Progress in human intestinal organoid research: applications to acute gastroenteritis viruses

Abstract

Acute gastroenteritis viruses, such as rotavirus, human norovirus, human astrovirus, human adenovirus, human sapovirus, represent significant threats to global public health. Research on these pathogens has long been hampered by the limitations of conventional models. Animal and cell-based systems, widely used in virological studies, show limited efficiency in supporting rotavirus replication, while noroviruses remain largely non-cultivable in these settings. Organoids—complex, three-dimensional multicellular structures derived from stem cells—exhibit organ-specific characteristics and spatial organization, making them promising tools for viral research. Intestinal organoids, in particular, recapitulate key features of the gut epithelium and have emerged as versatile platforms for investigating viral pathogenesis and developing intervention strategies. This review systematically outlines the cultivation and functional properties of human intestinal organoids, as well as the evolution and progress of their application in studying acute gastroenteritis viruses. However, current intestinal organoid models are primarily composed of epithelial cells and lack immune and other non-epithelial components, thereby limiting their ability to fully simulate host–pathogen interactions and immune responses following infection. Future efforts should focus on incorporating emerging technologies, such as CRISPR/Cas9 gene editing, to develop more physiologically relevant intestinal models that better mimic in vivo conditions.

Introduction

Acute gastroenteritis is a common gastrointestinal illness primarily characterized by symptoms such as diarrhea, abdominal pain, nausea, and vomiting. While typically self-limiting, severe cases can lead to dehydration and electrolyte imbalances, particularly in children and immunocompromised individuals. A variety of viruses are among its principal causative agents, such as rotavirus (RV), human norovirus (HuNoV), human astrovirus (HAstV), human adenovirus (HAdV), human sapovirus (HuSaV), being leading causes of the disease worldwide [1]. Studies on viruses are usually carried out using transformed cell lines or animal models. However, due to the unique structure and physiological functions of tissues in different parts of the intestine, it is difficult to fully simulate the structure and function of the human intestine and the process of disease occurrence and development using a single transformed cell line or animal model, and they have different genetic characteristics from normal cells, which leads to the limitations of the research results [2, 3] .

Organoids are organ specific cell cultures with a certain spatial structure formed by using adult stem cells or pluripotent stem cells for three-dimensional (3D) culture in vitro. They recapitulate the original organs in terms of cellular composition, structural organization, extracellular matrix characteristics, and physiological properties. In 2009, Clevers’ group identified Lgr5⁺ as a marker of mouse intestinal epithelial stem cells and showed that multicellular intestinal epithelial cultures, namely intestinal organoids, could be established from isolated Lgr5⁺ cells in vitro. Based on the mouse intestinal organoid culture method, human intestinal organoids (HIOs) were successfully cultured in 2011 [4]. HIOs are isolated from intestinal crypts, or formed by human embryonic stem cells (hESCs) or human pluripotent stem cells (hPSCs) under the condition of providing physical and chemical signals required for epithelial tissue and polarization. They usually contain crypt villus structures and various differentiated cell components, such as intestinal epithelial cells, goblet cells and enteroendocrine cells [5].

At present, HIOs is a new and widely used model for in vitro study of intestinal pathophysiology. Its application can promote the study of the infection mechanism of intestinal pathogens, and may realize the in vitro culture of specific pathogens, providing a new model for the study of new antiviral strategies. This article reviews the research progress in the cultivation, function, optimization and application of HIOs and the applications in studies on acute gastroenteritis viruses.

