] In comparison, CD patients displayed impaired neutrophil recruitment and attenuated macrophage activity in vitro.[ 145 , 146 ]
IBD patients also present dysbalanced innate lymphoid cell populations,[ 147 , 148 ] essential mediators of antimicrobial defense. This leads to increased cytokine release, exacerbating the inflammation and, thus, the disease severity. Upon inflammation, cells of the adaptive immune system are further activated and secrete proinflammatory cytokines, activating local and circulating leukocytes to migrate to the effector sites. CD and UC are characterized by unique T helper (Th) cell responses and differential inflammatory cytokine patterns,[ 149 ] potentially explaining the different disease phenotypes. During CD, a hyperactive Th1[ 150 ] and Th17[ 151 , 152 ] response after stimulation by APCs is the primary driver of the inflammation. In contrast, UC is characterized by an atypical Th2 cell‐type‐like cytokine profile.[ 153 ]
Collectively, this demonstrates that changes in microbial composition, epithelial barrier integrity, and underlying inflammatory conditions drastically affect intestinal homeostasis and health during IBD (Figure 1 ).
Figure 1.
Dysregulated host–microbiota interactions in IBD pathophysiology. An overview of the complex and dynamic interplay between host cells and luminal microbiota, which is affected by factors such as genetic predisposition or environmental influences (medication, chemicals, stress, lifestyle, and infections). IBD is associated with changes in microbial composition and metabolic activity within the luminal compartment of the intestine. Disruption of the mucosal epithelium is indicated by a reduced mucus layer due to goblet cell malfunction, decreased secretion of antimicrobial peptides following Paneth cell abnormalities, and compromised junctional complex integrity. In response to dysbiosis and epithelial barrier dysfunction, microbiota and microbial products can invade into the lamina propria resulting in an activation of the mucosal immune system. Interactions of translocated microbiota and resident immune cells such as macrophages and dendritic cells further exacerbate a disbalance in regulatory and effector T cell populations and lead to increased immune cell infiltration from the peripheral vasculature. This results in an amplified cycle of chronic inflammation in the intestinal mucosa. Created with BioRender.com.
4. In Vitro Models of IBD
Mouse models have been commonly leveraged for IBD research since they recapitulate the whole organismal complexity of the intestine and immune cell populations that are critical during the development of IBD. However, besides ethical concerns and a high need for resources, a major limitation of animal models is that they are too complex to independently dissect and control the factors that contribute to IBD. This can hinder the characterization of specific signaling pathways or metabolites that are involved in the disease. Therefore, there has been a great interest in developing in vitro models that can elucidate different aspects of human IBD. In addition, a failure to achieve clinical translation is a major limitation of animal models, such as mice. For instance, IL‐10 treatment was effective in reducing experimental colitis in rodent models.[ 154 , 155 , 156 ] Contrastingly, clinical trials in humans did not show significant benefits or very limited clinical effects of IL‐10,[ 157 , 158 ] probably due to different immunoreactivity and altered intestinal microenvironment. By combining human cells and tissues with more complex in vitro models, the risk of species‐specific discrepancies might be reduced and may improve the likelihood of successful clinical translation. Moreover, the majority of IBD induction methods in rodents are based on applying chemical agents such as dextran sodium sulfate (DSS),[ 159 , 160 ] 2,4,6‐Trinitrobenzenesulfonic acid,[ 161 ] and oxazolone,[ 162 ] which are artificial triggers of IBD‐like phenotypes and cause rather an acute form of the disease with self‐limiting inflammation compared to the rather chronic disease pattern and associated relapses observed in human IBD.[ 163 ] In the following section, we summarize and discuss current state‐of‐the‐art in vitro models and their utility for investigating IBD, focusing on OoC models (Table 1 ).
Table 1.
Overview of different in vitro models with their respective advantages, limitations, and applications for IBD research.
| Model | Advantages | Limitations | Applications |
|---|---|---|---|
| 2D mono‐cultures |
|
|
|
| Transwell models |
|
|
|
| Organoids |
|
|
|
| OoC |
|
|
|
4.1. Epithelial Cell Monolayers and Transwell Models
Statically cultured epithelial cell monolayers are the simplest and most reductionistic in vitro model of the intestinal epithelial barrier. Caco‐2 cells, derived from human colorectal adenocarcinoma, are a commonly used cell line to study the intestinal epithelium. Upon differentiation, these cells acquire characteristics similar to enterocytes of the small intestine, expressing a brush border and having the capacity to produce digestive enzymes and cytokines.
In IBD research, Caco‐2 cells have been used in microplate formats to assess, for example, bile salt uptake by enterocytes upon inflammation[ 164 ] or the effect of anti‐inflammatory molecules on enterocytes.[ 165 ] Most studies seed the intestinal cells into transwell inserts, resulting in a polarized monolayer with apical and basal compartments.[ 166 , 167 , 168 ] By providing a spatial separation of compartments in transwell models, barrier permeability can be measured via transepithelial electrical resistance or diffusion of fluorescent tracer molecules or specific enzymes, allowing to quantify changes in the intestinal barrier integrity,[ 169 ] a key feature of IBD. Thus, transwell models can be used to study several fundamental aspects of IBD and have improved our understanding of the disease, especially about the involvement of different cellular pathways.[ 167 , 169 , 170 , 171 , 172 ]
Besides Caco‐2, other cell lines can be leveraged to reproduce specific disease aspects. For example, HT29‐MTX and HT29‐16E, which are polarized goblet‐like cell lines, produce higher amounts of mucus compared to Caco‐2. This allows one to investigate how mucus alterations affect host–pathogen interactions during IBD.[ 96 , 173 ] Usually, these mucus‐producing cell lines have been cocultured with Caco‐2 cells to compensate for the low mucus secretion by Caco‐2.[ 174 , 175 ]
Static epithelial cell cultures are economical, easy to handle, reproducible, and suitable for high throughput. In addition, their low complexity allows for the detailed dissection of the role of behavior of individual components. For example, it was possible to investigate how proinflammatory cytokines secreted in an IBD model can inhibit a taurocholate transporter in the enterocytes.[ 164 ] Nevertheless, these models lack a fully differentiated intestinal barrier and interaction with immune cells, thus making it impossible to explore complex multicellular and, in particular, immunological aspects of IBD.
4.2. Scalable Complexity and Multicellularity
Advanced in vitro models achieve higher complexity by incorporating essential cell types, such as immune cells.[ 176 ] The immune system, and especially innate immune cells, plays an important role in the pathophysiology of IBD. For example, the accumulation of proinflammatory macrophages and monocytes in the intestinal tissue along with proinflammatory cytokine secretion has been associated with IBD. Therefore, incorporating these cells in in vitro models allows to more accurately reproduce the disease and to explore the molecular mechanisms of onset and development of inflammation during IBD, interactions with other components of the system (such as epithelial cells or microorganisms), and responses to applied treatments. In one example, macrophages were incorporated into a transwell model with Caco‐2 and HT29‐MTX (goblet‐like) intestinal cells to study leaky gut syndrome,[ 177 ] which is associated with IBD,[ 178 ] and to screen for anti‐inflammatory compounds.[ 179 ]
Besides immune cells, endothelial cells[ 180 ] and fibroblasts[ 181 ] can also be cocultured with epithelial cells in transwells. Growing evidence indicates alterations in the intestinal microvasculature and peripheral blood circulation during IBD.[ 182 ] In addition, endothelial cells mediate inflammation and orchestrate the recruitment of circulating immune cells, which are key processes in the onset and progression of IBD. Similarly, intestinal fibrosis is common in IBD patients, indicating the involvement of fibroblasts in the pathogenesis of IBD.[ 183 ] Therefore, models that incorporate endothelial cells or fibroblasts facilitate investigation of specific molecular aspects of IBD, such as the role of the intestinal microvasculature in tissue inflammation and barrier permeability and the effect of stromal factors on inflammation. Since all these aspects involve several cell populations, multicellular models are required to dissect them at a molecular scale.
