Differential protein expression, gene expression, cell migration, organization, survival, and cell signaling have been observed in organoid cultures comparative to monolayer cell culture (70–73). Defects in the function of multiple epithelial cell types have been demonstrated in IBD, underlining the need for a physiologically relevant model which includes multiple cell lineages (74–78). Gut organoids may also be co-cultured with non-epithelial cell lines of interest in order to more accurately represent the intestinal mucosal niche. Co-culture of gut epithelial organoids with cell lines such as macrophages and lymphocytes and with mesenchymal cells demonstrate promise in providing a more physiologically relevant model of the gastrointestinal mucosal environment (79–82).

Due to this ability to accurately simulate the intestinal microenvironment, intestinal organoids represent exciting models for investigating the host-microbial interactions which are key to the pathogenesis of IBD. Organoids have already been successfully utilized as a more accurate model for human virus infection. In a study by Saxena, fully differentiated cells present in organoid culture supported greater rotavirus viral load and replication than had been previously observed in monolayer culture; and infection of enteroendocrine cell types in addition to enterocytes with rotavirus was demonstrated (83). Organoids have also been used to investigate norovirus, which is difficult to cultivate in monolayer cell culture. Previously only successfully cultured in B cells, organoids permitted culture of norovirus in duodenal, jejunal, and ileal cell types with viral replication and growth occurring within (84). Current applications of organoids include disease modeling of SARS-CoV-2 in respiratory and small intestinal derived cell types, with viral infection, replication, and host viral response observed ex-vivo (85–87).

Organoid cultures have also been applied to simulate host-bacterial interactions. Salmonella, H. pylori, C. difficile, and pathogenic E. coli infection have all been modeled in organoid cultures (66, 88–91). In one study, gastric organoids which secrete mucous, include multiple epithelial cell types and retain the polarity of the in-vivo gastric epithelium have been successfully utilized as a model for host-microbe interactions in H.pylori infection (66). Interestingly, duodenal, ileal and colonic organoid cultures derived from different donors demonstrate a differential response to infection and differing patterns of bacterial adhesion, possibly due to the genetic variability based on the tissue of origin (89). A co-culture model developed to study the host-pathogen interactions of C. jejuni incorporates intestinal enterocytes, mucin-secreting goblet cells and dendritic cells, thus combining a mucus-secreting epithelial layer with cellular elements of the intestinal innate immune system (92).

As well as modeling invasive microorganisms, organoids can also be used to study interactions between the gut and commensal microbiota. In one study, microbiota were found to play a role in epithelial regeneration in murine small bowel organoids. The pattern recognition receptor NOD2, single nucleotide polymorphisms (SNPs) of which are highly associated with Crohn's disease, is highly expressed in mouse intestinal stem cells (93–96). Stimulation of NOD2 by MDP (peptidoglycan muramyl-dipeptide), a bacterial cell wall constituent, enhanced organoid survival and protected them from oxidative-stress mediated cell death (96, 97). Organoids derived from adult and fetal murine tissues have also been utilized to determine developmental expression patterns of components of the innate immune system, including NOD2, TLR4, and TLR5 (98). Exposure of murine intestinal organoids to gut commensal bacteria including Akkermansia muciniphilia and Faecalibacterium prausnitzii has been shown to induce changes in gene expression and transcription, particularly of genes responsible for lipid metabolism (99). Similarly, exposure to the organism Bacteroides thetaiotaomicron and cytokine signaling via IL-22RA1 induces upregulation of Fut2 and increased fucosylation, which in turn inhibits colonization by opportunistic Enterococcus faecalis strains (97, 100, 101). Finally, alterations in the microbiome have been associated with colonic neoplasia; colonic organoid models have been used to demonstrate a mutational profile induced by exposure to colibactin synthesized by genotoxic E. coli which is also associated with colorectal cancer in-vivo (102). Thus, organoid systems may be utilized to explore activity of the gut microbiome on the epithelium and mechanisms of homeostasis (Figure 1).

Unlike organoids derived from pluripotent stem cells, which rapidly accumulate mutations and epigenetic modifications, ASC-derived organoids are relatively genetically stable (103, 104). They retain the genetic profile and also the transcriptional and epigenetic landscape of the primary tissue from which they are derived (73, 105–107). While the majority of IBD is polygenic, some monogenic forms exist. These are mediated by specific genetic defects in epithelial dysfunction and stress response, defects in immune regulation of regulatory T cells or immunodeficiencies of phagocytic cells (108). Organoids represent useful models for studying these rare diseases, as well as other genetically determined intestinal disorders (109).

