Inflammatory Bowel Disease
ICD-10 Code K50-K50.9 and K51.0-51.9
What is the clinical spectrum of Inflammatory Bowel Disease?
Inflammatory Bowel Disease (IBD) is a chronic autoimmune disorder that includes two main disease forms, ulcerative colitis (UC) and Crohn’s disease (CD).
It is characterized by repetitive episodes of inflammation of the gastrointestinal tract (GI), intestinal barrier dysfunction, and increased risk of colitis-associated cancer, posing a significant social, economic and personal burden.
Prevalence of IBD in the Europe ranges from 1.5 to 331 per 100,000 population for CD and 2.4 to 432 per 100,000 population for UC. CD generally has a higher mortality rate compared to the general population and to UC 1.
IBD’s forms UC and DC differ in their location within GI, in clinical features and in therapeutic strategy 2, 3, 85. While CD can affect any part of the GI tract, UC is limited to the colon and the rectum.
Within CD, there is heterogeneity in location of pathological changes, producing variation in the disease phenotype.
For example, when it is the ileum that is impacted, complications such as stricture can arise - narrowing in the intestine caused by scar tissue, that forms as a result of long-term inflammation and healing cycles - producing symptoms of abdominal pain, cramps, bloating, nausea, constipation, and vomiting.
In addition, individuals with CD can also present with penetrating disease behavior, which is a severe form of CD characterized by inflammation masses across entire thickness of intestinal wall, abscesses, and fistulas - abnormal connections between different parts of the intestine and other tissues.
Stricturing and penetrating disease behaviour are typically present in 13–35% of CD patients at diagnosis.
When it is the colon that is impacted, extensive inflammation and ulceration may occur, causing abdominal pain, diarrhea, weight loss, and rectal bleeding.
In UC, the distribution of pathological alterations within the colon often correlates with the degree of severity of symptoms and treatment approaches.
In the proctitis form of UC, the inflammation is limited to the rectum, while in left-sided colitis the inflammation extends up to the splenic flexure. Pancolitis, in which the inflammation affects the entire colon, is considered as the most severe form of UC and is associated with a higher risk of complications.
In both CD and UC, it is the chronic inflammation that drives progression to severe clinical features, with about 25% of CD/UC patients progressing to severe forms after 5 years.
Ophthalmological, rheumatological, cutaneous, and hepatobiliary extra-intestinal manifestations (EIM) may also occur in parallel with the intestinal disease. In CD, EIMs are associated with female gender and age, whereas in UC they are associated with pancolitis.
The severity of IBD also varies according to the weight of genetic and environmental factors.
The subgroup of patients known as very early onset IBD (VEO-IBD), diagnosed in children younger than 6 years old, is commonly associated with a family history of shared monogenic risk factors 4. Children with VEO-IBD often experience poor growth, higher rates of hospitalization and higher rates of medication failure.
Sporadic IBD, that occurs without a clear genetic predisposition or family history, typically presents later in life than familial IBD. Sporadic IBD with an older-onset - usually after the age of 60, tends to have a less severe disease course and lower rates of EIMs compared to VEO-IBD 5.
What do we know about the etiology of Inflammatory Bowel Disease?
Inflammatory bowel diseases [IBD] - ulcerative colitis (UC) and Crohn’s disease (CD) are believed to be multifactorial and polygenic in origin, although their precise mechanisms remain to be unveiled.
The understanding that CD and UC are associated with dysregulated immune response, intestinal dysbiosis, and environmental triggers was largely derived from clinical trials, epidemiological studies, genetic susceptibility studies, stool and biopsy samples. Thus, IBD is believed to arise and progress through a complex interaction between internal (genetic) and external (diet, hazardous substances, medication, infection, smoking) triggering and contributing factors.
Genetic triggering factors
Genome-wide association studies (GWAS) have identified over 200 risk loci and more than 300 genes associated with IBD, such as mutations in nucleotide-binding oligomerization domain containing 2 (NOD2) gene, that can lead to a weakened immune response to bacterial infections, and mutations in genes regulating the IL-23 and IL-17 pathways that can result in mounting of an overactive Th17 response 6.
Comparison of concordance rates of UC and CD in monozygotic and dizygotic twins indicate that the genetic trait is less important in UC than in CD. Genes notably associated with UC play a role in major histocompatibility complex, immune regulation and epithelial barrier 7.
Nonetheless, the so far identified genetic variants in CD and UC alone are insufficient to predict disease extent, progression, EIM, and response to therapies 8.
The hypothesis that dysregulation of epigenetic mechanisms through environmental exposure plays a part in CD and UC is yet to be thoroughly explored.
Diet triggering factor
Dietary residues absorbed in the small intestine can influence the host's immune system directly via the nutrient content or indirectly via the gut microbiota 9, 10, 11. An unbalanced diet can cause damage to intestinal barrier directly through excess intake of saturated fats/refined sugars, deficiency in vitamins/minerals, and production of reactive oxygen species (ROS), further triggering an inflammatory response. By promoting or inhibiting the growth of microorganisms, nutrition affects the composition of the microbiota and the concentration of metabolites in the gut.
Microbiome dysbiosis contributing factor
A prospective inception cohort study of paediatric CD patients has allowed to predict the risk of stricturing and penetrating complications based on a combined analysis of genotypes, antimicrobial serologies, ileal gene expression, and faecal microbiota 12.
The study has identified age, race, disease location, and antimicrobial serologies as risk factors. It also showed that Ruminococcus gnavus was implicated in stricturing complications and Veillonella in penetrating complications, likely through their production of pro-inflammatory polysaccharides 13.
In UC, short-chain fatty acid (SCFA)-producing Ruminococcaceae and Lachnospiraceae species were shown to be depleted, whereas pro-inflammatory Escherichia coli and Fusobacteriaceae have been found in abundance 7.
Notably, SCFA metabolites of the gut microbiome enhance the function of tight junction proteins like ZO-1 and occludin, promote the expression of genes involved in maintaining the intestinal barrier by inhibiting histone deacetylases, stimulate MUC2 production, reduce the production of pro-inflammatory cytokines like IL-1β and IL-18 by inhibiting the activation of the NLRP3 inflammasome, regulate autophagy, promote differentiation of regulatory T cells (Tregs), and reduce differentiation of pro-inflammatory T helper 17 (Th17) cells 14.
Epithelial barrier damage contributing factor
Dysregulation of transcription of junctional proteins that are essential for epithelial barrier function, such as E-cadherin, β-catenin, and claudins, was found in both UC and CD with varying degrees of alterations that reflect differences in disease pathology 7.