Another improvement to emulate the intestinal microenvironment is the addition of structural scaffolds for the cells. These can be used to support cells in forming a 3D structure that mimics the morphology of villi and crypts in the intestine,[ 184 ] resulting in a more physiological behavior of either host cells or the microbiota, as demonstrated for the pathogenic bacterium AIEC.[ 185 ] In addition, culture under hypoxic conditions in static systems is also possible and has contributed to dissecting its effect on IBD.[ 186 ]
A further step toward increasing biological complexity of in vitro models is the integration of microbial communities. Nevertheless, bacteria tend to rapidly overgrow cell cultures and affect host cell viability.[ 187 ] Both the beneficial effect of commensal strains on IBD and the detrimental role of pathogens and pathobionts have been addressed in static models, but the integration of microorganisms is usually limited to short timespans, colonization with very low numbers, or experimental manipulations, such as the use of antibiotics or medium exchange.[ 131 , 185 , 188 , 189 , 190 ] These artificial conditions can hinder the study of physiologically relevant host–microbiota interactions.
4.3. Intestinal Organoids
Organoids are miniaturized, 3D tissue cultures derived from stem cells. They are based either on adult stem cells isolated from intestinal crypt tissue[ 191 ] or induced pluripotent stem cells (iPSCs).[ 192 ] While adult stem cells allow for more genetically stable organoids, the use of iPSCs can be exploited to generate other tissue‐associated cell types such as endothelial cells, immune cells, or mesenchymal cells, to recapitulate autologous cell–cell interactions.[ 193 ] Regardless of their origin, intestinal organoids recapitulate the differentiation of specific intestinal cell populations, like Paneth cells, goblet cells, or M‐cells.[ 191 ] These cells do not arise normally in cell line monocultures but are essential for maintaining intestinal homeostasis and emulating cell‐specific disease alterations.
Organoids from patients with active IBD implicated an inflammatory phenotype, with smaller size, inverted polarization, increased cell death, and reduced AJCs compared to organoids from non‐IBD patients.[ 194 ] Besides these phenotypical alterations, organoids preserve IBD‐specific genetic and epigenetic features, such as methylation patterns[ 195 , 196 ] or altered expression of AJCs.[ 197 ] This makes it a valuable tool to investigate patient specific phenotypes, in contrast to models based on cell lines.
Further studies have focused on recapitulating processes involved in mucosal inflammatory damage. Within this scope, IBD‐related cytokines such as IL‐17,[ 198 , 199 ] TNF‐α,[ 200 ] or IFN‐ γ,[ 201 ] either different types alone or in synergism with TNF‐α,[ 202 ] adversely affected cell viability and cell proliferation leading to stem cell dysfunction. Besides, the regenerative expansion capacity of intestinal stem cells was diminished after stimulation with IL‐22,[ 203 , 204 , 205 ] although organoid volume increased due to elevated differentiation of progenitor cells into specialized intestinal cell types.
Despite these notable advancements in the field, organoid models face limitations. First, the structure and functionality of the organoids depend on the origin of the cells and on the culture conditions, which can vary between laboratories and can lead to variances in their reproducibility. In addition, most intestinal organoids only include epithelial cells, even though other cell types are important to recapitulate and understand IBD. However, efforts have been made toward the integration of other cell types. For example, coculture of organoids with macrophages,[ 206 ] intraepithelial lymphocytes,[ 207 , 208 ] mesenchymal fibroblasts, and enteric nerves[ 209 ] better reflected cell–cell interactions in the intestine and inflammatory signaling during IBD. T cells were also integrated into organoid systems,[ 210 ] which showed that they are relevant contributors to IBD pathogenesis by inducing the expression of proapoptotic factors, particularly when costimulated with the bacterial endotoxin lipopolysaccharide (LPS). Additionally, lymphocyte–epithelial interaction was investigated when interfering with the anti‐β7 integrin subunit, which reduced T‐cell infiltration, apoptosis, and inflammation.[ 211 ]
Since iPSC‐derived organoids are generated by complex reprogramming, their susceptibility to cell contamination, tumorigenesis, and retainment of their fetal genetic signatures is frequently observed.[ 212 ]
An important limitation of using organoids for IBD research is the difficulty in accessing the intestinal lumen. Their inherited lumen‐enclosed and basal‐out morphology requires complicated injection procedures for transport studies, luminal compound exposure, and apical microbiota culture.[ 213 ] While recent studies already showed the possibility of exploring epithelial–microbial interactions like the administration of fecal supernatants to induce IBD conditions in healthy intestinal organoids[ 214 , 215 ] or the culture of pathogenic AIEC on organoid‐derived epithelial monolayers,[ 216 ] a stable coculture with microbiota is very challenging and often requires microinjection of the microbiota into the enclosed lumen. Apical‐out organoids could be used as a more convenient model to research host–pathogen interactions.[ 217 ] Nevertheless, enclosed organoids complicate the manipulation and analysis of both apical and basal compartments, which can be relevant, for example, to investigate differential cytokine secretion during inflammation.
The enclosed organoid morphology can be circumvented by generating 2D monolayers from 3D intestinal organoids.[ 218 , 219 ] This allows the integration of the differentiated cells with other in vitro models, providing a more convenient system for microbial culture. Nevertheless, static organoid cultures still face the limitations of microbial overgrowth and short cultivation periods.
4.4. Organ‐on‐Chip (OoC)
OoC models are advanced in vitro models that have raised interest in translational research due to a series of features that allow modeling of complex aspects of human diseases. According to the European Society of Organ‐on‐Chip, “an Organ‐on‐Chip (OoC) is a fit‐for‐purpose microfluidic device, containing living engineered organ substructures in a controlled microenvironment, that recapitulates one or more aspects of the organ's dynamics, functionality and (patho)physiological response in vivo under real‐time monitoring”.[ 220 ] OoC platforms are manufactured from different polymers and provide a varying growth area for cell culture, from a few mm2 to a few cm2. Within these chips, different cell sources of interest, such as cell lines, primary cells, and organoid‐derived cells can be cultured and subjected to mechanical forces like tissue perfusion or vacuum stretching, which improve cell differentiation and polarization.[ 221 ] By integrating independent cell culture compartments, tissue–tissue interfaces, and extracellular scaffolds, it is possible to mimic the microanatomy and physiology of the respective organ (Figure 2 ).
Figure 2.
OoC as a model platform to recapitulate IBD disease phenotypes in vitro. Schematic representation of an IoC model consisting of two individual cultivation compartments separated by a porous membrane as scaffold for cell adhesion. The depicted model consists of a vascular cell layer (endothelial and immune cells) and an idealized 3D epithelial cell layer. The IoC simultaneously allows to independently culture different cell types of the intestine and to dynamically perfuse these cellular compartments. This is not only important to generate a 3D tissue morphology with crypt‐ and villus‐like structures but also as a next step for maintenance of inoculated microbiota without accumulation of cellular debris and bacterial overgrowth. By applying different triggers such as proinflammatory cytokines (TNF‐α, IL‐1β, and IFN‐γ), pathogenic bacteria or bacterial lipopolysaccharide (LPS), nutritional deprivation, and chemical dextran sodium sulfate (DSS) into the indicated compartments (black arrows), IBD‐like phenotypes with clinical hallmarks can be induced. Created with BioRender.com.