Construction and application of HIOs

Construction of HIOs

The intestinal epithelium is not only the largest single barrier between the organism and the external environment, but also a multifunctional and homeostatic functional organ unit that integrates mechanical defense, digestion and absorption, immune sensing, and hormone secretion. The majority of intestinal epithelial cells are absorptive enterocytes responsible for digestion and absorption, while other cell types include small intestinal Paneth cells, goblet cells, hormone-secreting enteroendocrine cells, and microfold cells. Among these, small intestinal Paneth cells maintain stem cells and their functions through the Wnt pathway and Notch ligands. Lgr5⁺ stem cells located at the base of crypts can differentiate into a variety of highly specialized cells, which migrate and differentiate toward the tips of small intestinal villi or the surface of the colon, driving the continuous renewal of the intestinal epithelium [6, 7]. Studies using heterozygous Lgr5-lacZ mice have revealed that the expression of Lgr5 is diminished in nearly all tissues at birth, while in adult mice, its expression is restricted to rare and scattered cells in the eyes, brain, hair follicles, mammary glands, reproductive organs, stomach and intestines [7]. LGR5⁺ stem cells in the small intestine intercalate with Paneth cells at the base of small intestinal crypts. Most colonic crypts lack Paneth cells, but they contain secretory cells that express typical Paneth cell markers and Reg4—namely Reg4⁺ cells—which are adjacent to LGR5⁺ stem cells in colonic crypts. Similar to Paneth cells in the small intestine, Reg4⁺ cells can maintain LGR5⁺ stem cells in the colon [5, 8].

The maintenance of the structure and function of the intestinal epithelium relies on cell-cell interactions mediated by receptors, adhesion molecules, and other factors, with the extracellular matrix playing an important role in this process. Most undifferentiated epithelial cells undergo apoptosis when deprived of cell-matrix interactions. Matrigel is mainly composed of extracellular matrix proteins such as laminin, type IV collagen, and growth factors. Therefore, the proliferation and differentiation of HIOs require Matrigel support. The generation of HIOs also requires Wnt, Notch, and epidermal growth factor (EGF) signaling to sustain stem cell self-renewal and normal epithelial maintenance. Among these pathways, the Wnt and Notch signaling pathways regulate the proliferation and differentiation of crypts; Wnt agonist R-spondin 1 can enhance Wnt responses, while EGF signaling is associated with intestinal proliferation. Lgr5⁺ stem cells are highly dependent on Notch signaling. In addition, supplementation with Noggin during HIO culture can induce an increase in the number of crypts [6, 9–11].

HIOs can be categorized into small intestinal organoids and large intestinal organoids, each with different structural and functional characteristics and unique research values. Since the large intestine is primarily composed of the colon, large intestinal organoids mainly refer to colonic organoids.

Currently, there are two methods for culturing HIOs: derivation from adult intestinal tissues and from human pluripotent stem cells. The first method involves culturing intestinal crypts isolated during intestinal biopsy from patients; the second entails directed differentiation of in vitro hESCs or hPSCs into HIOs [12]. Intestinal crypts isolated from the epithelium need to be cultured in Matrigel containing cytokines such as Wnt3a, EGF, R-spondin1, and the BMP inhibitor Noggin, where they further expand to form HIOs consisting of several crypt domains surrounding a central lumen, with villus domains between the crypt domains. LGR5⁺ stem cells divide to generate self-renewing stem cell populations and differentiate into cells such as intestinal epithelial cells, which eventually squeeze into the lumen from villus-like domains, forming a structure similar to human intestinal epithelium [4]. HIOs can also be derived from the differentiation of in vitro hESCs or hPSCs, with a cultivation process mimicking embryonic intestinal development. hESCs or hPSCs are treated with activin A for 3 days to induce differentiation into FOXA2⁺/SOX17⁺ endoderm. Subsequently, FGF4 and WNT3a are used to convert the endoderm into CDX2⁺ mid/hindgut tissue, and 3D midgut/hindgut spheroids emerge from the monolayer epithelium attached to the tissue culture dish. These spheroids are then further cultured and expanded in Matrigel containing necessary growth factors to form villus-like organoids with polarized columnar epithelium. Composed of crypt-like domains, these organoids can differentiate into all cell types of intestinal epithelial cells (Fig. 1) [13–15].

Fig. 1.