Intestinal goblet cells produce mucus that forms a protective barrier against pathogens/toxins and helps modulate the immune response in the gut. IBD-associated genetic mutations, dysbiosis, prolonged use of antibiotics, persistent inflammation, and high fat/sugar diets can damage goblet cells and reduce mucus layer production, composition and integrity 15.
In both UC and CD, mucosal layer thickness was found to decrease, leading to increased susceptibility to inflammation and damage. It is, however, unclear whether the etiology of this pathological change - a decreased synthesis of mucin 2 or an increased mucin degradation - is the same in both IBD forms.
When the intestinal barrier is damaged, innate immune cells, including neutrophils, macrophages and dendritic cells, are recruited from the circulation through chemotactic gradients formed by cytokines, chemokines, growth factors, and bacteria-derived molecules such as SCFA.
Immune response contributing factor
Dysregulation of both innate and adaptive immune responses is believed to be involved in pathogenesis of IBD, however, the exact unfolding and chronology of events is unknown.
UC is associated with a Th2 response, whereas CD is characterized by a Th1/Th17 response, which explain the distinct clinical presentations and complications of UC and CD 7.
T-helper 2 (Th2) cells release IL-4, IL-5, and IL-13 cytokines, associated with a localized and superficial mucosal inflammation, while Th1 and Th17 cells release IFN-γ and TNF-α cytokines driving a deep transmural inflammation.
Analysis of intestinal biopsy samples from IBD patients show increased expression of complement pathways in CD but not in UC patients 16. This increase likely reflects the body's heightened innate immune response against pathogens and is consistent with CD’s bacterial-driven pathology and transmural inflammation. Excessive complement activity can amplify inflammation, contributing to complications and penetrating disease behaviour in CD.
In both UC and CD patients, the abundance of neutrophil and neutrophil extracellular traps (NET)-associated proteins in patients’ intestinal biopsies was increased 17, 18.
In homeostasis, neutrophils, recruited from the circulation following intestinal barrier damage, participate in the elimination of pathogens through phagocytosis, degranulation, ROS generation, and the release of NET.
However, in conditions typical of IBD - chronic inflammation, genetic mutations affecting apoptosis and clearance of neutrophils, and exposure to toxins - the process of subsequent clearance of neutrophils can be disrupted. In this context, neutrophils can cause injury to the epithelial barrier by releasing neutrophil elastase, matrix metalloproteinases, pro-inflammatory cytokines (IL-8, TNF-α, IL-1β), leukotriene B4, ROS, and NET, that excessively degrade the extracellular matrix (ECM) in the intestinal lining and mount an inflammatory response.
In homeostasis, the intestinal microbiota inhibits the migration of antigen-loaded CX3CR1high intestinal macrophages to mesenteric lymph nodes and presentation to T cells, thereby ensuring immune tolerance towards commensal bacteria.
Gut microbiome dysbiosis in IBD can disrupt this tolerance through change in metabolite-mediated signaling and increase in pro-inflammatory activation of CX3CR1high intestinal macrophages.
Improperly activated macrophages promote myofibroblast-mediated excess ECM production that is characteristic of intestinal strictures in CD 19, 20.
Similarly to other innate immune cells, intestinal dendritic cells (DC) promote tolerance to microbial and dietary components through pattern recognition receptors, but can be dysregulated in IBD through dysbiosis, diet/lifestyle and microbial dysbiosis.
Intestinal DC from UC patients show enhanced expression of CCR9, a chemokine receptor involved in the migration of immune cells to the gut and thymus, and of β7 integrin, a cell adhesion protein involved in the migration and homing of immune cells to the gut-associated lymphoid tissues.
Furthermore, in comparison to healthy individuals, DC of CD patients express more CD40, TLR2 and TLR4 receptors, and release more IL-6 and IL-12 cytokines, which may contribute to altered microbial recognition 21, 22.
In these circumstances, intestinal DC can participate in an inappropriate immune responses against harmless substances, release inflammatory cytokines such as IL-12, IL-6, and IL-18, and mediate the production of Th1 cytokines (IL-6, IL-8, and TNF-α) that exacerbate the auto-immune inflammation.
Oxidative stress contributing factor
Arising from a complex interplay of immune dysfunction, epithelial barrier disruption, microbiome dysbiosis and environmental/lifestyle triggers, oxidative stress contributes to a pathological loop that exacerbates the intestinal inflammation and tissue destruction in IBD 25.
Oxidative stress is generally defined as an overwhelm of the finely-tuned balance between reactive oxygen species (ROS) and antioxidant defenses in favour of ROS, leading to a series of dysfunctions 26. Excessive ROS production can cause oxidative modification of proteins/lipids, impairing the intracellular signaling pathways involved in stress response, immune modulation and cell survival (NF-κB, MAPK, PI3K/Akt).
By activating the expression of genes that code for antioxidant enzymes, such as glutathione S-transferases (GST), heme oxygenase-1 (HO-1) and superoxide dismutase (SOD), and by reducing NF-κB-driven transcription of proinflammatory cytokines, the epithelial transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) enhances intestinal wound healing and protects from apoptosis during oxidative injury 27. An increased level of Nrf2 protein was detected in epithelial cells of inflamed colonic biopsies derived from UC patients 28, suggesting an attempt to counteract oxidative damage.
Excessive non-specific oxidation of the Nrf2-Keap1 protein complex by ROS can lead to suppression of Nrf2 protective activity. The gene encoding NRF2 is highly polymorphic and its several polymorphisms have been associated with the development of UC 29.
How similar are human and animal digestive systems?
This is not an exhaustive list of species-specific differences, nor can one be made given their unknown full extent, but rather an example of how these differences impact the face, construct, predictive, and intrinsic validity of animal models.
Not all species-specific differences can be accounted for in animal models, as there are hundreds of them, their relevance for inflammatory bowel diseases (IBD) is unclear, and their interaction with other organ systems in the animal model makes it difficult to predict how they would have behaved within the human system.
Although gastrointestinal tracts in humans and species commonly used in animal experimentation are grossly composed of similar organs, they also present with specificities in anatomy and physiology that reflect distinct evolutionary paths, dietary needs, feeding patterns and digestive strategies.
Species-specific differences in intestinal structure, organisation, histology and function lead to inter-species divergences in microbiome/mucus/bile acids composition, surface areas interactions, absorption of orally administered compounds, susceptibility to IBD, and responses to treatments.