IoC models are chip‐based biological models mimicking the structural and functional characteristics of the human intestine in vitro. Most of the models emulate the intestinal microanatomy, including the intestinal mucosal barrier and surrounding tissues, such as the lamina propria or the adjacent vascularity. The application of microphysiological perfusion and mechanical forces, such as stretch and strain, recapitulate intestinal peristalsis in an in vivo‐like manner, which supports the formation of microvilli‐like structures and formation of a mucus layer.[ 221 ] Parameters such as nutrient availability, oxygen levels, microbiota abundance, or drug concentrations during treatment can be precisely controlled and adjusted through the perfusion system. Most IoC models consist of a two‐channel system with an epithelial compartment containing Caco‐2 cells as a surrogate for intestinal epithelial cells and a vascular compartment lined with endothelial cells. Many of these models include functional immune cell populations. In the following sections, we elaborate on how IoC models are leveraged, especially for emulating host–microbiota interactions, and how these models can contribute to an improved understanding of IBD.
Technical details on IoC design and setup, functioning, additional features, and translation have been extensively reviewed.[ 222 , 223 , 224 , 225 , 226 ] As a note, IoC models have focused on reproducing the epithelial lining of the intestine. Most IoC models feature intestinal epithelial cells with mucus and also include a vascular compartment, representative of the blood vessels in the submucosa. However, the complete morphological structure of the intestine, including mucosa, submucosa muscular layer, and serosa has not been replicated in vitro yet. Fully recapitulating the intestinal anatomy would require several types of tissues (epithelial, connective, muscle), each including several specialized cell types. Besides, defining a cell culture media and conditions where all cell types can thrive, integrating the different tissues in the right configuration, and getting them to function coordinately is a challenge beyond current capabilities. However, the current IoC configurations can already reproduce essential microanatomical features of the intestine to study infections, inflammation, and some therapeutic strategies.
4.4.1. Host–Microbiota Interactions in OoC
In vitro models are ideal for dissecting host–pathogen interactions at a molecular level due to their reductionistic approach. In comparison, OoC models are especially suitable for integrating a living microbiota since microfluidic perfusion can remove overgrowing bacteria, accumulating cell debris, and metabolic waste products while supplying the host cells with fresh nutrient‐rich medium, enabling homeostatic coculture conditions over more extended periods compared to static models. Several studies have successfully integrated living bacteria into OoC models,[ 227 , 228 , 229 , 230 , 231 , 232 ] circumventing the limitations under static culture conditions. Given the complexity and relevance of the microbiota in intestinal health and disease, there is a significant interest in reproducing a stable host–microbiota interface in vitro. Kim et al. established a pioneer IoC model for the long‐term culture of a commensal microbe.[ 228 ] A further study using the same OoC system managed to integrate a stable microbiota community composed of 200 unique operational taxonomic units with similar ratios compared to the human intestine.[ 233 ] This paves the way to integrate microbiota, or representative microbial communities, from IBD patients to unravel their contribution to the disease and investigate strategies to modulate it in an anti‐inflammatory manner. Therefore, it could be an interesting approach to compare the effects of microbiota from healthy and IBD patients and use their differences to identify the most harmful or beneficial species, as previously done to identify protective strains against Salmonella typhimurium.[ 234 ]
In IoC models, it is possible to recreate physiological oxygen gradients along the crypt‐villus axis. Since many of the bacterial communities in the intestine are anaerobic, reproducing such an interface in OoC models can allow one to study microbial growth and dynamics in a more physiologically relevant way. This has been achieved by maintaining the apical (intestinal) compartment under anoxic conditions and supplying a highly oxygenated medium through the perfusion into the vascular compartment. Moreover, these interfaces have already been advanced to study the role of specific species known for their role in IBD. For example, a hypoxic IoC was colonized with F. prausnitzii,[ 235 ] which is highly sensitive to oxygen. Transcriptomics analysis and liquid chromatography enable the dissection of bacterial colonization and metabolism, revealing a high production of SCFAs and the upregulation of anti‐inflammatory pathways.
Microbial dysbiosis is a relevant hallmark of IBD. However, the role of different phyla, species, or metabolic pathways in the disease has not been adequately elucidated. As summarized in the section “Microbiota,” descriptive studies with human patients have provided knowledge on intestinal microbial communities that are depleted or enriched. Dissecting the contribution of individual strains during IBD pathogenesis could help not only to understand the driving disease factors of IBD but also to design personalized therapies.
Finally, OoC models also offer the possibility of integrating and studying pathobionts.[ 236 ] By avoiding the tissue damage caused by microbial overgrowth, the molecular mechanisms of infection in a physiologically relevant setting can be dissected in detail.[ 231 , 237 , 238 ]
Studies with patients have provided plenty of descriptive data about microbial and metabolomic changes in IBD patients. However, these studies lack mechanistic insights since they are usually limited to correlational analysis. In this context, OoC models are proposed as a preclinical in vitro platform to bridge this gap. OoC models allow one to control and manipulate microbial communities and underlying metabolic changes can be recorded in real‐time. Similarly, the microenvironment and metabolite pool can be adapted in OoC models to investigate how specific alterations affect the composition and balance of the microbiota. OoC IBD models monitor host responses and health (for example, by measuring IBD‐relevant disease hallmarks such as inflammation, mucus alterations, barrier permeability, or cell death). Thus, combining these approaches could be advantageous in establishing causative links between changes in the microbiota, metabolome, and disease progression.
Nevertheless, the culture of microbiota in OoC platforms has so far been limited to one or two weeks.[ 228 , 239 ] This is an important advancement compared to static models, in which host–microbiota cocultures are maintained for a few days but are far from several months that might be necessary for a chronic and relapsing disease like IBD. Interestingly, in these experiments, the epithelial cells seemed viable and functional, indicating the feasibility of keeping such cocultures even for longer periods of time. However, it is necessary to determine physiological inoculation densities for each species and to identify suitable medium compositions in which neither overgrowth, microbial devitalization, nor compositional changes (if multiple microbial strains are inoculated) are induced.
5. Modeling IBD with OoC
Microphysiological OoC systems emulating the human intestine are increasingly relevant for IBD disease modeling. Since IBD is a multifactorial disease with still unknown causes, reproducing it in vitro is complicated. However, many studies have induced IBD‐like phenotypes in IoC models using diverse approaches with different degrees of complexity (Figure 2, 3 ).
Figure 3.
