Human intestinal organoids: Modeling gastrointestinal physiology and immunopathology — current applications and limitations

Human intestinal organoids are an ideal model system for studying gastrointestinal physiology and immunopathology. Altered physiology and mucosal immune response are hallmarks of numerous intestinal functional and inflammatory diseases, including inflammatory bowel disease (IBD), coeliac disease, irritable bowel syndrome (IBS), and obesity. These conditions impact the normal epithelial functions of the intestine, such as absorption, barrier function, secretion, and host–microbiome communication. They are accompanied by characteristic intestinal symptoms and have significant societal, economic, and healthcare burdens. To develop new treatment options, cutting‐edge research is required to investigate their etiology and pathology. Human intestinal organoids derived from patient tissue recapitulate the key physiological and immunopathological aspects of these conditions, providing a promising platform for elucidating disease mechanisms. This review will summarize recent reports on patient‐derived human small intestinal and colonic organoids and highlight how these models have been used to study intestinal epithelial functions in the context of inflammation, altered physiology, and immune response. Furthermore, it will elaborate on the various organoid systems in use and the techniques/assays currently available to study epithelial functions. Finally, it will conclude by discussing the limitations and future perspectives of organoid technology.


Introduction
For many decades, research on intestinal epithelial cell (IEC) biology was hampered by a lack of relevant primary in vitro cell culture models.This changed fundamentally after the description of intestinal Lgr5 + crypt base columnar (CBC) stem cell (SC) derived organoids by the group of Hans Clevers in 2009 [1].The genera-tion of these primary cell cultures has revolutionized the study of intestinal epithelial functions and gastrointestinal (GI) disease.
Chronic inflammation is a hallmark of many diseases of the lower GI tract (small intestine [SI], colon, and rectum), such as inflammatory bowel disease (IBD; Crohn's disease [CD] and ulcerative colitis [UC]), and coeliac disease, while alterations to the mucosal immune system are associated with functional disorders such as irritable bowel syndrome (IBS) and metabolic conditions such as obesity.Organoid models have been successfully generated from the intestinal tissue of patients with these conditions [2][3][4][5].Human intestinal organoids retain key aspects of their tissue of origin, providing a platform for elucidating molecular and physiological disease mechanisms, with the potential to revolutionize drug discovery and personalized medicine [6].
This review will provide a summary of recent publications that use patient-derived intestinal organoids to study diseases of the GI tract.Each section will address how organoids can be used to study a specific intestinal epithelial function in the context of inflammation or altered immune activation, with a particular focus on the techniques and assays employed.Note that this review will only describe studies that use adult human intestinal SCs; for a review that covers induced pluripotent SCs and embryonic SC-derived organoids, please see Rossi et al. [7].Finally, we discuss the limitations and future perspectives in this rapidly advancing area.

Lower GI epithelial structure and organoid models Epithelial cell subsets of the lower GI tract
The GI epithelium serves multiple functions, including absorption, barrier function against luminal content, secretion, and supporting cell-cell and host-microbiome communication.The various IEC subsets have specialized roles that facilitate this homeostatic functioning.These include Lgr5+ CBC-SCs, enterocytes, goblet cells (GC), enteroendocrine cells (EEC), Paneth cells (PC), and tuft cells (TC) [8].
Structurally, the intestinal epithelium is covered in invaginations (crypts), at the base of which reside the CBC-SCs [8].The epithelium is continuously renewed through a cycle of tightly regulated proliferation, differentiation, and cell death.Proliferation of the CBC-SCs is responsible for this regeneration.As CBC-SCs migrate toward the lumen they differentiate into other IEC subtypes.The absorptive enterocytes take up ions, nutrients, and water from the lumen, whereas EECs, GCs, PCs, and TCs belong to the secretory lineage [8].EECs release peptide hormones that regulate metabolism, GI motility, and satiety in response to dietary and microbial stimuli.GCs secrete mucin proteins, forming the mucous membrane that protects the mucosa from direct contact with the microbiota.PCs are typically found in the SI, located at the base of the crypt, intercalating CBC-SCs.PCs secrete antimicrobial peptides and growth factors that shape microbiota composition and support the crypt [8].The mechanosensing TCs sense luminal content and secrete both immunomodulatory and neuromodulatory components [9].All IECs can produce chemokines and cytokines that regulate immunity and recruit immune cells into the tissue.Aberrant cell populations and defects in functionality of the various IEC lineages have been strongly associated with GI inflammatory disorders [8,[10][11][12].

Organoid models for studying normal GI functions
The development of intestinal organoids has provided a physiologically relevant in vitro model whereby SCs undergo proliferation, differentiation, and maturation, resulting in similar cell populations and tissue architecture as seen in vivo [13,14].Moreover, they are responsive to physiologically relevant stimuli, including dietary components, bacteria, drugs, and cytokines [4,[15][16][17].