Species-specific differences in gastrointestinal anatomy and physiology
Species-specific differences in gastrointestinal (GI) anatomy and physiology are shaped by dietary strategies, evolutionary pressures, and metabolic demands 23, 24. Since IBD affects nutrient absorption, immunity, microbiome, and mucosal healing in different regions of the GI tract, these physiological and anatomical differences have major implications for the ability of model organisms to faithfully recapitulate IBD.
For instance, IBD is associated with a decreased gut motility, contributing to microbial dysbiosis and inflammation 30. In humans, the hormone motilin produced in the duodenum triggers periodic contractions to clear residual food particles and prevent bacterial overgrowth. However, rodents, who lack the gene encoding motilin, have different gut motility patterns compared to humans, making it difficult to gather human-relevant insights on the mechanisms of pathological alterations of gut motility 31.
Furthermore, segmental contractions in the rodent colon differ from the peristaltic waves observed in humans, impacting how external triggering factors (environmental toxins, drugs) interact with the gut mucosa and the gut microbiome.
In contrast to humans, mice and rats feed almost incessantly and mostly during the night, leading to lower pH in the rodent stomach compared to humans 32, 33. Variations in intestinal pH can influence the solubility and absorption of certain drugs and/or favor the presence of metabolizing enzymes and microbiota that are not typical for the human GI tract, resulting in inter-species differences in drug metabolism and composition of the gut microbiome 34.
Species-specific differences in gut microbiome
Qualitative and quantitative inter-species differences in GI microbiome have a major impact on the activity of drug-metabolizing enzymes, on intestinal pH, on gut inflammation, and on integrity of the gut barrier, thereby affecting predictive value of animal models of IBD.
The study of the microbiome has historically relied on mouse studies, based on coarse comparisons at higher taxonomy hierarchy level with a poorly characterized mouse GI microbiota.
However, metagenomics analysis of gut microbiome at lower taxonomy level have since shown major inter-species differences in GI microbiome composition, quantity, and function between humans and other species 35, 36, 37, 38.
Notably, the Faecalibacterium genus, that plays a protective role in IBD patients, has a high relative abundance of over 10% in humans, whereas it is hardly detected in mice. Faecalibacterium is decreased in patients with IBD, especially in ileal CD 39.
Within the same phylum Firmicutes, the genus of Roseburia, that is altered in IBD compared to healthy individuals, has a relative abundance of 5-10% in humans versus 1-5% in mice. As for the Bifidobacterium genus, of which alterations were equally observed in IBD, it has a relative abundance of 1-5% in humans and 0.01-1% in mice.
Initiatives were taken to humanize mice with human microbiota and to inter-cross inbred strains. However, it was found that the GI microbiome in mice varied immensely depending on the housing conditions, even more so than on the genetic background 35.
Previously, DNA sequencing of fecal samples of mouse strains of diverse genetic, provider, housing and diet backgrounds, and comparison to the human gastrointestinal genome, has demonstrated that only 4% of mouse GI microbial genes were shared with human GI microbial genes 40.
Rats tend to have a higher abundance of the phylum Bacteroidetes compared to mice. Mice, on the other hand, have a higher abundance of the family Muribaculaceae within the phylum Bacteroidetes 32.
Although the gut microbiota in humans was closer to that of NHP than that of rodents, significant differences in microbiome remain between the two species. For example, African green monkeys show an opposite microbial taxa and genes responses to a typical high-protein, high-fat, low-fiber Western diet compared to humans, suggesting that NHP are not an adequate model for studying the effect of the human gut microbiota on host metabolism and microbiome-associated human diseases 41.
The so-called human microbiota-associated (HMA) mouse models do not replicate patients' microbiome 42, possibly as a result of adaptations of microbes to the mouse intestinal environment. Human-based research has shown that the gut microbiome intervenes in shaping both the innate and adaptive immune system from infancy through adulthood through human host-specific interactions between human host-specific immune system and human host-specific microbiota.
Therefore, it is not surprising that HMA mice have a low immune maturation with reduced numbers of adaptive and innate intestinal immune cells when compared with mice that harbor a murine microbiota. Since the engrafted human microbiota in mice does not recapitulate the dysbiosis nor the immune responses characteristic of IBD, the use of HMA animals to model IBD is not likely to be of benefit to patients.
Species-specific differences in feeding patterns
Species-specific nutritional needs, physiology, and behavioural patterns play a role in inter-species differences in dietary content, microbiome composition, and susceptibility to IBD.
For instance, because of their higher energy demands, small animals necessitate a short food retention time. To maintain a population of gut miocrobiota all while allowing food particles to pass on rapidly, rodents partly recycle their microbiota through proximate colon folds. These folds act as mucus traps and enable the transport of the microbiome and the mucus back to the colon 43.
The need to recycle nutrients is also reflected in the practice of coprophagy, characteristic of rodents and several other species (dogs, horses, some NHP). Since coprophagy is not considered as normal behaviour in humans, extrapolation to humans of the dietary effects on intestinal inflammation in rodents may lead to inaccurate assumptions 44.
Species-specific differences in xenobiotics metabolizing enzymes
There are significant inter-species differences in expression, activity, and substrate specificity of gastrointestinal phase I (CYP) and phase II (UDP-glucuronosyltransferases - UGT, sulfotransferases - SULT) enzymes that metabolize xenobiotics 45, 46, 47.
In preclinical models, these may produce misleading hypotheses on the mechanisms of intestinal inflammation and barrier function disruption following exposure to xenobiotics (medication, smoking, pollutants, processed foods).
For instance, non-steroidal anti-inflammatory drugs (NSAID), were found to exacerbate intestinal inflammation in humans 48, by inhibiting cyclooxygenase COX1 enzymes, responsible for producing prostaglandins that stimulate the protective mucus layer production. Because of inter-species differences in activity of COX1/COX2 enzymes, the toxic effects of NSAID were not captured in animal testing.
Moreover, animals are not representative of human-relevant inter-individual variations in metabolism of xenobiotics, such as in gene polymorphisms, and expression levels of individual CYP enzymes 49. For example, human-based studies have shown that genetic polymorphisms in the main NSAID metabolizing enzyme, CYP2C, were associated with inter-individual differences in metabolic clearance, plasma elimination half-life, and plasma concentrations of NSAID 50.
Substrate specificity, tissue distribution, relative abundance, and functional activity of drug transporters were also found to vary across species 51, further compromising the predictive value of animal testing.