Exemplary OoC models for modeling and investigating IBD. A) Schematic illustration of a multicellular three‐lane IoC in the OrganoPlate (Mimetas). The cells were seeded in the adjacent perfusion channels, with an extracellular matrix (ECM) channel in between. The epithelium consisted of Caco‐2 and HT29‐MTX‐E12 cells, whereas the vascular compartment included THP‐1 monocytes and MUTZ‐3 dendritic cells. Both channels were perfused with a proinflammatory cocktail of TNF‐α, IL‐1β to induce IBD‐like conditions. Reproduced with permission.[ 242 ] Copyright 2020, Elsevier. B) Experimental setup of a two‐compartment IoC consisting of Caco‐2 (top) and human umbilical vein endothelial cells (HUVECs, bottom) separated by a porous membrane. Cells were subjected to perfusion on both sides and vacuum pressure for stretch motions. IBD‐like conditions were induced by application of proinflammatory cytokines in different concentrations and either in the HUVEC compartment or in both the Caco‐2 and the HUVEC compartment. Intestinal barrier dysfunction was measured by parallel administration of a permeability tracer. Reproduced under terms of the CC‐BY license.[ 241 ] Copyright 2023, The Authors, published by Public Library of Science (PLOS). C) Perfused IoC model containing intestinal epithelial cells and endothelial cells with circulating immune cells (PBMCs). LPS was used to mimic the presence of the microbiome in the intestinal lumen and to stimulate PBMC attachment and infiltration to induce inflammation. Reproduced with permission.[ 245 ] Copyright 2016, National Academy of Sciences. D) Representation of an IoC incorporating intestinal epithelial cells and circulating PBMC. The combination of LPS and DSS in the presence of circulating PBMCs induces a colitis‐like phenotype by disrupting the 3D tissue architecture, leading to villus atrophy. Reproduced with permission.[ 246 ] Copyright 2018, National Academy of Sciences. E) Outline of an intestinal OoC model consisting of enterocytes, HUVECs, and macrophages. The gut microbiome is partly recapitulated through the presence of pathogenic E. coli 11775, which results in intestinal inflammation in the model. Reproduced under terms of the CC‐BY license.[ 247 ] Copyright 2022, The Authors, published by Frontiers. F) Promotion of an inflammatory disease state by nutritional deprivation (‐N/‐T) in an IoC model. Reproduced under terms of the CC Attribution 4.0 International License[ 248 ] Copyright 2022, The Authors, published by Springer Nature. G) Emulating biopsy‐derived human colonoids into an OoC model with adjacent microvascular endothelial cells. Both compartments are separated by a membrane and are constantly perfused and stretched through a vacuum channel. Exposure to IFN‐γ or IL‐22 increases intestinal barrier permeability, resulting in a leaky gut. Reproduced with permission.[ 249 ] Copyright 2021, Elsevier.
5.1. Induction with IBD‐Associated Cytokines
The most common form of inducing IBD in OoC models is the application of single proinflammatory cytokines or cytokine cocktails, as inflammation is the primary driver of IBD. Beaurivage et al. demonstrated that stimulating Caco‐2 cells in an IoC with IL‐1β, TNF‐α, and IFN‐γ led to disrupted barrier functionality and an increased secretion of interferon gamma‐induced protein‐10, IL‐8, and CC‐chemokine ligand‐20,[ 240 ] which are inflammatory chemokines involved in lymphocyte and neutrophil recruitment during IBD. Notably, the increase in chemokines was predominantly measured at the basolateral side, highlighting the advantage of a compartmentalized model to dissect the inflammatory triggers and immune cell attraction site. Furthermore, the disease phenotype was not exacerbated when the cytokines were applied for a longer duration, indicating a direct and rapid phenotype induction after the initial trigger without dependency on the treatment duration. Interestingly, treatment with the NF‐κB pathway inhibitor (5‐(p‐Fluorophenyl)‐2‐ureido)thiophene‐3‐carboxamide (TPCA‐1) reduced the inflammation. By knocking down the inflammatory effectors MYD88 and RELA, the initial cytokine release induced by the cytokine cocktail was mainly prevented. This showcases the potential of this OoC model for anti‐inflammatory treatment strategies and target discovery.
Multicellular OoC models have allowed the investigation of tissue‐specific reactions observed in the human intestine. For example, stimulation of a vascularized IoC model with TNF‐α and IFN‐γ increased proinflammatory responses in the vascular compartment compared to the epithelial compartment.[ 241 ] This model provided insights into nonimmune cell cytokine secretion mechanisms and effects. Another IoC considered other cell types involved in IBD and further included goblet‐like cells (HT29‐MTX‐E12), Tohoku Hospital Pediatrics‐1 (THP‐1) monocytes, and the human acute myeloid leukemia cell line MUTZ‐3 as immune cell surrogates.[ 242 ] Here, exposure to TNF‐α and IFN‐γ resulted in increased apical and basal IL‐8 production and decreased barrier integrity, while preventive application of TPCA‐1 again attenuated the inflammatory conditions. Similar results were achieved in this model compared to the Caco‐2 model despite increasing complexity and multicellularity, although the most complex model also considered immune cell cytokine secretion.
5.2. Induction via Bacterial Involvement
To mimic the microbial involvement in IBD, many established OoC models use LPS as a stimulus alone or in combination with cytokines to induce an IBD‐like phenotype. LPS is a bacterial endotoxin present in the cell walls of gram‐negative bacteria and causes inflammation, mainly mediated via TLR4 signaling.[ 243 ] The recognition of LPS by TLR4‐expressing myeloid cells such as monocytes, dendritic cells, and macrophages is an essential mechanism during IBD pathogenesis, leading to increased inflammation.[ 244 ] Therefore, the integration of functional immune cell populations within OoC models is a requirement to fully recapitulate inflammatory processes in the intestinal mucosa upon LPS translocation.
A pioneering study by Kim et al. demonstrated that applying supraphysiological LPS in an OoC model consisting of Caco‐2 cells and human peripheral blood mononuclear cells (PBMCs) caused a robust proinflammatory response with increased TLR4 expression.[ 245 ] LPS administration in a vascularized chip model incorporating endothelial cells and PBMCs also resulted in intercellular adhesion molecule‐1 (ICAM‐1) expression on the endothelial surface. This increased PBMC attachment, reflecting crucial pathophysiological immune cell recruitment stages in IBD. Another study demonstrated that administration of TNF‐α and LPS induced TLR pathway activation, proinflammatory cytokine release, and reduced barrier integrity in an IoC model.[ 250 ] The same study reported an enhanced expression of inflammation‐related genes, and the autophagy‐related gene ATG16L1, which can harbor a risk allele associated with CD.
A further study used a mixture of LPS and N‐formylmethionine‐leucyl‐phenylalanine (fMLP) to stimulate extracellular matrix (ECM)‐embedded THP‐1 cells in a Caco‐2/THP‐1 tubular chip model.[ 251 ] Luminal exposure to LPS/fMLP in the Caco‐2 tubule did not induce inflammation, similar to a homeostatic intestine with an intact epithelial barrier. Direct stimulation of THP‐1 monocytes resident in the ECM tubule with LPS/fMLP led to the secretion of proinflammatory cytokines. Interestingly, IL‐6 and IL‐8 secretion was associated with intestinal epithelial cells in the model, even in the absence of immune cells, demonstrating differential cell‐specific inflammatory responses.
5.3. Induction of IBD by Applying Chemicals
Applying barrier‐disrupting chemicals, such as DSS, is a commonly used method to induce experimental colitis in animal models.[ 159 , 252 ] Therefore, using DSS to induce IBD in OoC models[ 246 , 247 ] is a valuable approach to compare results obtained in both models. Application of DSS and LPS in an OoC model consisting of Caco‐2 cells and PBMCs resulted in a colitis‐like IBD phenotype with compromised barrier integrity, disrupted AJCs, diminished mucus layer, and villus atrophy.[ 246 ] Increased production of reactive oxygen species, elevated proinflammatory cytokine release, and PBMC infiltration toward the basolateral side of the epithelium were proposed as underlying mechanisms for developing the disease. Compared to previous studies in which cytokines were used as a trigger to induce inflammation, this study suggested that a permeable mucosal barrier and associated microbial translocation could precede immune cell infiltration and chronic inflammation.
5.4. Induction of IBD via Nutritional Deficiencies
Besides these widely used methods, a recent study showed that nutritional deficiencies of niacinamide and tryptophan in the intestinal culture medium can promote IBD‐like conditions in an organoid‐based chip model.[ 248 ] The study demonstrated that nutritional deficiency could cause villus atrophy, disruption of barrier function, and changes in nutrient absorption via dysregulation of several genes involved in transport, metabolism, and inflammation. This implicates an environmental influence of nutritional deficiency rather than inherent genetic features of the disease.