Intestinal organoid research has enhanced our understanding of the signaling pathways regulating IEC behavior and fate [18].In vivo, most growth factors governing this process are secreted by PCs and underlying mesenchymal cells [18].These growth factors, including Wnt, R-spondin, epithelial growth factor (EGF), and bone morphogenetic protein (BMP), play pivotal roles in maintaining, proliferating, and differentiating cells into distinct progenitors [18,19].Wnt and R-spondin are crucial for maintaining the stemness of CBC-SCs, whereas EGF promotes their proliferation.Conversely, villus cell populations secrete BMP, prompting CBC-SC differentiation.To establish a BMP gradient along the crypt-villus axis, mesenchymal cells surrounding CBC-SCs secrete BMP inhibitors such as Noggin and Gremlin1, leading to a controlled stimulation of cell differentiation [18].
Utilizing our understanding of these pathways, organoid models are exposed to enriched culture media containing the requisite growth factors: Wnt, R-spondin, EGF, and Noggin, thereby mirroring the in vivo SC niche microenvironment [8,[18][19][20].The primary distinction in culturing SI organoids (enteroids) and colonic organoids (colonoids) lies in the slight variation in IEC subsets.While in the SI, PCs secrete growth factors utilized by CBC-SCs, colonic CBC-SCs depend on mesenchymal cell-derived niche growth factors.Consequently, colonic organoids may necessitate supplementary factors for extended culturing [20].The heterogeneity of niche inter-and intracellular signaling between the SI and colon is discussed in greater detail in Sasaki et al. [20].
Organoid cultures take advantage of the construction of the intestinal mucosa in vivo, embedding crypts within a medium mimicking the basement membrane [1], their native ECM.The ECM is a 3D network of proteins that provides both structural and biochemical support to cells while guiding tissue growth and differentiation.For organoid culture, commercially available substrates such as Matrigel or basement membrane extract are used.These products contain high concentrations of the ECM proteins collagen and laminin; however, their precise composition remains undisclosed [21].Instead of whole crypts, single CBC-SCs can be used to generate organoids [1].This is not common practice as it sensitizes the cells and requires additional growth factors/inhibitors [22].However, single-cell dissociation is necessary for efficient genetic modification and clonal expansion of organoid lines [22].
The anatomical differences between organoids and the in vivo intestine varies depending on the specific model.Traditional organoids are closed 3D structures, with the apical membrane facing inwards toward an internalized lumen, and the basolateral surface associating with the ECM.More recent advancements in organoid technology have developed "apical-out" organoids with a reversed polarity [23].This conformation allows for access to the apical membrane reflecting in vivo interactions with luminal content.Organoid monolayers are generated similarly to traditional organoids, crypts or organoid fragments are seeded onto a transwell membrane coated in ECM and cultured with media containing essential niche growth factors [24].Fig. 1 summarizes how organoids can be utilized, and Table 1 lists the wide range of assays compatible with organoids to study intestinal epithelial functions.

Organoid models for studying colorectal cancer
Although the focus of this review is on non-neoplastic lower GI disease, it is important to briefly describe the use of patient organoids as models of colorectal cancer (CRC).CRC organoids are generated from patient tumors and retain the highly heterogeneous histopathological and genetic characteristics of their parental tissues [25].Organoids are used to model the various stages of cancer progression from initiation to metastasis [26,27], and to study drivers of colorectal carcinogenesis including chronic inflammation [28].CRC organoids have been shown to be highly predictive of patient response to anticancer therapies [29], and can be utilized to identify new CRC biomarkers and develop novel treatment strategies for CRC subtypes with poor prognosis [30,31].Organoid technology has enabled the in vitro study of diverse CRC subtypes and different disease stages; and has the potential to revolutionize drug development and patient treatment.For a more comprehensive review of the use of GI cancer organoids for basic and translational research please refer to the study by Lau et al. [32].

Absorption
Most nutrient absorption in the gut occurs in the SI, although some vitamins and minerals, as well as water, are absorbed in the colon [33].The colon is relatively flat, whereas, to facilitate absorption, the small intestinal epithelium has small finger-like projections protruding into the lumen, called villi [8].GI organoids retain these tissue-specific morphological features, with differentiated colonoids being spherical and differentiated enteroids having protruding villus-like structures.GI absorption is influenced by multiple factors, including tissue morphology (surface area and villus height); GI motility and transit time; metabolism; epithelial signaling; and membrane nutrient transporter availability [34][35][36].In GI conditions with characteristic inflammation, many of these factors are affected (e.g.villus atrophy and blunting; increased gut transit time; and altered epithelial metabolism), potentially resulting in altered epithelial physiology and absorption [37][38][39].Furthermore, micronutrient deficiencies due to malabsorption are common during active inflammation in IBD [40].The influence that inflammation can have on GI physiology is understood [39,41], but evidence of the effect of inflammation on cellular absorption in the intestinal epithelium both in healthy and disease states is sparse.