Additionally, inter-species differences in inflammatory responses may suppress or induce different enzymes than in humans and inter-species differences in microbiome may produce divergences in host enzyme expression.
Species-specific differences in stress response capacity
Inter-species differences in organisms' ability to detect, respond to, and adapt to stressors negatively affect predictivity of animal research, and notably in studies that investigate the role of environmental triggers (pollution, chemicals, drugs) in IBD pathogenesis.
Xenobiotics are detected by xenobiotic receptors, also known as xenosensors, that control the expression of genes encoding metabolizing enzymes and transporters by acting as transcription factors. Some of the key xenosensors expressed in human intestine and liver include the aryl hydrocarbon receptor (AhR), pregnane X receptor (PXR), and constitutive androstane receptor (CAR).
Xenosensors show marked inter-species differences in their activation by xenobiotics 52, 53. For example, in contrast to mouse PXR, rifampicin and SR12813 are potent agonists for human PXR, leading to induction of P450 enzymes followed by increased metabolism and clearance of co-administered drugs.
Humanized mouse models expressing hPXR or hCAR do not recapitulate the human-specific transcriptional and post-translation regulation, that are crucial for detoxification activity of human PXR and CAR isoforms, limiting their physiological relevance.
Generated by alternative splicing, 3 isoforms of PXR and 15 isoforms of CAR have been identified in humans. These human-specific isoforms vary in their sequence and structure, and thereby in their ligand responsiveness and activity.
The response to toxicants commonly involves activation of protective Nrf2 pathways, and subsequently, the activation of expression of genes encoding ROS-neutralizing antioxidant enzymes like GST and SOD. In the context of IBD, by activating the expression of antioxidant and cytoprotective genes, a robust Nrf2 response can help mitigate the damage from chronic inflammation 26, 27.
Some animal species, such as rats, have a more robust basal and adaptive Nrf2-mediated stress response compared to humans, contributing to erroneous conclusions on safety of drugs tested in animals 54. Since the same pathways are involved in intestinal epithelial resilience and inflammatory regulation in IBD, inter-species differences in threshold of activation and robustness of stress response are likely contribute to differences in phenotype and pathophysiology between IBD animal models and IBD patients.
While historically known for regulating detoxification of xenobiotics, PXR and CAR are also recognized as key players in endobiotic regulation 53 – bile acid detoxification, glucose and lipid metabolism, steroid hormone balance, and immune modulation. Thus, species-specific differences in endobiotic activity of various PXR and CAR isoforms are also susceptible to play a part in poor face and predictive validity of IBD animal models.
Species-specific differences in metabolic rates
Species-specific differences in metabolic rates may increase the susceptibility to IBD in experimental animals through an increased production of ROS or, on the contrary, decrease it through a better nutrient absorption and metabolism of xenobiotics.
Metabolic rates of mammals are adapted to their body sizes, physiology, diet, behaviour, environment and activity levels 55. For instance, in comparison to humans, mice, rats and rabbits have higher metabolic rates, while pigs and sheep have lower metabolic rates. Human metabolism also differs significantly from that of other primates. In spite of their higher physical activity levels, NHP have lower metabolic rates, presumably due to higher energy demands of the human brain 56.
Species-specific differences in mucins
Mucins play a key role in maintaining GI health on several levels - by providing lubrication for food and stool progression, by participating in activation/inhibition of immune signaling pathways, and by protecting the intestinal epithelium from toxins/pathogens.
In IBD, defects in mucin production and function can lead to a compromised barrier, exacerbating inflammation and disease progression 57.
The role of mucins has been extensively studied in animals, however, such experiments are highly invasive, technically challenging, and not directly translatable to humans.
The main mucin types found in the intestines and in the stomach, Muc2, Muc5b, and Muc5ac, overlap between humans, rats, and mice. Nonetheless, there are major species-specific differences in mucins' composition, glycosylation patterns, expression levels, and functional roles 35.
Changes in glycosylation patterns can impact the protective mucus layer in the gut. For instance, decreased glycosylation has been observed in the intestinal mucus of IBD patients, which might lead to increased bacterial contact with the epithelium, potentially triggering inflammation.
Dysregulation in mucin synthesis and secretion can compromise the mucosal barrier, making it easier for pathogens to invade and cause inflammation.
Due to inter-species variation in mucin function, specific mucins may play a more pivotal role in preserving gut barrier integrity in some species than in others.
Species-specific differences in mucus-associated GI microbiome can affect mucus adhesion, degradation, and overall layer thickness.
In addition, differences in the immune systems of humans and rodents can affect how mucins modulate immune responses and intestinal inflammation.
Species-specific differences in bile acids
Bile acids (BA) act as signaling molecules that interact with the farnesoid X receptor (FXR) and the G protein-coupled bile acid receptor 1 (GPBAR1) that are highly expressed in the ileum and the colon.
FXR is believed to have a protective effect on IBD, by maintaining the integrity of the intestinal barrier and by suppressing the production of pro-inflammatory cytokines and chemokines. Its exact role remains, nonetheless, to be elucidated in a human-based system, since animal studies on FXR have shown inconsistent and unreliable results 58.
Malabsorption of BA in the ileum is frequently observed in IBD patients, particularly those with CD, and can lead to diarrhea, abdominal pain and bloating 59.
The gut microbiota can modulate the BA pool size and composition through conversion of primary BA to secondary BA, and amino acids conjugation.
Reciprocally, BAs were found to shape the gut microbiota through regulation of the growth and the survival of specific bacterial species.
Patients with IBD develop an altered BA composition characterized by an increase in primary BA and a decrease in secondary BA, probably as a consequence of gut dysbiosis 60.
The BA pool composition in rodents is very different in humans.
For instance, in mice and rats, the primary BAs - cholic acid (CA), chenodeoxycholic acid (CDCA) are converted to the primary BAs - α-muricholic acid (α-MCA) and β-MCA. Muricholic acids are more hydrophilic than the secondary BAs - deoxycholic acid (DCA) and litocholic acid (LCA), and therefore tend to be less cytotoxic, since they are less likely to solubilize lipid membranes than the hydrophobic BAs. However, muricholic acids are less common in humans, because the liver enzyme responsible for their synthesis, CYP2C70, is rodent-specific 61. This species-specific difference has implications for the capacity of BAs to induce epithelial damage and for interactions between the BAs and the gut microbiota.
The liver and the gut microbiome can hydroxylate LCA into the less toxic hyodeoxycholic acid (HDCA). However, this metabolic pathway is more active in rodents than in humans, contributing to inter-species differences in BA toxicity 62.