5.5. Addressing Genetic and Epigenetic Changes in IBD with OoC
Collectively, these models could recapitulate a majority of important IBD characteristics and provided essential insights into cell‐specific involvement and the distribution of disease‐causing factors. Nevertheless, the transferability and extrapolation of results remain questionable due to the use of tumorigenic epithelial cell lines. Therefore, OoC models have emerged that include stem‐cell‐derived intestinal cells as epithelial cell sources.[ 253 , 254 , 255 , 256 , 257 ] This approach requires complex model refinements such as medium composition, cell ratios, matrix composition, and consistent cell availability. In addition, dissociation and fragmentation of the organoid structure are necessary to integrate the cells into the OoC device. However, it offers the possibility to recapitulate the patient's genetic and epigenetic signatures. This allows investigating the molecular implications of specific genetic variations in IBD and the design of personalized therapies.
A human primary cell gut‐on‐chip model incorporating organoid‐derived intestinal epithelial cells as a monolayer and macrophages[ 258 ] was leveraged to reproduce inflammatory IBD conditions. Compared to statically cultured intestinal organoids, intestinal cells cultured within the dynamically perfused OoC model exhibited increased expression of genes related to intestinal functionality, cell‐specific differentiation markers, and maturation markers, similar to in vivo. Application of LPS and IFN‐γ in this model triggered pathways involved in cytokine secretion and bacterial response even without macrophages, whereas synergized secretion of IL‐6 and IL‐12p70, mainly at the basal side, was significantly induced when macrophages were present. Another study by Apostolou et al. described an organoid‐derived model containing colonic crypt‐derived epithelial cells and microvascular endothelial cells.[ 249 ] They found increased permeability, apoptosis, and altered AJC expression in response to IFN‐γ application. In addition, stimulation with IL‐22, a pleiotropic IBD‐related cytokine, induced apoptosis and disruption of epithelial barrier functionality. The loss of barrier functionality could be rescued by blockade of IL‐22 with its soluble receptor IL22BP.
While integrating complex cell sources involves more laborious handling processes and lower throughput, these challenges are moderate compared to those of animal experiments. Chip models based on primary intestinal tissue in high‐throughput microplate formats are already available,[ 259 , 260 ] although they suffer from lower physiological complexity without incorporating immune cells, which are key effector cells in IBD. Different in vitro models should, therefore, be combined to utilize their individual advantages.
It is important to note that to date, no OoC model can fully recapitulate the biologically complex interactions of the mucosal immune system. Moreover, closer attention should be drawn to the interaction between differentiated epithelial cell populations such as Paneth cells or goblet cells and immune cells, as their dysfunctionality is closely linked to the IBD severity.
5.6. Circulating Immune Cell Recruitment in IBD‐on‐Chip
Several IoC models integrate a vascular compartment lined with endothelial cells. This can be used to mimic pathogen dissemination, intravenous drug administration, or to integrate circulating immune cells. This is particularly of value for IBD research since processes such as neutrophil infiltration from the vasculature into the epithelial tissue are frequently observed.
Using IoC this phenomenon could be reproduced and studied with functional neutrophils that were able to exert inflammatory effects on the intestinal parenchyma.[ 251 ] By integrating primary neutrophils in an IoC in which inflammation had been induced via LPS and fMLP, it was demonstrated that the inflammatory milieu signaled neutrophils to infiltrate into the tissue.[ 251 ] As neutrophil invasion upon mucosal inflammation (together with other circulating immune cells) is an essential hallmark of IBD,[ 142 , 143 ] this study underscores the importance of cellular complexity and the possibility of studying cellular migration processes. As immune cell infiltration has been used as a marker for disease progression in mice,[ 261 , 262 ] it is relevant to extrapolate data from human in vitro models to these animal results. These studies underline the features of OoC models that can be leveraged to gain insights into IBD pathophysiology in a human context, which can be used both in a reductionistic way and further to address complex cell interactions.
OoC models can potentially simulate temporal changes in immune responses and dynamics of specific immune cell populations. Due to the separation of perfused compartments, immune responses of resident subepithelial mucosal immune cells as well as the activation and recruitment of peripheral circulating immune cells can be investigated in OoC systems. Using labeling and imaging of specific immune cells,[ 246 , 251 ] fluctuations in immune cell phenotypes and their migration toward inflammatory stimuli, can be monitored and quantified.[ 263 ] Moreover, integrating immunosensors for live measurement of cytokine release within OoC platforms[ 264 , 265 ] is a promising approach to track immune cell activation in a time‐dependent manner. These aspects are particularly important for IBD, where variations in immune activity greatly contribute to the disease initiation and progression. In addition, a recent study demonstrated that individual innate immune cell populations derived from THP‐1 cells and peripheral blood mononuclear cells (PBMCs), including adaptive immune cells, secrete differential cytokine responses after stimulation with LPS and IFN‐γ in an OoC‐based IBD model.[ 266 ] This system allows the introduction of different immune cell types and inflammatory stimuli at specific chip sites, enabling the precise investigation of individual cell type contributions in intestinal inflammation and the neutralizing effect of the anti‐inflammatory antibody Infliximab.
Current OoC models for IBD still face several limitations that challenge their applicability. Most induction methods to trigger IBD rely on the outlined stimulation with cytokines, bacterial molecules, or chemical agents to selectively induce inflammation. However, these induction methods are neither standardized nor do they fully recapitulate systemic responses in vivo. Despite very promising developments in the field of IoC models, a complete recapitulation of the intestinal microenvironment, for example, the morphological architecture of different intestinal sections, inclusion of all relevant cell types, presence of a microbiome, and stable oxygen gradients remain noticeable hurdles. Further, it is very challenging to model the plasticity and dynamics of the human immune system, particularly disease‐type‐specific lymphocytes[ 147 , 148 ] and their characteristic cytokine profiles.[ 149 ] Terminally, there are as yet no long‐term chronic models available that can discriminate between different disease severities or disease stages of IBD.
6. OoC for IBD Treatment Evaluation
Models that can accurately recapitulate IBD, including both molecular and phenotypical hallmarks, can also be utilized to investigate the effectiveness of IBD treatments.
IBD patients are usually treated with 5‐aminosalicyclic acids, corticosteroids, immunomodulators, biologics and different small molecules to counteract the inflammation.[ 267 ] Nevertheless, many patients show a primary nonresponse or a secondary loss of response to these drugs.[ 268 ] Up to 10% of patients with UC finally have to undergo surgical colectomy due to a medically refractory flair or due to the diagnosis of dysplasia or malignancy as a consequence of longtime inflammation.[ 269 , 270 ] Up to 80% of CD patients require surgery during their lifetime, mainly because of fibrostenotic disease or fistulas.[ 271 , 272 ] However, all these options do not provide curative treatment or disease prevention.
Due to the close relationship between host and microbiota and the aforementioned alterations in microbiota composition in IBD, novel treatment approaches aim at improving the intestinal microecology (Figure 4A).
Figure 4.