In this section, we will provide examples where organoids from both control and patient tissues have been utilized to advance our understanding of epithelial absorption and conditions with associated absorptive defects.Research into the effect of dietary components on human enteroids to advance our understanding of intestinal absorption [16,42,43] has employed assays to measure uptake, transport, and metabolism kinetics for labeled carnitine, palmitate, glucose, and fructose, as well as drug uptake, both in whole and fragmented enteroids.Similar models have been established using labeled dodecanoic acid, a saturated fatty acid, in apical-out enteroids [23].Biotin deficiency is a defect in multiple diseases, Ramamoorthy et al. [42] used enteroid monolayers to show that alcohol and its metabolites significantly inhibit the absorption of biotin.These findings underscore the value of organoids as a model for studying nutrient absorption and metabolism kinetics in the GI tract, under controlled environmental conditions.
Coeliac disease is characterized by impaired SI absorption, primarily due to villous atrophy following an immunological response, believed to be initiated by T cell interactions with gliadin [4].Recent studies using enteroid monolayers and enteroids generated from patient biopsies revealed that at baseline the epithelium had low-grade inflammation, decreased proliferation, and altered differentiation compared to controls [4,44].These novel findings highlight an epithelial-specific role in coeliac disease for initiating an innate immune response and promoting possible defects in absorption [4,44].
Obesity is characterized by altered metabolism and activity of glucose membrane transporters such as sodium-glucose linked cotransporter-1 [45][46][47] and has been associated with chronic low-grade systemic inflammation and intestinal immune cell infiltration [48][49][50][51].While the exact contribution of the SI in obesity is poorly understood, recent publications have identified distinct obesity subsets within patient cohorts using obese and overweight enteroid monolayers compared with lean controls [32].Subsets of obese and overweight organoids retain characteristics indicative of the obesity phenotype, including heightened gluconeogenesis, increased glucose absorption and elevated protein expression of the glucose transporter sodium-glucose linked cotransporter-1 compared with lean controls [5,34].This suggests that targeting epithelial-specific metabolic reprogramming may serve as a potential pharmacological intervention to control nutrient uptake in the context of obesity.
Short bowel syndrome is a severe malabsorption disorder that can result from small intestinal diseases such as CD or necrotizing enterocolitis after resection to remove severely inflamed tissue, leaving the tissue with drastically reduced capacity for absorption due to decreased epithelial surface area [52].Recent research has shown promise for using human organoids as a tool for tissue regeneration in patients with short bowel syndrome, as xenotransplanted human enteroids were successfully grafted into the murine colon, expanding absorptive capabilities [43].Human intestinal organoid models and assays for studying epithelial functions.(A) Traditional organoids are embedded in an ECM, their lumen is internalized, and the apical membrane is difficult to access.Apical-out organoids are generated by removing organoids from the ECM and culturing them in suspension, this promotes a reversed polarity, with the apical membrane facing the media environment, allowing for direct manipulation.Organoid monolayers are produced by seeding SCs onto transwell membranes and culturing them until fully confluent, facilitating accessibility to both basal and apical membranes.The various organoid models contain all the adult IEC types typically found in vivo.By modifying certain media components, it is possible to enrich for SCs or promote the differentiation of specific IEC subsets [99].(B) Absorption can be measured by incubating apical-out organoids with a fluorescent fatty acid analog and then imaging with confocal microscopy [23].(C) FITC-dextran is a fluorescently labeled polysaccharide used to measure epithelial paracellular permeability, an indicator of barrier function.FITC-dextran flux from the media environment into the organoid lumen can be measured by live fluorescent microscopy [15], and flux across organoid monolayers, from the apical to basal compartment, is determined by measuring media fluorescent intensity with a plate reader [13].TEER can also be used to assess transwell monolayer barrier function; it is calculated by measuring the impedance between two electrodes caused by the presence of the

Barrier function
The intestinal epithelium functions as a critical physical barrier, regulating absorption, and preventing harmful substances from entering the body.A disrupted barrier is a distinct feature of mucosal inflammation, and a characteristic of GI inflammatory diseases such as coeliac disease and IBD [3,14,44,53].The implications of a compromised barrier in vivo include increased paracellular diffusion, and potential exposure of immune cells to luminal content, including microbiota, initiating a perpetuating inflammatory response [14].Our understanding of factors affecting barrier function has advanced considerably with the use of organoids [13,54].
Multiple studies using a broad range of readouts (TEER, permeability assays, and barrier function markers) on control/IBD organoids and organoid monolayers subjected to inflammatory stimuli (e.g.IFN-γ and TNF-α) or lipopolysaccharide [LPS]) consistently demonstrate reduced barrier function compared with untreated samples [13,15,57,58].Notably, the loss of barrier function caused by inflammatory disease persists after culture, with UC colonoid monolayers exhibiting diminished baseline barrier function compared with controls [3].Furthermore, d'Aldebert et al. [57] found that control colonoids stimulated with an inflammatory cytokine cocktail and untreated IBD colonoids, showed a similar reduction in the protein levels of tight junction markers (ZO-1 and occludin) when compared with unstimulated control samples.This highlights the destructive effect inflammation has on integral components of the intestinal epithelial barrier function in both "healthy" and IBD organoids.