In humans, most primary BAs are glycine conjugated, while in rodents, the majority of primary BAs are taurine conjugated 63.
In contrast to humans, in which CDCA is the most potent endogenous FXR agonist, the taurine-conjugated α/βMCA exert antagonistic FXR activity in rodents, which might explain the higher synthesis rates of BAs in rodents compared to humans 64.
This inter-species divergence has functional consequences for BA metabolism, signaling, and toxicity, likely contributing to the failure of preclinical studies to predict the response to treatment in IBD patients.
Species-specific differences in gene expression regulation
Non-coding RNA, including microRNA (miRNA) and long non-coding RNA (lncRNA), play a key role in gene expression regulation through epigenetic, transcriptional and post-transcriptional mechanisms.
In IBD, dysregulation or insufficient miRNA-mediated repression can favor excessive immunological response and inflammation 7.
In spite of presence of conserved regions, sequences and expression profiles of miRNA and lncRNA were found to vary across species 65, 66, likely contributing to inter-species differences in the regulation of expression of the genes that maintain the epithelial barrier and modulate the immune reactivity.
Species-specific differences in immune system
Innate and adaptive immunity responses play a crucial role in the pathogenesis of IBD.
Extensive differences between humans and mice were demonstrated in the structure of innate and adaptive immunity, including in balance of leukocyte subsets, defensins, Toll receptors, inducible NO synthase, the NK inhibitory receptor families Ly49 and KIR, FcR, Ig subsets, the B cell (BLNK, Btk, and λ5) and T cell (ZAP70 and common γ-chain) signaling pathway components, Thy-1, γδ T cells, cytokines and cytokine receptors, Th1/Th2 differentiation, costimulatory molecule expression and function, Ag-presenting function of endothelial cells, and chemokine and chemokine receptor expression 67.
In addition, it was shown that transcriptional responses to acute inflammatory stresses of different etiologies in mouse models do not correlate with human acute inflammatory diseases 68.
Major species-specific differences were also found in transcriptional regulation, chromatin state and higher order chromatin organization. Notably, cis-regulatory sequences next to immune-system-related genes have shown the most divergence, as they have apparently undergone more rapid evolution than others 69.
It was suggested that using animal models with a humanized immune system might improve translatability to humans, however, such an approach would face persistent, insurmountable challenges: the role of the human immune system in IBD is complex and not fully understood, the equivalence of humanized animals to the human immune system was never demonstrated by objective measures 70, and the cross-talk between the human immune system and the rest of human organ systems cannot be recapitulated in animals.
Additionally, the development and adaptation of the immune system in response to the environment cannot be recapitulated in humanized animal models either. This missing aspect is crucial since, as twin studies show, the human immune system is more shaped by the environment than by genes 71.
Face validity - How well do animal models replicate the human disease phenotype?
No single animal model faithfully recapitulates the clinical and histopathological characteristics of inflammatory bowel diseases [IBD] - ulcerative colitis (UC) and Crohn’s disease (CD).
Several animal species, including rodents, rabbits, dogs, pigs and non-human primates (NHP), were used to create animal models of IBD via chemical induction, adoptive T cell transfer, and genetic engineering methods.
Mice, rats, NHP or pigs do not naturally suffer from IBD in the wild, while cats and dogs may experience IBD-like symptoms like chronic diarrhea, vomiting and weight loss due to a weakened immune system, diet, parasites, or bacterial infections.
Chemically-induced animal models
Typically, administration of chemical incitants in vivo produces an acute response, unless incitants are repeatedly administered to mimic long-term exposure 44.
Dextran Sodium Sulfate (DSS) is commonly employed to simulate UC in rodents. DSS directly damages the epithelial barrier of the colon, leading to colonic inflammation with granuloma, bleeding, diarrhea, and weight loss. Depending on the dose and route of exposure, pancolitis, proctitis, and left-sided colitis forms of UC are captured by this method 72, 73.
2,4,6-Trinitrobenzene Sulfonic Acid (TNBS) induces transmural inflammation characteristic of CD, with corresponding symptoms of severe diarrhea, weight loss, and rectal prolapse. While rectal delivery of TNBS primarily induces colonic inflammation, its oral delivery can induce inflammation in both colon and ileum, but without isolated ileal involvement that is a hallmark of certain forms of CD 74, 75.
A major limitations of this method is the difficulty in producing irreversible IBD-like phenotypes in vivo. Indeed, contrarily to humans, mice recover once the administration of the chemical compound is stopped. In addition, the early events associated with the induction of IBD-like inflammation are not be captured in these chemically-induced animal models.
Adoptive T cell transfer animal models
The most common approach to T cell transfer, that involves transferring naïve Treg-depleted CD4+CD45RBhigh T cells from wild-type mice into Rag-KO or SCID immunodeficient mice, induces chronic colonic inflammation characteristic of UC 75.
An important limitation of the T cell transfer approach is that it does not recapitulate transmural inflammation, intestinal fibrosis and strictures characteristic of CD 73.
Genetically-engineered animal models
In the past 30 years, several dozen genetic knock-out (KO) mouse strains, such as IL-10 KO, STAT3 KO and XBP1 KO, were engineered to induce colitis/ileitis 44, 73.
The IL-10 KO mouse only partially mimics the IBD phenotype since no weight loss was observed and the colitis development was highly variable. A non-steroidal anti-inflammatory drug, piroxicam, is commonly used in this model to accelerate the onset of colitis.
NOD2 KO mice exhibit some features of IBD, such as increased susceptibility to colitis, but lack other key aspects like granuloma formation and transmural inflammation.
Spontaneous animal models
The SAMP1/YitFc mouse strain, obtained by selective inbreeding, develops ileitis spontaneously with several similarities to human CD 73. Nevertheless, SAMP mice do not display colitis and the time needed for full disease penetrance can exceed 30 weeks in this model.
Mice that overexpress tumor necrosis factor (TNFΔARE mice) show CD-like chronic ileitis and granuloma formation. However, other common features of CD, such as strictures and fistulas are not represented.
It is also important to mention that EIM are not fully recapitulated in animal models of IBD. Only a few rodent models manifest multifocal inflammation, with colitis–arthritis models being the dominant phenotype available 76.
TNFΔARE mice 73 display axial spondyloarthritis (SpA) sacroiliitis, Achilles tendon enthesitis, and peripheral arthritis.
Genetically engineered rats that have been modified to express the human HLA-B27 gene that is associated with SpA in humans develop colitis, SpA, gastritis, psoriasis, and epididymitis 44.