Novel treatment strategies for IBD using IoC platforms. A) Schematic overview of treatment strategies in OoC IBD models to improve the intestinal microecology. OoC models can interfere with IBD conditions with different strategies. Prebiotics such as dietary fibers from nutrition act as substrates for beneficial bacteria in the intestine to promote their growth, thereby restoring the microbial balance. Probiotics such as Lactobacillus, Bifidobacterium, and synthetically engineered strains are administered within OoCs as therapeutic agents to reduce inflammation and to restore the intestinal barrier integrity. Postbiotics, for example SCFAs, secondary bile acids, and tryptophan metabolites, are produced by living microbiota via metabolization and exert anti‐inflammatory and regenerative functions, which can be evaluated in OoCs. B) OoC platforms can facilitate the transition toward personalized medicine in IBD treatment. Incorporating patient‐derived cell sources such as immune cells from the blood or intestinal epithelial cells from biopsies enhances the physiological complexity of the model and their predictability. OoCs further allow testing of fecal microbiota transfer as personalized treatment for IBD or the perfusion of fecal filtrate suspensions to investigate metabolite profiles. Created with BioRender.com.
6.1. Microbiota‐Based Approaches: Pre/Pro/Postbiotics
Prebiotics are fermentable carbohydrates, such as oligosaccharides and inulin, that can improve intestinal barrier functionality, promote growth of beneficial bacteria, and interfere with inflammatory conditions.[ 273 , 274 , 275 , 276 ] Clinical data already demonstrated evidence that high inulin intake can control inflammation in UC patients with pouchitis after colectomy.[ 277 ] A contemporary study highlighted that inulin, which can be metabolized to SCFAs by microbes, can be encapsulated with the probiotic E. coli Nissle 1917 (EcN) within microparticles for improved delivery.[ 278 ] Subsequent fermentation of the inulin gel increased the probiotic colonization in a gut‐on‐chip model containing Caco‐2 cells. In addition, these microparticles efficiently ameliorated experimental colitis in mice by enhancing SCFA production to exert anti‐inflammatory effects. A further study by Jing et al. demonstrated that applying chitosan oligosaccharide in the intestinal compartment of an IoC (mimicking oral administration) protected intestinal and vascular barrier integrity by inhibiting E. coli 11775 invasion in a DSS‐induced enteritis model.[ 247 ] Chitosan oligosaccharide treatment promoted the expression of a functional mucus layer and reduced NF‐κB‐dependent inflammatory signaling and TLR4 expression by limiting E. coli invasion and adherence.
Probiotics are living microorganisms known to transmit beneficial effects to the host. E. coli Nissle 1917 is a probiotic strain, which is successfully used in clinical practice to maintain remission in patients with UC as effectively as with standard mesalazine.[ 279 ] This probiotic is a standard treatment option and reimbursed by the health insurances. Incorporating individual probiotic strains or mixtures has already been established in OoC IBD models due to their protective effects on barrier integrity and mucosal inflammation. A fundamental study indicated that VSL#3, a bacterial mixture containing eight probiotic strains such as Lactobacillus spp., Bifidobacterium spp. and Streptococcus spp., had protective functions in an inflamed gut‐on‐chip model.[ 245 ] By applying the mixture in this Caco‐2/PBMC OoC model, damage induced by a pathogenic enteroinvasive E. coli infection was partly reduced and barrier integrity restored. Furthermore, this study was continued by inducing characteristics of UC with DSS and LPS in a Caco‐2/PBMC OoC model.[ 246 ] Interestingly, the beneficial effects of VSL#3 were temporal dependent. While the administration of pretreatment before the induction of IBD reduced the inflammatory cytokine response and increased mucus production and barrier function, postadministration of VSL#3 exacerbated the disease due to microbial translocation and unfavorable PBMC stimulation. So far, it has been unclear why probiotic administration has only strong beneficial effects for patients in the remission stage but not for patients with active IBD.[ 280 , 281 ] These results provide very important suggestions for the timing of probiotic treatment and possible side effects during severe disease stages and highlight the opportunity for real‐time monitoring in OoC.
Probiotics have been further coadministered with prebiotics to synergistically promote probiotic colonization and growth and foster the production of beneficial postbiotics. Using such a synbiotic consisting of Bifidobacterium longum and oligofructose‐enriched inulin reduced tissue lesions and release of proinflammatory IL‐1β and TNF‐α in patients with UC.[ 282 ] Most current studies show decreased cytokine release, increased SCFA production, reduced pathogen growth, and maintenance of immunological homeostasis when synbiotics are administered to UC patients.[ 283 ]
Recent studies have also shown an adaptation of an aerobic and anerobic interface in their OoC models to provide near‐physiological culture conditions for probiotic cocultures. Here, it was shown that probiotic Lactobacillus plantarum HY7715 suppressed an LPS‐induced decrease in barrier integrity in a Caco‐2/HUVEC chip model.[ 284 ] Additionally, a pathomimetic model using Caco‐2 cells was stimulated with a proinflammatory cocktail to induce inflammatory disease conditions.[ 285 ] Herein, the addition of either the probiotic Lactobacillus rhamnosus GG or VSL#3 induced epithelial barrier restoration, increased mucin 2 (MUC2)‐secreting cells, and suppressed inflammatory signaling molecules such as p65, pSTAT3, and MYD88 preventing potent cytokine release.
Another approach is directly administering bacteria‐derived postbiotics, such as SCFAs, in OoC models. In particular, SCFAs contribute to regulating the mucosal immune response by stimulating, for example, regulatory T cell (Treg) differentiation, neutrophil migration, antibody secretion by B cells, M2 macrophage polarization, and maintenance of the intestinal barrier function.[ 286 ] Therefore, a physiomimetic gut‐liver model was used to explore the effects of SCFA on the gut‐liver axis.[ 287 ] Exposure of SCFAs in the gut model reduced innate immune cell activation, mainly through the downregulation of peroxisome proliferator‐activated receptor and NF‐κB signaling, and improved metabolic function in the liver model. However, in models with circulated CD4[+] T cells, SCFAs aggravated liver injury by inducing increased differentiation of Treg and Th17 cells toward Th1 and Th2 effector cells with increased IL‐13 production. Remarkably, SCFAs effectively reduced inflammation in the absence of acute T cell inflammation, leading to the speculation that SCFAs can exert pleiotropic effects upon different severity stages of UC. In relation to this, the efficacy and therapeutic effectivity of SCFAs can vary considerably across different disease stages of IBD. Beneficial effects on intestinal homeostasis, such as maintaining microbiota diversity, anti‐inflammatory responses, and preserving the integrity of the mucosal barrier, may be more effective in preventing the onset of the disease. In contrast, during active inflammation, SCFA uptake and oxidation can be impaired,[ 106 , 288 , 289 ] leading to a dampening of the beneficial effects and potential resistance to SCFA treatment. Moreover, the paradoxical immunomodulatory effects of SCFAs might depend on the administered concentration and immunological milieu. While SCFAs promote anti‐inflammatory responses by stimulating IL‐10‐producing T cells and expanding Tregs during homeostasis,[ 286 ] they promote CD4[+] T cell differentiation into proinflammatory effector cells under inflamed conditions potentially exacerbating the active inflammation.[ 287 , 290 , 291 ] Nevertheless, the positive effects of SCFAs during the remission phase of IBD by preventing inflammatory flares have already pointed out.[ 292 ] These fluctuations in the efficacy microbiota‐based therapies across different disease stages need careful consideration prior to clinical implementation.
These models highlighted the role of probiotic microbiota and microbial‐derived metabolites in interfering with IBD disease conditions. A very exciting and recent development is the generation of next‐generation synthetic microbial communities, particularly probiotics, to modulate both the intestinal microecology and the release of therapeutic molecules. These engineered strains can either act as biosensors for active IBD, secretors of biotherapeutics, or as sense and response systems following biomarker release.[ 293 ] Studies by Nelson et al. in a gut‐on‐chip model already outlined the potential of engineered synbiotic strains from E. coli Nissle 1917 to convert phenylalanine into trans‐cinnamic acid to treat phenylketonuria[ 294 ] and induce tryptophan metabolism after sensing cortisol for increased neurotransmission and modulation of cognitive function.[ 295 ] These models delineated the potential of synthetic microbiota administration to develop IBD‐targeted therapies further. While many synthetic strains have already been beneficial in treating IBD in mice,[ 293 , 296 , 297 ] they should be further tested and validated in human chip‐based models to facilitate clinical translation.