Similarly, coeliac enteroids and enteroid monolayers at baseline show decreased barrier function compared with controls [4,44].Freire et al. [44] demonstrated that gliadin or gliadin metabolite stimulation of enteroid monolayers resulted in a further decrease in barrier function accompanied by increased proinflammatory cytokine secretion, which can be partially rescued by butyrate or lactate treatment.These findings underscore the association between inflammation and impaired barrier function but also extend to the impact of dietary antigens capable of triggering an inflammatory response, such as gluten metabolites in coeliac disease.
Several studies have demonstrated that impaired organoid barrier function can be improved by the addition of various drugs, cytokines, and nutrients.This illustrates their potential for high throughput drug screening both at a population level and from a personalized medicine approach for diseases with altered barrier [13,55,56].Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling contribute to the hyper-inflammatory status in IBD as reported both in animal models and patients, with JAK-inhibitors such as tofacitinib and upadacitinib approved by the FDA for their treatment [59,60].Ileal and colonic organoids and organoid monolayers derived from control and IBD tissue were used to show that tofacitinib rescues cytokine-induced barrier dysfunction [13,15].IL-27 has previously shown therapeutic potential in murine models of IBD [58].Using human colonoids/colonoid monolayers and cell line cultures, its capacity to promote mucosal healing and intestinal barrier restoration was evaluated by analyzing the gene expression of key barrier proteins and TEER [58].In a series of related studies, the multimineral extract, Aquamin, derived from red marine algae, was shown to improve barrier function in both control and UC colonoids compared with untreated controls [54][55][56].When human UC colonoids were treated with Aquamin morphometric analysis showed modestly increased lumen diameter and wall thickness, and proteomic analysis showed increased barrier function markers, ion transporters, and mucin, compared with controls, suggesting a potential benefit for increased mineral consumption in UC [55].These multifaceted approaches show promise for clinical application in improving barrier function in IBD.

Epithelial secretion
Chronic intestinal inflammation is associated with alterations to epithelial secretory cell quantities, differentiation, and functionality [10][11][12].In UC, colonic GC differentiation is defective resulting in a thinner mucous layer [11].UC colonoid monolayers retain this phenotype, displaying a thin and uneven mucous layer compared with non-IBD monolayers [3].This was not caused by a reduction in total GC numbers.Instead, GCs had a defective response to mucous-secreting stimuli (cholinergic/cAMP signaling) and were unable to release their apical mucous compartments, which was confirmed by electron microscopy.This reduction in mucin secretion may promote the colonic barrier dysfunction observed in UC.COX-2/PGE2 signaling has been associated with patients with UC who are primary anti-TNF-α nonresponders.In SC enriched non-IBD colonoids co-treatment with PGE2 enhanced the TNF-α induced reduction in colonoid cell monolayer [13].(D) Organoid secretory function can be assessed by immunoassays.The spatial distribution and number of secretory cell types can be determined using lineage markers, and secretion of mucins, antimicrobial peptides, and chemokines/cytokines can also be measured [3,63,65,69].(E) Organoid regeneration assays measure the ability of single intestinal SCs to regenerate whole organoids following dissociation [83].(F) Typically, immune/mesenchymal cell subsets interact with the epithelium through the basolateral membrane.For co-culture, non-epithelial cells can be embedded with organoids in the ECM [88], or cultured in the basal compartment of an organoid monolayer [85].Having access to the apical membrane is essential to accurately model certain host-microbe interactions, the internalized apical membrane can be exposed to microbes by fragmenting organoids [97], apical-out organoids can be used [23], or microbial cells can be cultured in the apical compartment of a transwell [17].and specific activity of signaling pathways by homogeneous time-resolved fluorescence (HTRF) [78,97].Microscopy to determine the spatial distribution and quantity of different epithelial populations [65].Morphometric analysis to measure organoid size, crypt (bud) number, crypt depth, villus length, wall thickness, or lumen diameter [57].Gene silencing/editing techniques such as small interfering RNA KD (siRNA KD), short-hairpin shRNA knockdown, and clustered regularly interspaced short palindromic repeats (CRISPR) [65,79].Functional assays -BF assays: permeability dyes such as FITC-dextran/Lucifer yellow or trans-epithelial electrical resistance (TEER) for epithelial integrity [13,15]; absorption assays: uptake or metabolism of nutrients using radio-labelled additives or commercial colorimetric/immunofluorescent assays [16,23]; D-R assays: cell death dyes Annexin V (apoptosis), propidium iodide (PI) or SYTOX nucleic acid stain (necrosis); organoid regeneration assay; proliferationbased assays, such as EdU or Ki67; cell viability (metabolic) assays, such as CellTitre-Glo (ATP), or MTT (3-(4,5-dimethylthiazol-2-yl)-2-5diphenyltetrazolium bromide) [44,61,69,74,78,79]; ROS dyes to assess oxidative stress [79].