SKG mice, that carry a mutation in the ZAP-70 gene, spontaneously develop rheumatoid arthritis-like autoimmune arthritis but do not show symptoms of IBD 77. Supplementary induction by intraperitoneal injections of 1,3-β-glucan is necessary for Ileitis and EIM such as arthritis and uveitis to manifest.
In addition, these animal models of IBD do not replicate the heterogeneity and complexity of clinical features seen in human SpA.
Construct validity - How well do the mechanisms of disease induction in animals reflect the currently understood etiology of the human disease?
Not only is there no evidence supporting the relevance of animal models to human inflammatory bowel diseases (IBD) - ulcerative colitis (UC) and Crohn’s disease (CD) - but existing research suggests the opposite.
The polygenic and multifactorial nature of UC and CD cannot be captured in animal models. In addition, it is not feasible to independently dissect in animals the cause-effect contributions of these pathophysiological factors to development of IBD.
Experimental methods of induction of IBD in animals are very different from complex and combined causes of natural triggers in humans. Pathways that underly development of natural human IBD are thus likely to diverge from pathways triggered experimentally in animals.
In combination with an incalculable number of species-specific differences in digestive systems, the use of animals to model IBD is therefore very likely to lead to poor understanding of disease mechanisms, target misidentification, pursuit of misleading hypotheses and failures in clinical trials.
Chemically-induced animal models
The method of chemical induction in animal models is technically simple but often leads to severe toxicity, high mortality, and significant differences in pathogenesis compared to human diseases 44, 73, 74, 75.
In real-world scenarios, DSS and TNBS are not ingested by IBD patients. In humans, IBD does not occur following a single acute exposure or a repeated exposure to a toxicant, but by a combined effect of genetic susceptibility, environmental factors, immune system dysfunction, and microbiome dysbiosis over time 6, 7, 9, 35, 37, 38, 71.
In addition, DSS and TNBS compounds induce acute inflammation, while human IBD is characterized by a chronic, relapsing-remitting pattern, limiting the study of early onset mechanisms, mechanisms of gradual progression, and mechanisms of relapse in animal models of IBD.
The exact mechanisms responsible for chemically-induced IBD in animals are in themselves poorly understood 73.
As demonstrated by the fact that anti-TNF and anti-IL-12/23p40 antibodies have shown efficacy in treating IBD patients, but not in treating chemically-induced colitis animal models, mechanisms that underly chemically induced IBD-like symptoms in animals differ from those in humans with IBD 78.
Genetically-engineered animal models
Typically, genetically engineered IBD animal models trigger intestinal inflammation through impairment of the epithelial barrier and of the innate immune response signaling, usually without evidence that experimentally targeted genes are in effect involved in IBD 44, 73.
For example, the C57BL/6 mouse expressing a dominant negative N-cadherin was engineered 30 years ago. However, today we know that the gene coding for cadherin-11 is not mutated but overexpressed in CD and UC patients 79, pointing to a dysregulation of gene expression.
Genetic knock-out is frequently employed to model IBD, however, complete loss of gene function is rarely observed in IBD patient populations 6, 7.
In addition, the oftentimes highly conserved inbred mouse strains typically study the effect of a single gene at a time, while human IBD is polygenic with an important inter-individual variability in genetic susceptibility.
Spontaneous animal models
The SAMP1/YitFc and TNFΔARE mouse strains 73 do not contain the genetic mutations identified in IBD patients 6, 7, 8.
The lack of human-specific and patient-specific genetic backgrounds, human immune system, and human microbiome 9, 37, 38 makes such model unlikely to shed light on human-relevant mechanisms of IBD. This major limitation also holds true for non-human primates that develop spontaneous colitis and subsequent colon cancer following extended periods of confined captivity.
Adoptive T cell transfer animal models
Since the method of T cell transfer makes use of immunodeficient mice 73, 75, it does not recapitulate the complex interplay between the human immune system, the human microbiome and the human genetic background. 6, 7, 35, 37, 38, 9, 71.
In absence of these human-specific components that play a crucial role in IBD induction and development, adoptive T cell transfer cannot be an adequate method for investigating the mechanisms of IBD.
Predictive validity - How well do animal models predict safety and efficacy of therapies in patients?
Unsurprisingly, given the poor face and construct validity of animal models of inflammatory bowel diseases (IBD), ulcerative colitis (UC) and Crohn’s disease (CD), the overwhelming majority of treatments developed through animal research failed to produce benefits for patients.
For example, a Lactococcus lactis strain secreting IL-10 was found to decrease DSS-induced colitis in mice 80 and in pigs, but has failed to show evidence of efficacy in phase 2 clinical trials 81.
The anti-IL-17A monoclonal antibody, secukinumab, not only failed to demonstrate clinical benefit in trials but has, in some cases, exacerbated symptoms in CD patients 82.
Currently there is no cure for IBD. Treatment strategies for induction and maintenance of remission in UC and CD focus on reducing the inflammatory response and altering the immune system's activity. Remission of IBD is defined as complete mucosal healing, normalization of blood markers and disappearance of symptoms.
Although UC and DC have medications in common, that often overlap with treatments for other autoimmune diseases, the efficacy of treatments can vary. Indeed, the heterogeneity in clinical presentations and mechanisms of UC and CD underscores the need for a personalized approach to treatment 83.
Historically, anti-inflammatory aminosalicylates (5-ASA) (like mesalamine) and corticosteroids (like prednisolone), are employed to reduce inflammation 84. These treatments provide improvement of symptoms but do not change the overall disease course in UC and CD patients.
5-ASA was more effective than placebo in both induction and maintenance treatment of UC, although the effect of 5-ASA in treating induction or relapse of CD was not consistent. Unlike 5-ASA, corticosteroids are not used as a long-term treatment to maintain remission because they can cause potentially serious side effects, such as osteoporosis and cataracts.
A common side effect of immunomodulators, such as tacrolimus and azathioprine, that reduce CD4+/CD8+ T cell proliferation and activation, is increased vulnerability to infections. Indeed, infections are common in IBD patients on immunomodulator and biologic therapy, with the highest risk observed in patients on combination therapy 85.
Monoclonal antibodies, such as infliximab, that target proinflammatory cytokine tumor necrosis factor-α (TNF-α), were introduced in the late 1990s to induce and maintain remission in CD. Infliximab and adalimumab were not originally developed for IBD but for rheumatoid arthritis, and their effectiveness in suppressing inflammation had enabled their approval for UC and CD 86.