It is possible to culture intestinal organoids derived from patients with active IBD (110, 111). In one study, IBD colonic organoids demonstrated a distinct phenotype to those derived from control tissue, with a smaller size, increased cell death, abnormal cell polarization, and poorer budding capacity (110). Interestingly, they also expressed reduced quantities of the tight junction proteins ZO1, Occludin, and Claudin-1 as well as alterations in the expression of adherens junction and desmosomal proteins. These altered expression patterns persisted when the inflammatory stimulus was withdrawn (111). The phenotype and altered transcriptional profile noted in the IBD-derived organoids was inducible in the control organoids with administration of pro-inflammatory cytokines (TNFα, IL-1, and IL-6). Hibiya et al. demonstrated that murine colonic organoids which are exposed to chronic inflammatory stimuli (TNFα, IL-1β, IL-6, LPS, flagellin) underwent upregulation of the NFκB signaling pathway, which persisted after stimuli removal (112). These organoids also underwent transformation to an undifferentiated state, along with upregulation of genes related to oxidative stress and carcinogenesis (Smox and CD151), suggesting their potential utility as a model to study the epithelial changes which occur in colitis-induced carcinogenesis. Vermeire et al. also generated CD and UC-derived organoids which were subsequently exposed to TNF and flagellin, resulting in modulation of expression of the SARS-CoV-2 receptor ACE2. These changes were restored to baseline with anti-TNF treatments (113). Other studies utilizing patient-derived organoids from pediatric IBD patients demonstrated alterations in DNA methylation and transcriptional profiles, which correlated with treatment outcomes (114). Finally, a study by Jardine et al. successfully used colonic organoids generated from patients with TTC7A deficiency to perform high-throughput drug screening for candidate therapeutic agents (115). Loss of TTC7A causes intestinal epithelial apoptosis and immune defects which presents clinically as very early onset IBD. Thus, primary organoid cultures from inflamed tissue seem to represent an applicable model for investigation of the epithelial and mucosal abnormalities which occur in IBD (Figure 1).

In the small intestine the mucosa of the gut is folded into villi and microvilli to maximize available surface area for absorption. Bioengineering techniques such as 3D printing and laser ablation allow the creation of scaffolds which recreate this intestinal topography. These can be directly seeded with epithelial organoids or used as molds to create hydrogel-based porous copies which reproduce the microanatomy of the gut (116). Alternatively, bioink comprising cell aggregates or organoids may be imprinted along with the desired biomaterials (hydrogels, matrix components) onto the scaffold via a computer-aided transfer process (117, 118). This allows for the creation of highly accurate and reproducible models with each component—organoids, biomaterial, and scaffold—spatially aligned at the desired patterns, gradients, and densities set by the modeling software. Such methods will help to address both reproducibility and scaling-up of organoid cultures into larger tissue constructs. Some 3D gut models aimed at investigating the pathophysiology of inflammatory bowel disease are already in use (119).

Aside from gut anatomy, intestinal motility and luminal flow are physiologic functions of the gut which are difficult to mimic ex-vivo. These can be simulated via epithelial cell-lined microfluidic platforms, sometimes referred to as a “gut-on-a-chip” (120–122). Such platforms permit recapitulation of flow patterns, mechanical deformation, shear stresses, and peristaltic activity with greater accuracy than has been possible previously (117, 123–125). Organoid-lined laser-ablated microchips with active perfusion of media components have also been developed, which permit simulation of intestinal homeostasis and cell turnover with a reduced need for passaging (126). These platforms are being utilized to further investigate the gut-microbiome relationship by inoculation with bacterial cultures and examining the effect of the physical environment on intestinal host-microbe interactions (127–132) (Figure 1).

The concept of mucosal healing as key to sustained remission of IBD has become increasingly prominent in recent years. This denotes absence of all mucosal ulceration at endoscopy, rather than resolution of clinical symptoms and serum biomarkers of inflammation alone (133, 134) Mucosal healing correlates with improved long-term clinical outcomes, including steroid use, hospital admissions and need for surgery in both CD and UC (135–139). The European Crohn's and Colitis Organization lists mucosal healing as a therapeutic target in its 2017 consensus guidelines for both UC and CD (140, 141). Local transplant of organoids to aid mucosal healing has been proposed as a potential therapy in IBD to aid epithelial regeneration and achieve mucosal healing (142). Studies using murine colitis models have demonstrated that human small bowel and colonic organoid cultures can engraft onto the ulcerated mucosa and reconstitute the normal crypt-villus architecture (58, 143, 144). More recently, patient-derived small intestinal organoids have been successfully expanded ex-vivo and engrafted into mice, with the ultimate aim of creating autologous small intestinal transplants to treat intestinal failure (145) (Figure 1).

Despite the advantages described above, there are limitations associated with the use of organoids. Comparative to two-dimensional models they are more costly and less accessible, and require specialized medium to be maintained in culture. Matrigel and similar matrices in which they are typically cultured are expensive and increase the difficulty of manipulation. Particular studies such as transport and luminal exposure studies require injection of organoids which is a technically difficult and labor-intensive procedure; alternatives such as computer-assisted injection are again expensive and not readily available. Access to human tissue for generation of primary organoid cultures can be limited (78, 146). They are typically derived from epithelial tissues and so other components of the intestinal niche, including immune and mesenchymal elements, are underrepresented (146). As they are three-dimensional structures, this presents difficulty for investigations requiring access to the apical and basolateral surfaces. For this purpose they may be dissociated into 2D structures; however this disrupts their crypt-villus architecture and terminates their culture cycle (146). Finally, reproducibility of organoid cultures is challenging, as constructs of differing sizes and morphology result when they are grown in-vitro.