These applications highlight the use of IoC models as a platform for IBD treatment evaluation. OoC models offer a promising platform for investigating host–microbiota interactions due to the perfusion, real‐time monitoring, and experimental controllability.
Although a complete recapitulation of the human physiological complexity is far away, key features of the intestinal microenvironment, such as crypt‐ and villus‐like morphology, enhanced barrier functionality, improved cell differentiation and polarity, can be replicated in perfused OoC models in a more in vivo‐like manner compared to static 2D cultures.[ 23 ] Systemic responses as in vivo cannot be fully depicted to date. However, multiorgan models[ 287 , 298 ] have already provided the first insights into crossorgan reactions. Compared to holistic animal models, OoC models permit the uncoupling of individual factors and mediator cell types, which are necessary to establish causal links for IBD and to find therapeutic intervention points.
The integrated perfusion in OoC devices further provides independent treatment administration routes into the intestinal lumen (oral) and blood circulation (intravenous). Nonetheless, it should be mentioned that treatment application and bioavailability of drugs in OoC models can be influenced by adsorption to the chip periphery (chip base body, microfluidic equipment, tubing).[ 299 ] Therefore, materials should be tested for their adsorptive capacities and free drug, or metabolite concentrations should be determined to exclude excessive surface binding.
To date, there is no validated method for IBD treatment evaluation in OoC models and the reproducibility varies due to different cell sources and biological complexities, varying OoC systems, the origin of microbiota strains, and low to medium throughput. Moreover, the technical complexity can limit the widespread adoption of OoC models. In terms of protocol harmonization, material consistency, cell source standardization, throughput, and assay standardization, commercial adoption and collaboration across multiple stakeholders could standardize these processes in a more reproducible way.
6.2. Intestine‐on‐Chip to Advance Clinical Translation
Despite the seminal insights obtained using these models, clinical translation is extremely difficult due to the lack of systemic complexity and full recapitulation of the human microbiome. The combination of microbiota with patient‐derived material to generate an autologous model containing different cell types from the same donor and from different intestinal regions could contribute to a deeper understanding of in vivo processes Figure 4B.
6.2.1. Fecal Microbiota Transplantation
The integration of stool samples such as fecal microbiota transplantation (FMT) into OoC models has been discussed to emulate interactions with the human microbiome. FMT involves the transfer of stool from a healthy donor into the intestine of a diseased recipient to restore a healthy and balanced microbiome. FMT has already been approved to treat C. difficile infection in patients and has already been tested in clinical trials as a potential IBD therapy. While clinical trials for CD showed negative data in terms of efficacy, accumulating evidence suggests FMT as a valuable treatment option for UC.[ 300 , 301 ] However, there is variable patient responsiveness and questionable long‐term safety. This is mainly due to significant differences in the microbial diversity of the donor, the immune responsiveness of the recipient, variable stool preparations, and the administration route and timing.[ 302 , 303 ] Furthermore, increased contamination risks and the occurrence of infections were reported. These questions might be addressed by pretesting FMT in patient‐derived IoC models to uncouple specific host–microbiota interactions and potential time points for clinical administration and to define effective inoculation densities for the patient. The successful cocultivation of intestinal Caco‐2 cells and a donor‐derived fecal microbiome has already been demonstrated for up to two days in an IoC model[ 304 ] without affecting the epithelial barrier functionality.
Recently, a translational framework for integrating patient‐derived samples and the microbiome for disease modeling and FMT validation was proposed.[ 305 ] However, keeping the microbial communities viable and stable over a longer period of time greatly depends on the right culture conditions and microbial species, which could be a limiting challenge in these platforms. Therefore, the application of stool filtrates[ 306 ] containing noncellular components of intestinal microbes like proteins, metabolic products, DNA, and bacteriophage signatures could be a possibility to investigate the influence of microbial metabolites or other mediators on host integrity and immunity without transferring undefined bacterial strains. Combining this approach with metabolomic analysis could help to identify specific microbial metabolites, which are essential to prevent inflammatory disease conditions and restore the intestinal health state.
6.2.2. Design of Personalized Therapies Using OoC
As previously discussed, OoC models can be built using primary cells derived from adult stem cells or iPSCs instead of cell lines. These personalized OoCs could serve as “mini‐avatars” to test personalized therapies for IBD patients, for example, immunomodulatory drugs.[ 258 ] The cultivation of the organoids after fragmentation as a 2D monolayer with adjacent vascularization has already been demonstrated in several studies[ 249 , 253 , 307 ] and enables the application of pharmaceutical drugs into the OoC perfusion without injection into the enclosed lumen. Collectively, OoC models consisting of patient‐derived intestinal cells could be useful to assess patient‐specific drug responses and the influence of genetic polymorphisms on drug metabolism and pharmacokinetics.
Taking the model a step further, the patient's microbiome could be obtained from stool samples and integrated into the IoC as it was already demonstrated for a Caco‐2‐based IoC.[ 304 ] By including controlled microbial communities, it is also possible to identify causative links of host–microbiota alterations in IBD. This has already been demonstrated in cell line‐based IoC models,[ 246 , 247 ] but not yet in organoid‐based chip models. Since it is well known that the intestinal microbiota is also involved in drug metabolism,[ 308 ] OoC models with integrated microbiota could further be leveraged to study both efficacy and toxicity of IBD drugs or drug metabolites. Herein, it was shown that, for example, an inactive metabolite of the cancerogenic drug irinotecan was metabolized and reactivated by E. coli into its toxic metabolite in a gut‐liver chip model.[ 298 ]
Circulating immune cells could further be isolated from the patient's blood and integrated into the chip to emulate the complex immune responses that occur during IBD more accurately. The combination of organoids with endothelial cells and immune cells and the patient's microbiome enables the possibility of preclinical drug screening and testing of alternative approaches such as FMT in an in vivo‐like setting.
However, OoC standardization and validation are essential for integrating IBD models into biomedical research, drug development, and clinical translation.[ 309 ] In terms of internal model robustness and reproducibility, the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ Consortium) Microphysiological Systems Affiliate represents a consortium of pharmaceutical and biotechnological companies to implement and qualify OoC models. Particularly the publication of the organotypic paper series provides considerations for standardized internal characterization and validation of OoC for gastrointestinal toxicity and diseases,[ 310 ] highlight the importance to replicate enterocyte/immune/microbiome interactions in IBD and providing standardized function and assay suggestions, such as measuring intestinal barrier function, mucus secretion, and immune reactivity. This paper series provides a comprehensive basis for organ and drug toxicity modeling, nevertheless it should be extended with recommendations for disease modeling and induction, technical OoC parameters, and feasible assays to assess clinical biomarkers regarding IBD.
To address general standardization and validation of OoC, the CEN‐CENELEC Focus Group Organ‐on‐Chip has just recently published a new standardization roadmap.[ 311 ] This includes the development of terminology standards, reporting guidelines, technical key aspects, and alignment with regulatory frameworks to advance the OoC field. Furthermore, the focus group has recommended a submission to the International Organization for Standardization, which, once approved, could promote regulatory compliance and accelerate the translation of OoC research for the healthcare sector.