Based on real-world data, remission rates in IBD patients after one year of therapy with anti-TNF-α agents is up to 45% 87. However, approximately one third of IBD patients are primary non-responders to anti-TNF-α induction therapy, meaning that they do not show clinical improvement during induction therapy and do not achieve remission. In addition, another third go on to become secondary non-responders during TNF-α inhibitor maintenance therapy 88, meaning that, in spite of an initial response to treatment, this response diminished after a certain time, often leading to disease relapse.
In 2014, the chimeric anti-α4β7 integrin monoclonal antibody vedolizumab, that prevents T cells from migrating to the gastrointestinal mucosa, was approved by the FDA for treatment of moderate-to-severe UC and CD in patients who exhibited an unsatisfactory response to anti-TNF-α biologics and immunomodulators 89.
Two years later, the FDA had approved the fully human anti-cytokines IL-12/IL-23 monoclonal antibody ustekinumab for CD patients who did not experience improvement with immunomodulators, corticosteroids or TNF blockers 90. In 2019, ustekinumab was also approved for patients with moderate-to-severe UC.
In 2025, it is the humanized IgG4 monoclonal antibody that targets the p19 subunit of interleukin-23, mirikizumab-mkrz, that was approved by the FDA for treatment of UC and CD, despite potentially severe and life-threating side effects 91.
Importantly, although biologics were effective in certain IBD patients, clinical remission rates have proven variable depending on the severity and the location of the disease, the genetic background, and the previous history of treatments. Furthermore, sensitization with formation of anti-drug antibodies was observed, which is a broad limitation of monoclonal antibody therapies across many disease areas 92.
Recent therapeutic leads include new small molecule drugs, such as Janus kinase inhibitors (JAK) tofacitinib and upadacitinib, that interfere with JAK-STAT signaling pathway, thereby reducing the production of pro-inflammatory cytokines and dampening the immune response.
Upadacitinib was approved by the FDA in 2021 for treatment of adults with moderate to severe CD who exhibited an inadequate response or intolerance to one or more TNF blockers. A year later the approval was extended for treatment of UC 93.
Sphingosine-1-phosphate (S1P) receptor agonists, such as the newly approved ozanimod, have shown evidence of efficacy in moderate to severe UC cases where the therapeutic approach with 5-ASA, corticosteroids, immunomodulators or biologics was insufficient, albeit with several severe side effects 94.
Of note, JAK inhibitors are not recommended for use in pregnancy, which is problematic since symptoms of IBD can be exacerbated in women during this period.
The discrepancy between chemically-induced damage to epithelial cells in IBD animal models and the intricate natural causes in IBD patients has also hindered the development of safe and effective therapies for treating the epithelial barrier damage. As a result, there are currently no therapeutic interventions available that promote the restoration of integrity and function of intestinal epithelial cells 92.
More than half of patients with CD develop strictiring complications over their lifetime, and yet, therapies against this complex, dynamic and multi-factorial pathology are missing because findings from animal models of fibrosis have not translated to effective anti-fibrotic IBD therapies 95, 96.
Ethical validity - How well do animal experiments align with human ethical principles?
Preclinical
Ethics is a human-specific philosophical concept. Animal experimentation is unethical in essence by human standards, since it involves physical constraint, psychological suffering and deprivation of freedom, social interactions, natural environment, and life purpose.
In addition to this baseline, animal experiments inflict severe clinical harm in animals 97:
Table S4: Severity classification of clinical signs
Abdominal distention: up to severe abdominal distention causing a tense/painful abdomen and/or dyspnea
Vomiting: up to severe with frequent vomiting causing dehydration/weight loss
Diarrhea: up to severe with watery or bloody diarrhea resulting in dehydration
Body weight (BW) loss animals not in growth phase: up to severe with 15-20% BW loss within 3 days
Body weight rodents < 10 days: up to severe with weight loss in 24h
Body weight growing animals/juvenile: up to severe with growth stop > 7 days
Table S5: Severity classification of chemical disease models
DSS/TNBS/Oxazolon mouse model of colitis: causing up to severe clinical signs
Table S13: Severity classification of genetically altered (GA) lines
GA strains with inflammatory bowel disease (IBD): up to severe clinical signs
Clinical
While there is no consensus on whether an unethical act can be justified by a pursuit of a hypothetically ethical outcome, it was suggested that animal research was necessary to advance safe and effective treatments for human diseases.
However, statistics consistently show that clinical success rates of drugs developed and tested in animals is very low, raising the question of whether it is ethical to put the health of patients at risk.
Over 2011-2020, the Phase I to II transition success rate, that primarily focuses on assessing the safety and tolerability of drugs, was 46.7% for gastroenterology, below the 52% average of all indications 98, exposing individuals to severe adverse drug events, such as liver failure and tumors.
Moreover, the overall likelihood of approval from Phase I to Approval for gastroenterology disease group was only 8.3%, stressing the low predictive validity of animal models of human gastroenterology diseases.
Intrinsic validity - How well do animal models capture the clinical heterogeneity of the human disease?
Animal models of inflammatory bowel diseases do not recapitulate the inter-individual heterogeneity in clinical features, pathophysiology, and responses to treatment.
Extrinsic validity - How well does animal experimentation generate reliable and reproducible outcomes?
It is often argued that although animal models have severe limitations, animal research enables to gather insights that may be valuable. However, the basic precondition for a hypothetical benefit is not met since the majority of animal experiments is irreproducible 99.
Contributing factors include flawed experimental design, variation in animal strains and experimental conditions, and lack of transparency on methodology and results of animal studies.
In a bid to improve the quality of reporting of animal experiments, the ARRIVE - Animal Research: Reporting of In Vivo Experiments - guidelines, were published in 2010 and updated to ARRIVE 2.0 in 2020 100.
Nonetheless, and in spite of significant investment in dissemination, various incentives and training of animal researchers, the Arrive guidelines remain poorly implemented 101, 102.
As a result of weak relevance, rigor and reliability of animal studies, erroneous and misleading hypothesis are generated, animal and human lives are needlessly sacrificed and dozens of billions of dollars are annually wasted 103.
Beyond the problem of experimental design and reporting of animal studies, there are deep-seeded cultural reasons that are not likely to be addressed any time soon, such as the "publish or perish" culture that discourages from unbiased analysis and reporting of negative results and rewards exaggerated and overhyped claims 104.
In Summary
Inflammatory bowel diseases [IBD], ulcerative colitis (UC) and Crohn’s disease (CD), are autoimmune disorders of the gastrointestinal system posing a significant personal and economic burden.