The cultivation of HIOs: there are currently two methods for HIOs cultivation: the first is derived from adult intestinal tissue, which is cultured from intestinal crypts isolated during patient intestinal biopsy; The second type is derived from human stem cells, which differentiate into HIOs through in vitro hESCs or hPSCs

Functions and applications of HIOs

While not bona fide human organs, HIOs recapitulate key features of native organs in multiple dimensions. They encompass most of cell types found in in vivo intestinal tissues after differentiation and exhibit physiological functions analogous to those in situ, including the digestion and absorption of nutrients as well as the regulation of water and electrolyte homeostasis. Notably, they do not contain intestinal neural or immune cell components (Table 1) [16].

Table 1.

The application of HIOs

Application	Objectives
Physiological study of intestinal epithelium	To simulate human intestinal development and investigate the impact of key signaling pathways on the maturation and regulation of different epithelial cell types [17, 18].
	To study physiological functions such as intestinal permeability and metabolic stability [19].
Research and modeling of disease mechanism	To study the mechanism and treatment of inflammatory bowel disease, cystic fibrosis and other intestinal diseases [20–22].
Intestinal microbiological research	To study the interaction between intestinal tract and intestinal symbiotic bacteria or pathogens [23, 24].
Drug development and screening	To develop and screen drugs for intestinal infection, inflammation or metabolic diseases, and test toxicity and efficacy [25, 26].
Regenerative medicine and tissue repair	To explore the mechanism of intestinal epithelial regeneration and the possibility of stem cell transplantation to promote the repair of intestinal injury [27, 28].

Application of HIOs in RV research

Human RV are the most significant cause of virus-associated diarrhea in children under 5 years of age. They primarily infect enteroendocrine cells to produce enterotoxins, leading to gastroenteritis and subsequent severe diarrhea [29, 30]. As double-stranded RNA viruses, RVs consist of 11 highly variable gene segments, resulting in extensive genomic diversity among patient-derived RV strains (Fig. 2).

Fig. 2.

Schematic diagram of the application of HIOs in the study of acute gastroenteritis virus

Support of RV cultivation by HIOs

Most in vitro studies on RV pathogenesis have been conducted in cultured cells, primarily using simian RV strains to infect homologous simian kidney cell lines or heterologous human colon adenocarcinoma cell lines. This approach is partly attributable to the limited availability of untransformed human small intestinal cell lines suitable for culture [31]. Conventional two-dimensional (2D) cell models used for virus propagation fail to fully recapitulate the dynamic changes and interindividual variations seen in patients. Moreover, even following adaptation to cell culture, viral titers generally remain low. Therefore, there is a clear need for a stable in vitro model derived from human cells to advance the study of RV infection.

In 2012, Finkbeiner et al. demonstrated that HIOs support RV replication, including laboratory isolates and 12/13 clinical RV isolates, indicating that HIOs can be used to establish models for studying RV and other gastrointestinal viruses [32]. Saxena et al. found that HIOs infected with RV can detect physiological luminal dilation, indicating that HIOs can be used for pathological and physiological studies of RV infection [33]. Studies on HIOs indicate that RV infection occurs in differentiated intestinal epithelial cells and intestinal endocrine cells [34].

Development of anti-RV drugs

HIOs can be applied to the research and development of anti-RV drugs. Yin et al. utilized the human intestinal cell line Caco-2 and primary HIOs to confirm that RV infection induces the upregulation of interferon-stimulated genes in organoids. They found that IFN-α, ribavirin, and mycophenolic acid can inhibit RV replication in HIOs. When evaluating the response of RV strains from patients, different responses to IFN-α and ribavirin were observed, suggesting that HIOs may be applicable for personalized assessment of antiviral drugs [35, 36]. Yin et al. have also demonstrated that 6-thioguanine, which is widely used as an immunosuppressant in the treatment of inflammatory bowel disease (IBD), can effectively inhibit RV replication by suppressing Rac1 activation. Thus, it represents a potential therapeutic option for IBD patients infected with RV or at risk of RV infection [37].