In terms of external validation and predictability, the Microphysiological Systems Database contains in vitro and in vivo data of chemical compounds,[ 312 ] which allows extrapolation and validation of data generated from OoC models to the existing reference data. This approach could be very relevant to benchmark results from IBD treatment data in OoC models to established in vivo models and clinical data.
While these developments are still ongoing, they will be instrumental to increase the validity of OoC‐based IBD models and the translational potential of personalized treatment approaches, which could improve the quality of life of the patients.[ 313 , 314 , 315 ]
7. Conclusion
IBD is a very complex multifactorial disease with currently only symptomatic but no curative treatment. Despite many approaches, it remains very challenging to fully reproduce IBD pathophysiology both in vitro and in vivo to test new therapeutic approaches in a human‐relevant and physiological manner. A major hallmark of IBD is microbiota dysbiosis, which is particularly difficult to study in conventional in vitro models. Perfused OoC models have proven to be a suitable platform for improved host–microbiota culture in vitro. In addition, these models can recapitulate different IBD phenotypes and associated clinical hallmarks. IoC models combine the advantages of reductionistic in vitro models but can provide the desired complexity by integrating multiple human‐derived cells, such as intestinal organoids. Further, these models offer the opportunity for host–microbiota coculture and real‐time investigation in a controllable and prolonged manner. Therefore, OoCs can be regarded as complementary translational tools to question results from animal models and, consequently, to improve IBD treatment efficacy.
These characteristics put OoCs in the spotlight to test experimental treatment strategies such as probiotics, prebiotics, and postbiotics or even synthetically engineered strains for specific disease targeting. Moreover, OoC models can also be leveraged to advance translational medicine, for example, by combining biopsy‐derived cell material and FMT in OoC platforms. Pretesting microbiota transfer in human cell‐based OoC could not only enhance the prediction of effectiveness but could further be used to unravel contributing factors and microbial products of the transferred microbiota to interfere with IBD conditions. Although standardization and validation of these approaches are still in process, there is great future potential to improve both fundamental mechanistic research and treatment discovery for personalized medicine.
Conflict of Interest
A.S.M. holds equity in Dynamic42 GmbH. A.S.M. consults Dynamic42 GmbH. B.H. consults CureVac. The rest of coauthors declare no conflict of interest.
Acknowledgements
This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy—EXC 2051—Project‐ID 390713860 (R.A.R., B.H., A.S.M.). R.A.R. acknowledges project funding from the Carl Zeiss Foundation. T.K. was funded by the Thüringer Aufbaubank (Germany)—2021 SD0018 and 2023 SD0029. M.S.G. was supported by the DFG Emmy Noether Program (project no. 434385622/GR 5617/1‐1). M.S.G. and B.H. are supported by the DFG Collaborative Research Centre (CRC)/Transregio (TRR) 124 FungiNet with project number 210879364 (subprojects C1 and C2). B.H. is further supported by the DFG Priority Program SPP2225 “Exit strategies of intracellular pathogens (project 446404928), the Leibniz Association Campus InfectoOptics SAS‐2015‐HKI‐LWC and the Leibniz Research Alliance INFECTIONS, grant number SAS‐2021‐1‐FZB.” A.S.M. is further supported by the DFG Collaborative Research Centre CRC 1278 “PolyTarget” (project ID 316213987, subproject Z01, A05). A.S.M. and B.H. are further supported by the German Federal Ministry of Education and Research (BMBF) within the funding program Photonics for Life Sciences, Leibniz Center for Photonics in Infection Research (LPI), Subproject LPI‐BT4, contract numbers 13N15714 and 13N15716. J.S. is supported by the Hermann Strauss Scholarship from the German Crohn's Disease/Ulcerative Colitis Association (Deutsche Morbus Crohn/Colitis ulcerosa Vereinigung; DCCV e.V.).
Open access funding enabled and organized by Projekt DEAL.
Biographies
Tim Kaden is a doctoral candidate at Dynamic42 GmbH and Institute of Biochemistry II, Jena University Hospital, Germany. He received his B.Sc. in Biology and his M.Sc. in Molecular Medicine from the Friedrich‐Schiller‐University Jena. He evaluated drug‐induced liver injury in a microphysiological liver‐on‐chip model for his Master thesis project. His research focuses now on intestine‐on‐chip models to investigate inflammatory bowel disease (IBD), development of IBD treatment strategies, host–microbiota interactions, and sensor integration for real‐time measurements in organ‐on‐chip.
Raquel Alonso‐Román is a doctoral candidate at the Leibniz Institute for Natural Product Research and Infection Biology—Hans Knöll Institute, in Jena. She received her B.Sc. in Biotechnology from the University of Zaragoza and her M.Sc. in Biomedicine from the University of Barcelona. She has worked on the interphase between 3D in vitro models of the human intestine and microbiology to research infections in a physiologically relevant manner and explore therapeutic options. Her main focus is the human fungal pathogen Candida albicans.
Johannes Stallhofer is a consultant in the Department of Gastroenterology, Hepatology, and Infectious Diseases at Jena University Hospital. A board‐certified specialist in internal medicine, he earned his medical degree from Ludwig‐Maximilians‐Universität (LMU) Munich, where he was awarded a scholarship from the German National Academic Foundation. Since completing his M.D. thesis, his research has focused on the pathophysiology and treatment of inflammatory bowel diseases (IBD). For his work in this field, he received the Ismar Boas Prize from the German Society for Gastroenterology, Digestive and Metabolic Diseases (DGVS) and the Hermann Strauss Scholarship from the German Crohn's Disease/Ulcerative Colitis Association (DCCV).
Mark S. Gresnigt obtained his Ph.D. in Medical Sciences from Radboud University Nijmegen (2015). Subsequently, he spent research periods at the Aberdeen Fungal Group (2015) and the University of Colorado (2015 and 2016) and worked on immunometabolism in fungal infections and sepsis at Radboud University. With an Alexander von Humboldt fellowship (2018) he joined the lab of Bernhard Hube at Leibniz‐HKI, where he established a C. albicans‐infection‐on‐chip in collaboration with Alexander S. Mosig. In 2020, he started an Emmy Noether Junior Research Group on Adaptive Pathogenicity Strategies at the Leibniz‐HKI, working at the interface between immunology and biology of candidiasis.
Bernhard Hube earned his Ph.D. in Microbiology at the University of Göttingen, followed by postdoctoral work at the University of Aberdeen and the University of Hamburg. In 2000, he became Head of Division “Mycology” at the Robert Koch Institute Berlin, later joining Friedrich Schiller University Jena as Professor in 2006 and heading the Department of Microbial Pathogenicity Mechanisms at Leibniz‐HKI. He has co‐authored >300 publications dealing with human pathogenic fungi, particularly Candida species. He discovered one of the first virulence genes, encoding a secreted aspartic proteinase, in C. albicans, while his recent research focuses on the fungal peptide toxin candidalysin.
Alexander S. Mosig is a research group leader at Jena University Hospital at the Institute of Biochemistry and the Center of Sepsis Control and Care. He studied Biochemistry and Molecular Biology at Friedrich Schiller University Jena and holds a Ph.D. in Cell Biology. His research focuses on developing Organ‐on‐Chip systems to study host–microbiota interactions, with a particular emphasis on the host immune system. His work aims to advance the understanding of inflammatory and infectious diseases and improve drug testing platforms for developing new therapeutic strategies.
Kaden T., Alonso‐Román R., Stallhofer J., Gresnigt M. S., Hube B., Mosig A. S., Leveraging Organ‐on‐Chip Models to Investigate Host–Microbiota Dynamics and Targeted Therapies for Inflammatory Bowel Disease. Adv. Healthcare Mater. 2025, 14, 2402756. 10.1002/adhm.202402756
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