regenerative capacity [61].This suggests that COX-2/PGE2 signaling could be compensating for the activity of TNF-α in primary non-responders to sustain inflammation.However, in differentiated colonoids PGE2 treatment increased MUC2+-GC numbers.This study illustrates the complexity of the inflammatory intestinal microenvironment and the context-dependent effects of stimuli.Another example of this complexity is IL-17, which plays a paradoxical role in IBD.It is a well-known cytokine that promotes disease pathogenesis; however, targeting IL-17 using biologics can exacerbate patient symptoms [62].Treatment of enteroids with IL-17a increased the percentage of cells positive for GC and EEC markers, which correlated with the expression of the transcription factor ATOH1, a regulator of secretory cell differentiation [63].This may partially explain why therapeutic targeting IL-17 promotes intestinal dysfunction.Enterochromaffin cells are a subtype of EECs.They are the primary producers of 5-hydroxytryptamine (5-HT) in the intestine, a neurotransmitter that regulates the enteric nervous system and intestinal motility.Enterochromaffin hyperplasia and dysregulated 5-HT production are linked with IBS diarrhea.Treatment of non-IBS colonoids with the neurotrophic factor nerve growth factor increased 5-HT producing cell numbers [64].This provides mechanistic data to support the recent evidence that has associated nerve growth factor signaling with the development of earlylife stress-associated intestinal disorders such as IBS.
Metabolic disorders such as obesity are associated with alterations to microbiome composition.PCs regulate the microbiome through the production of antimicrobial peptides such as defensins.The effects of a high-fat diet were modeled by treating colonoids with the long-chain fatty acid palmitic acid, this resulted in an increased expression of fatty acid binding protein (FABP4) while decreasing defensin 5 expression.This was likely caused by FABP4-mediated ubiquitination of the transcription factor PPARγ, a master regulator of lipid metabolism [65].This reduction in colonic defensin 5 expression induced by fatty acid exposure could partially explain the microbiome alterations linked to metabolic disorders.
IECs can secrete chemokines and cytokines that recruit immune cells and regulate the mucosal immune response.Type I IFNs are known to resolve infection and promote restitution of the epithelium [66]; however, emerging evidence suggests that type I IFN signaling is involved in the development of anti-TNF-resistant IBD [67].Recent organoid studies have demonstrated how type I IFN can drive inflammation by inducing the release of proinflammatory cytokines.ISG15 is a type I IFN-regulated protein that has cytokine-like activity when secreted in its free form.The expression of ISG15 is increased in the colonic epithelium of patients with active UC and CD [68].Non-IBD colonoids stimulated with type I IFN, TNF-α, or the TLR3 ligand Poly (I:C) secrete higher levels of free ISG15 [68].Co-treatment of peripheral blood mononuclear cells with free ISG15 and IL-12 was shown to increase secretion of IFN-γ.Type I IFN treatment of non-IBD colonoids, in combination with TNF-α, induced release of the proinflammatory cytokines IL-1β/IL-18 through a novel mechanism independent of the inflammasome [69].
Intestinal organoids can be used as a model system to investigate novel treatments that promote homeostatic secretory functions and reduce inflammation.Aquamin was shown to increase the production of mucins and trefoils (peptides that interact with mucins to stabilize the mucous layer) in UC colonoids [55].Two bioactive plant-derived compounds, andrographolide, and isoliquiritigenin, could synergistically increase gene expression of the AMP human β-defensin 3 in healthy colonoids [70].Similarly, the JAK inhibitor tofacitinib prevents type I and II IFN-induced secretion of inflammatory cytokines and chemokines from non-IBD colonoids [69].

Cell death and regeneration
Chronic intestinal inflammation is associated with increased levels of IEC death, increased release of damage-associated molecular patterns, and a reduction in the regenerative capacity of the epithelium [71].This is thought to lead to a breakdown in barrier function and a loss of immune regulatory mechanisms.Organoids are sensitive to several proinflammatory stimuli that promote cell death.They also contain a heterogeneous population of stem and progenitor cell types, making them ideal for studying epithelial regeneration post-injury.
Alterations in cytokine signaling promote IEC death and drive IBD pathology [72].Organoids can be used to study the immunomodulatory and cytotoxic effects of IBD-associated cytokines and to identify therapeutics to prevent epithelial cell death.Colonoids treated with IL-17a undergo growth arrest and a form of inflammasome-dependent immunogenic cell death called pyroptosis [73,74], which can be rescued using caspase 1 and iNOS inhibitors.TNF-α treatment increases the protein level of mixed-lineage kinase domain-like protein, the molecular executioner of the inflammatory cell death necroptosis, in CD enteroids [75].CD enteroids homozygous for mutations to the IBD genome-wide association studies autophagy gene ATG16L1 (ATG16L1 T300A ) are sensitive to TNF-α-induced cell death, which is rescued by necroptosis and JAK inhibitors [76].Type I, II, and III IFNs and TNF-α synergize to induce cytotoxicity in ileal, ascending colon, and sigmoid colon organoids [77].Our group has demonstrated that this is a non-canonical form of cell death which is independent of apoptosis, pyroptosis, and necroptosis [69,78].We also determined that the JAK inhibitors tofacitinib and upadacitinib can rescue this synergistic cell death [69,78].