Complications arising from IBD can lead to significant morbidity and mortality, requiring early diagnosis, careful monitoring and effective treatment.
The in vivo modeling fails to recapitulate the underlying mechanisms and clinical features of IBD, all while producing severe clinical signs in animals.
For a very long time - and still today - the path to developing safe and effective therapies for IBD has been fraught with clinical failures, due to species-specific differences in the gastrointestinal physiology, the immune systems, the microbiome, the metabolism, and the genetic background.
Currently there is no cure for UC and CD, and the existing treatment options show a variable efficacy, underscoring the need for personalized approach.
Human-based tools are needed to elucidate heterogenous disease mechanisms, identify new therapeutic targets and adjust treatments to patient subtypes.
How is Human-Based In Vitro the Answer to Advance Biomedical Research into Inflammatory Bowel Diseases
*To model heterogenous familial and sporadic IBD phenotypes, using patient-derived iPSC, GI tract tissues/gut organoids/GI-on-chip 105, 106, 107, 108.
*To identify genetic variants of CD and UC that are predictive of disease phenotype, stricturing and penetrating disease behaviour, extra-intestinal manifestations, and response to therapies, by gene editing (base editing, overexpression, knock-out, etc.) and gene perturbation (CRISP interference, siRNA) in human 3D GI tissues/organoids/gut-on-chip.
*To identify epigenetic drivers of IBM (diet, toxicants) and to test potential epigenetic therapies for IBD, using epigenetic editing (CRISPR activation/repression, lncRNA) in healthy human/IBD patient-derived 3D tissues/organoids/multi-organs-on-chip.
*To model human-specific inter-individual differences in IBD pathophysiology, by exposing the culture medium of healthy human 3D tissues, genetically edited for GWAS-identified variants, to nutrients, pathogens, smoke, and chemicals, and by measuring resulting inflammation (cytokine assays), oxidative stress (DCFH-DA staining), gene expression change (RNA-seq), mitochondrial membrane potential (TMRM staining), and other endpoints.
*To identify molecular subtypes of inflammatory, stricturing and penetrating UC and CD, by morphologic, functional and multi-omics characterization of a biobank of intestinal organoids derived from UC and CD patients of different sex, age, diagnosis, ethnic and genetic backgrounds 109.
*To elucidate human-specific mechanisms of UC and CD, by analysing differential gene expression, GO term enrichment, cytokine assays, NF-κB reporter assays, etc., in patient-derived iPSC/3D tissues/organoids/gut-on-chip 19, 110.
*To investigate immune responses in chronic intestinal inflammation, by using human intestinal-immuno-organoids/intestine-lymph-node-on-chip combined with autologous tissue resident immune cells (DC, macrophages, memory T cells) 111, 112 and non-resident immune cells (neutrophils) 113.
*To determine the contribution of each cell type to UC and CD phenotype, by using chimeric bioprinted tissues containing one or more diseased cell types in the context of a healthy background of other cell types - intestinal epithelial cells, fibroblasts, paneth cells, goblet cells, tuft cells, enteroendocrine cells -, in healthy and IBD patient--derived GI tract 3D tissues 114.
*To identify novel biomarkers of IBD subtypes, disease progression and responses to treatments, by multi-omics analyses of multiple types of biomolecules in large scale IBD patient-derived gut 3D tissues/organoids/(multi)organs-on-chip 115, 116.
*To elucidate mechanisms of IBD-associated extra intestinal manifestations, by studying common/distinct molecular pathways and temporal relationship between mucosal and extraintestinal inflammation, using patient-derived multi-organs-on-chip with intestine connected to affected organs (skin, liver, muscle) 117.
*To investigate how dysregulation of the human intestinal mucus production and composition affects epithelial barrier function, inflammation and IBD progression, using human healthy/IBD patient-derived colon-on-chip 118.
*To study the role of specific intestinal regions - ileum, cecum, colon, rectum -, anatomical locations within these regions, and residual microbiomes within locations, by leveraging human healthy/IBD patient-derived iPSC/adult intestinal stem cells.
*To study how the temporal cell type-specific accumulation of epigenetic changes influences the development of IBD, by using 3D intestinal tissues/organoids/(multi) organ-on-chip derived from IBD patients of different age, sex, geographic and ethnic background.
*To study the role of the human microbiome in IBD pathogenesis: microbiome-host interactions, pathogenic-beneficial inter-microbial dynamics, nutrient absorption and metabolism, role of individual gut microbes, pathogenic signaling pathways, alterations in host proteome, immune tolerance, etc., by using immunocompetent IBD patient-derived tissues/gut microbiome-on-chip 116, 119, 120, 121, 122.
*To determine the impact of dietary/allergenic components on gut microbiome, bile acids metabolism, mucus degradation and intestinal inflammation, by using human (multi)-organ-on-chip /IBD patient-derived in vitro intestine and ex vivo fermentation systems 123, 124.
*To identify new therapeutic targets for UC and CD and to high-throughput screen drug candidates in human tissues/organoids/(multi)-organs-on-chip 125, 126.
*To reliably predict human-relevant drug bioavailability and hepatic clearance, including in pregnant IBD patients, using patient-derived gut-liver-on-chip 127.
*To investigate different drug delivery strategies that can selectively deliver therapeutics to the GI tract and thereby minimize systemic toxicity, using multi-organ-gut-chip.
*To select the most safe and effective drugs for each IBD and EIM molecular subtype, by screening of novel and of clinically approved repositionable drugs in IBD patient-derived organoids/(multi)-organs-on-chip.
*To assess safety and efficacy of combination treatments in IBD patient-specific organoids/(multi)organs-on-chip, in a personalized medicine approach.
Last Updated: September 2025
Although in vitro methods have inherent limitations, their relevance to human biology far exceeds that of animal research.
Animal model organisms were never comprehensively compared to humans and scientifically validated. Complementing in vitro methods with animal experiments is not effective for human patients, because species-specific differences prevent reliable integration and translation of results to humans.
While animal research benefits from experimenting on a complete organism, model organisms fail to replicate the interplay of thousands of human-specific features, from molecular level to organism level, and are therefore not representative of the complete human organism.
To address the challenges of individual human-based in vitro models, they can be integrated with other human-based in vitro methods, AI-driven analysis, clinical data, and real-world patient data.
Have you leveraged in vitro methods in unique ways? We would love to hear how! Join the conversation to exchange ideas, collaborate and inspire new directions in human-based science!
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