Saxena et al. established a RV infection model using HIOs and found that exogenous IFN treatment could limit RV replication, with type I IFN exhibiting superior inhibitory effects. However, RV replication was not affected by endogenous IFN, suggesting that type I IFN derived from epithelial sources may play a critical role in suppressing RV replication [38]. In a study by Chen et al., two dihydroorotate dehydrogenase (DHODH) inhibitors, brequinar (BQR) and leflunomide (LFM), effectively inhibited RV replication in both Caco-2 cells and primary HIOs by inhibiting DHODH and consuming intracellular pyrimidine, thereby exerting antiviral activity [39]. They also confirmed using Caco-2 cells and HIOs that the anticancer drug gemcitabine effectively inhibits RV infection by suppressing pyrimidine biosynthesis, supporting its potential use in treating RV-infected cancer patients [40].

Host-RV interactions

Yin and colleagues used both Caco-2 cell model and primary HIOs in their study to investigate the effect of PI3K-Akt-mTOR signaling pathway on RV infection. They found that blocking PI3K, mTOR, and 4E-BP1 in this pathway can produce anti RV activity [41]. Ding’s team used a genome-wide CRISPR-Cas9 screen to find host factors that RV relies on, along with new regulators of the innate immune system. This work uncovered a fresh role for STAG2: when STAG2 is exhausted, it triggers a series of biological behaviors, including damage the host’s genomic DNA, let the cell identify microchromatin in the cytoplasm, and fire up the cGAS-STING-IRF3 signaling pathway, ultimately leading to the inhibition of RV. This result had also been validated on HIOs [42]. Liu and others used MA104 cells and HIOs to show that RV infection can cause the interaction between glyceryl diester O-acyltransferase 1 (DGAT1) and K48 polyubiquitinated RV non structural protein NSP2, leading to the degradation of DGAT1. And they also noted that silencing or knocking out DGAT1 makes RV replicate better [43].

Application of HIOs in HuNoV research

HuNoV is one of the main causes of foodborne diseases and acute gastroenteritis across all age groups, accounting for one-fifth of all viral acute gastroenteritis worldwide. It is estimated that HuNoV infections reach 685 million cases annually globally, resulting in 200,000 deaths, including 50,000 children [44]. The difficulty in in vitro cultivation of HuNoV has significantly hindered the progress of related research.

HIOs enable in vitro cultivation of HuNoV

Prior to the application of HIOs, research on HuNoV was primarily conducted using surrogate systems. These included viruses that are genetically related to HuNoV and amenable to cultivation, such as murine norovirus (MNV), feline calicivirus, and swinepox virus. MNV can be propagated in murine RAW264.7 cells and is commonly used to investigate viral infection, antiviral drugs, and norovirus inactivation. However, mice cannot be infected with HuNoV due to the discrepancy between the entry receptors of MNV and those of HuNoV. In addition to these, other surrogate tools for studying the infection mechanism of HuNoV include the GI.1 norovirus replicon and virus-like particle (VLP) systems, which can recapitulate HuNoV replication and adsorption at the technical level, respectively [44].

In 2016, Ettayebi et al. developed a new HuNoV culture system, achieving the first time in vitro culture since the discovery of HuNoV in 1968 by using HIOs. This also provided a reference framework for studying human host-pathogen interactions involving pathogens that were previously unculturable in vitro. Additionally, they found that the replication of certain HuNoV strains requires the presence of bile [45]. The Costantini’s research group used the HIOs model to observe the replication of the six GII genotypes of HuNoV, including three GII.4 variants, and its replication depended on the viral load and genotype of the inoculum. In addition, repeated infection within 1 year showed that the viral titers of the three GII.4 variant strains continued to increase, indicating the stability of the HIOs model [46]. According to the study of CO JY et al. on controlling epithelial polarity, Mirabelli et al. established a HuNoV infection model in HIOs undergoing differentiation and spontaneous polarity reversal, which has the advantages of shorter experimental time, higher infection rate and reproducibility [47, 48].