The regenerative capacity of the intestinal epithelium is altered by prolonged inflammation.Organoid models of chronic inflammation can be used to understand the molecular events that underpin this process and identify mechanisms to reverse it.Colonoids exposed to a cocktail of TNF-α, IL-1β, and the TLR5 agonist flagellin for 60 weeks display increased expression of apoptotic markers, elevated ROS production, and decreased colonoid regenerative capacity [79,80].Using this model, Schlafen 11 (SLFN11, a putative DNA/RNA helicase involved in cell death) was identified as a novel negative regulator of epithelial healing and confirmed to be increased in the colonic tissue of patients with UC [79].The model was also used to determine that a reduction in telomere length is associated with chronic inflammation and that telomerase activators can partially reverse the inflammatory organoid phenotype [80].Chronic damage of IECs can alter their functionality post-regeneration.For example, colonoid monolayers subjected to multiple alternating cycles of air-liquid interface and submerged culture (submergence damage) lose sensitivity to stimulation with bacterial ligands and acquire a more differentiated phenotype [81].
For long-term remission in IBD, the complete regeneration of the intestinal epithelium is required ("mucosal healing").However, the IBD microenvironment causes intrinsic changes to CBC-SCs that can alter their regenerative capacity.CD enteroids generated from inactive mucosa treated with TNF-α display Lgr5+ SC dysfunction [75,82].This leads to a reduction in enteroidforming efficiency compared with non-IBD enteroids, which can be rescued by PGE2 co-treatment [75].Unexpectedly, Suzuki et al. [83] found that untreated enteroids generated from CD-active tissue have higher levels of enteroid-forming efficiency compared with CD-inactive and non-IBD enteroids.

Epithelial cell-cell interactions
Appropriate cell-cell communication between the epithelium and other intestinal cell populations is essential for maintaining homeostasis and coordinating immune responses.Alterations to intercellular signaling networks that occur in intestinal chronic inflammatory disorders promote pathological processes and sustain inflammation [84].Intestinal organoid co-cultures can be used to study crosstalk between the epithelium and other isolated intestinal cell types in a simplified context.
Innate immune cells support epithelial homeostasis and are the first line of defense against infection when the intestinal monolayer is breached.Protocols have been developed for the non-autologous (cell populations obtained from different patients) co-culture of peripheral neutrophils, monocytes, and macrophages with enteroid and colonoid monolayers [85,86].Monolayers are established on the apical side of a transwell, followed by transwell inversion and seeding of immune cells on the basolateral membrane surface.The immune cells are allowed to adhere and the transwell is returned to its original orientation.Using this approach, it was shown that monocyte-derived macrophages promote differentiation of the intestinal monolayer and partially restore the loss of epithelial barrier integrity caused by enterotoxigenic E. coli infection [85].
T cells are known to drive the pathogenesis of intestinal inflammatory disorders due to excessive cytotoxicity and production of pro-inflammatory cytokines.T cells and organoids have been co-cultured indirectly using transwell systems (with T cells cultured in the basolateral chamber) [87], or directly by embedding both populations in an ECM [88,89].Using the indirect coculture method, it was demonstrated that secretion of inflammatory mediators by T cells is sufficient to destroy colonoid monolayers [87].An autologous co-culture system (cell populations obtained from the same patient) was developed to study disease and patient-specific interactions between mucosal lymphocytes and enteroids [88].Direct co-culture of CD CD3+ T cells, but not non-IBD CD3+ T cells with enteroids resulted in an increased epithelial expression of pro-apoptotic caspase 3.This phenotype was enhanced by the stimulation of the co-culture with the microbial TLR4 LPS, illustrating the potential proinflammatory role of microbiota-derived pathogen-associated molecular pattern molecules in the pathogenesis of CD [88].Blocking lymphocyte-epithelial interactions with the anti-β7 integrin subunit monoclonal antibody Etrolizumab (phase III candidate biologic for the treatment of IBD) reduced T cell infiltration, caspase 3 expression, and production of proinflammatory cytokines [88].Yokoi et al. [89] used nonautologous co-culture to investigate the potential colitogenic activity of a newly identified CD-specific population of intestinal CD103 + CD4 + CD161 + CCR5 + tissue-resident memory T cells.Direct co-culture of healthy intestinal organoids with this CD-T cell-population induced epithelial cytotoxicity as measured by LDH release.Cytotoxicity was dependent on treatment with an IBD relevant cytokine cocktail (IL-7, IL-12, IL-15, IL-18) and was fully rescued by an IFN-γ neutralizing antibody.This work provided functional evidence of a pathological role for CD4 + tissue-resident memory T cells in CD, a cell type rarely characterized in IBD.