Host genetic susceptibility to HuNoV

The human histo blood group antigens (HBGAs) is an important factor for host susceptibility to HuNoV, and its high variability is determined by genetic factors. HuNoV recognizes and binds to HBGAs in a strain specific manner. After binding, the virus will enter the cell and replicate in the cytoplasm. The expression of HBGA and mutations in the capsid protein may both influence the susceptibility of the host to HuNoV [49]. The expression of HBGAs can be affected by fucosyltransferase 2 (FUT2) gene in intestinal epithelial cells [50]. To explore the role of FUT2 in HuNoV replication, Haga et al. constructed FUT2-knockout HIOs. The results showed that FUT2 expression could affect the binding of HuNoV to the surface of HIOs cells and the susceptibility to HuNoV. A previous study found that overexpression of the FUT2 gene in transformed cell lines such as Huh-7 increased viral binding to cells but did not enhance susceptibility to HuNoV [51]. Therefore, in conjunction with the study by Haga et al., the expression of FUT2 is a necessary but not sufficient condition for the replication of HuNoV in HIOs [52].

Host-HuNoV interactions

Intestinal epithelial cells are the first line of defense of the host against HuNoV invasion, and limit virus replication through the innate immune response. The work of Hosmillo et al. revealed that interferon induced JAK/STAT signaling pathway and RNA polymerase II-mediated restriction of HuNoV replication in intestinal epithelial cells, providing new insights into the way to control HuNoV infection and enhance the robustness of HuNoV culture [53].

HuNoV inactivation and environmental monitoring

Historically, the difficulty in in vitro culturing HuNoV has hindered the development of prevention and control strategies. The evaluation of the effectiveness of inactivation and disinfection procedures can only be carried out through the use of alternative viruses and other methods. Thus, HIOs have also been applied to assess the efficacy of disinfection methods in inactivating HuNoV [46]. Multiple research teams, using HuNoV-infected HIOs, found that gamma radiation, heat treatment, alcohol-based disinfectants containing electrolytes, and sodium hypochlorite can all inactivate HuNoV [45, 46, 54].

Environmental monitoring is crucial for preventing HuNoV transmission. Noroviruses are typically transmitted person-to-person via the fecal-oral route but can also spread through environmental contamination, such as contact with polluted food, water, or infectious aerosols generated by vomiting [55]. When water sources like seawater become contaminated, shellfish farmed in coastal areas can accumulate the virus. Due to the high stability and infectivity of HuNoV particles in external environments such as water, humans can be infected even with small viral loads in shellfish [56].

As an emerging HuNoV culture system, HIOs have also been used to detect HuNoV in environmental water sources, assess the persistence of infectious HuNoV in freshwater and seawater, and predict a higher likelihood of detecting infectious HuNoV in contaminated water using HIOs [57]. Overbey et al. optimized HIOs culture protocols for environmental HuNoV monitoring, finding that a 250 µL inoculum volume improves the detection of low-concentration HuNoV in environmental isolates [58].

Development of anti-HuNoV drugs

The screening of anti-HuNoV drugs is primarily conducted through MNV culture systems, in vitro enzyme activity assays (polymerase assays or protease assays), and computer screening. Following the discovery of HIOs’ applicability in HuNoV cultivation, HIOs have also been utilized for the screening and evaluation of anti-HuNoV drugs.

Hayashi et al. screened compounds with anti-HuNoV activity from a viral compound library using the HIOs cultivation system and found that dasabuvir, an anti-hepatitis C virus drug, could also inhibit HuNoV infection in HIOs. Therefore, they concluded that dasabuvir is a novel anti-HuNoV drug [59]. Simultaneously, Hayashi et al. screened 22 crude drugs from traditional Japanese medicine “Kampo” using the HIOs model and identified Ephedra herb as a significant inhibitor of HuNoV infection [60].