Intestinal graft-versus-host disease is a complication that can occur after patients receive allogeneic hematopoietic cell transplants (allo-HCT), with donor T cells attacking host tissue.Intestinal graft-versus-host disease and CD share several pathological features, including associations with the ATG16L1 T300A variant [76].IBD patient enteroids are more sensitive to tissue damage when directly co-cultured with allogenic compared with syngeneic T cells [76].It was also demonstrated that ATG16L1 T300A homozygous enteroids are more susceptible to allogenic T cell mediated cytotoxicity, suggesting that autophagy protects against allo-HCT-induced cell death.
Mesenchymal cells and endothelial vasculature support the intestinal epithelium by supplying essential growth factors and nutrients.A triple co-culture system using enteroids, mesenchymal, and endothelial cells was established using a commercially available microfluidic cell culture device [90].When cultured in the presence of FGF and vascular endothelial growth factor, the triple co-culture promoted the development of SC-enriched organoids [90].

Epithelial cell-microbial interactions
The intestinal epithelium must maintain an effective barrier to prevent microbial (pathogenic) translocation while simultaneously allowing bidirectional communication between the microbiota (commensal) and the host.Inappropriate host-microbe communications and alterations to microbiome composition are thought to contribute to the development of inflammatory disorders [91].Intestinal organoids are ideal for modeling disease-specific interactions between intestinal microbes and the epithelium.
Fecal supernatants can be used as a reductionist in vitro model of the microbiome.Several studies have analyzed the response of organoids to fecal supernatants derived from patients with various GI conditions.Treatment of healthy colonoid monolayers with IBS fecal supernatants increased gene expression of immune regulatory, proinflammatory cytokines, and epithelial junctional complexes while decreasing TLR5 expression [92].Arnauts et al. [93] attempted to determine if epithelial cell defects or the microbiota are responsible for barrier dysfunction in UC.Using colonoid monolayers they found that UC fecal supernatants disrupted barrier function independent of patient origin (UC vs non-IBD).However, UC colonoid monolayers were more sensitive to the UC-derived microbiota in the context of combined cytokine and flagellin-induced inflammation.In a comparative study, healthy apical-out colonoids were shown to have differential transcriptional responses to fecal supernatants from patients with UC, IBS, and CRC [94].Overall, these studies suggest that interactions between the intestinal epithelium and luminal microbes are altered in GI conditions and that these changes are disease-specific.
Other studies have focused on individual species of bacteria within the microbiome that may drive inflammation in intestinal disorders.Fusobacterium nucleatum is a gram-negative opportunistic pathogen associated with IBD.The >50 kDa fraction of F. nucleatum conditioned media induced TNF-α secretion and increased NF-κB, extracellular signal-regulated kinase 1/2, and cAMP response element-binding protein signaling in colonoid monolayers [95].Adherent-invasive E. coli (AIEC) is a pathobiont that has been implicated in the pathogenesis of CD.A detailed methodology was established to study the invasion of colonic epithelium by AIEC using healthy colonoid monolayers [17].This protocol can be used to study mechanisms of AIEC pathogenicity and to characterize newly isolated IBD-associated strains.Klebsiella pneumoniae is a UC-associated gram-negative bacterium belonging to Enterobacteriaceae.K. pneumoniae was found to induce epithelial pore formation in healthy colonoid monolayers in a strain-specific manner [96].Epithelial pore formation would enable bacterial translocation in vivo, leading to the stimulation of underlying immune cells.Using colonoids, it was determined that LPS from K. pneumoniae can bind to caspase 4 and that infection induces secretion of the potent immunoregulatory cytokine IL-18 [97].This suggests that K. pneumoniae LPS can activate the epithelial noncanonical inflammasome pathway and trigger a pyroptotic inflammatory cascade.These two studies are excellent examples of how organoid-microbe co-cultures can be utilized to identify potential microbial mechanisms that promote IBD pathogenesis.

Limitations and future perspectives
Advances in organoid technology and its application have increased the possibility of examining more complex biologi-cal questions using primary cells.However, like any model system, there are limitations and drawbacks that must be considered.These include technical issues, problems with variability/reproducibility, and considerations regarding the level of physiological relevance.
Organoids are large, complex, tissue-like structures.For these reasons, they can be technically difficult and time-consuming to work with compared with traditional in vitro cell culture models.Most bioassays and commercially available kits have been optimized for use with cancer cell lines grown as flat monolayers on plastic culture dishes.The ECM present in organoid cultures can interfere with downstream assays, consequently, it must be dissociated before the isolation of protein/nucleic acid and for immunofluorescence processing [98,99].Organoids are more resistant to lysis than traditional cell cultures, so it may be necessary to optimize lysis protocols for efficient extraction.Due to their size and complexity, 3D-microscopy modalities such as confocal or light sheet microscopy are required for effective organoid imaging [100].
Organoid lines are generated from individuals, therefore, there is genetic and environmental variability even before commencing culture.This variability is increased by the culture environment the organoids are subjected to.It has been demonstrated that alterations in the transcriptional profiles of organoids are more associated with culture conditions, growth media, and donor variability than disease status [13].There are several aspects of organoid culture that increase variability and cause reproducibility issues.The universally implemented commercial ECM substrates (e.g.Matrigel) have undetermined concentrations of components, suffer from batch-to-batch variability, and contain animal components.Alternatively, defined synthetic hydrogels can be used [101]; however, there is limited availability, and they lack inherent biological activity.The most common approach for sourcing niche growth factors is to produce conditioned media in-house from mouse fibroblast L-cells.Conditioned media production is problematic due to batch-batch and site variability [102] as well as the use of different L-cell lines between labs [99,102].The alternative is to use defined media such as the commercially available media from STEMCELL technologies; however, it is expensive and a "black box" in terms of its composition.Commercial recombinant proteins can be used, but they have been demonstrated to lack biological activity compared with conditioned media [103].However, engineered surrogate Wnt recombinant proteins have been recently developed to address this lack of activity [104].Growth factor-free culture media supplemented with chemical inhibitors is possible [105], but these inhibitors are likely associated with off-target effects.
Interpretation of data and reproducibility is made more difficult by the omission of important experimental details in the published literature.The inflammatory status, specific intestinal region, and in some cases even the disease state of patient tissue is not always reported.The cell seeding density of organoids and duration of culture are also often not included in the reports.This may be due to the difficulty in accurately normalizing organoid cell numbers.Only viable SCs can generate a new organoid after passaging; however, organoid cells are highly sensitive to dissociation-induced cell death (anoikis), and they contain differentiated cells incapable of generating new organoids.Reproducibility can be improved by using a rho-associated coiledcoil containing protein kinase inhibitor to prevent anoikis; and expanding organoids in an SC enriched state before seeding and differentiating for an experiment [69].
Organoid models do not perfectly replicate all aspects of the in vivo intestine, so caution is recommended when considering their use.Typically, they lack other nonepithelial intestinal cell types and the microbiota.The tissue architecture and crypt-villus gradient of growth factors found in vivo are missing, as well as mechanical stimuli such as physiological fluid flow and peristalsis.It has also been demonstrated that organoids in culture partially lose the transcriptional and epigenetic inflammatory signature of their source tissue over time [106,107].
A new revolutionizing advancement in this area is the development of organ-on-a-chip, a more sophisticated 3D-cell culture system that can incorporate intestinal organoids.These systems utilize microfluidics to measure different readouts in real time, allow for the co-culture of multiple cell types and microbes, and integrate mechanical stimuli [108].However, this technology is still in its infancy and time will reveal the suitability of organ-ona-chip systems for studying GI physiology and disease.

Conclusion
This review highlighted and summarized recent publications using human organoids to model diseases of the lower GI tract with inflammatory or immunomodulatory components, with a particular focus on epithelial functions and associated techniques/assays employed to date.These primary cell systems provide physiologically relevant models that maintain many disease characteristics.Although organoids do not fully recapitulate the complexity of the intestinal tissue, they reflect a major progression from previous cell lines, often cancer models.However, the future looks bright, with frequent advances in organ-on-a-chip technology and organoid co-culture systems that incorporate nonepithelial cell types and the microbiota.

Figure 1 .
Figure 1.Human intestinal organoid models and assays for studying epithelial functions.(A) Traditional organoids are embedded in an ECM, their lumen is internalized, and the apical membrane is difficult to access.Apical-out organoids are generated by removing organoids from the ECM and culturing them in suspension, this promotes a reversed polarity, with the apical membrane facing the media environment, allowing for direct manipulation.Organoid monolayers are produced by seeding SCs onto transwell membranes and culturing them until fully confluent, facilitating accessibility to both basal and apical membranes.The various organoid models contain all the adult IEC types typically found in vivo.By modifying certain media components, it is possible to enrich for SCs or promote the differentiation of specific IEC subsets[99].(B) Absorption can be measured by incubating apical-out organoids with a fluorescent fatty acid analog and then imaging with confocal microscopy[23].(C) FITC-dextran is a fluorescently labeled polysaccharide used to measure epithelial paracellular permeability, an indicator of barrier function.FITC-dextran flux from the media environment into the organoid lumen can be measured by live fluorescent microscopy[15], and flux across organoid monolayers, from the apical to basal compartment, is determined by measuring media fluorescent intensity with a plate reader[13].TEER can also be used to assess transwell monolayer barrier function; it is calculated by measuring the impedance between two electrodes caused by the presence of the