Since HuNoV requires binding to HBGAs before entering cells, drugs that can block HuNoV from binding to HBGAs may inhibit HuNoV infection. Based on the fact that 2′-fucosyl lactose (2′FL) binds to the same site as HBGAs in some HuNoV strains, Patil et al. evaluated the effect of 2′FL on GII.4 Sydney HuNoV using HIOs and found a significant reduction in HuNoV replication. Therefore, Patil et al. suggested that 2′FL holds promise for further development as a therapeutic agent against HuNoV infection [61].

Application of HIOs in research on other acute gastroenteritis viruses

Human astrovirus

Human astroviruses (HAstV) are small, non-enveloped positive-strand RNA viruses and represent one of the important pathogens causing viral acute gastroenteritis in children, accounting for 2–9% of non-bacterial diarrhea cases in children [62]. Kolawole et al. utilized HIOs to investigate HAstV infection parameters and antiviral host responses. RNA sequencing analysis of HIOs infected with the HAstV VA1 strain revealed that interferon-mediated antiviral responses constitute the primary host response to VA1 infection. Compared to Caco-2 cells, VA1-infected HIOs exhibit a stronger IFN response and greater sensitivity to exogenous interferons, which can inhibit viral infection, whereas endogenous interferons only restrict but cannot completely block viral infection [63]. Triana et al. performed single-cell sequencing and multiplex single-molecule RNA FISH analysis on HIOs infected with HAstV1, and found that HAstV can infect all types of intestinal cells, and different types of intestinal cells can exert antiviral effects by upregulating the expression of different interferon stimulated genes after HAstV infection [64].

Human adenovirus

Human adenoviruses (HAdV) are non-enveloped double-stranded DNA viruses. Infections by the HAdV-F subgenus can cause acute gastroenteritis and represent one of the common pathogens leading to viral diarrhea in infants. Holly et al. successfully cultured human adenoviruses (HAdV) and their clinical isolates using HIOs. Among them, HAdV-5p, HAdV-16p, and HAdV-41p can infect undifferentiated HIOs, while HAdV-41p can also infect differentiated HIOs. Additionally, they found that interferons can inhibit the replication of HAdV in HIOs [65].

Human sapovirus

HuSaV is a positive-sense, single-stranded RNA virus belonging to the Caliciviridae family, the same as HuNoV. HuSaV can cause acute gastroenteritis in individuals of all age groups worldwide, with symptoms, severity, and duration varying among individuals [66]. Since its discovery in 1976, HuSaV has failed to be cultured in vitro. It was not until 2020 that Takagi et al. successfully cultured HuSaV in the HuTu80 and NEC8 cancer cell lines, but stable in vitro models for HuSaV cultivation remain limited [67]. In 2023, Gabriel Euller-Nicolas et al. successfully cultured HuSaV in a HIOs model, unaffected by its secretory state or group blood antigen expression, suggesting that HIOs models are suitable for studying host interactions [68]. Matsumoto et al. utilized the HIOs model to investigate conditions for inactivating HuSaV through heat and alcohol, finding that HuSaV exhibits similar sensitivity to heat-induced inactivation and alcohol treatment as HuNoV [69].

Coronavirus

Zhou et al. demonstrated that HIOs exhibit high sensitivity to MERS-CoV, with the virus capable of sustained replication within these models [70]. Lamers et al. identified that HIOs contain intestinal cells with elevated angiotensin-converting enzyme 2 (ACE2) expression, which can be infected by both SARS-CoV and SARS-CoV-2 [71]. Zhao’s team compared the susceptibility of HIOs to these two coronaviruses, revealing that SARS-CoV replicates more actively, triggers a weaker IFN response, and demonstrates greater resistance to IFN. Consequently, SARS-CoV spreads more robustly in HIOs [72]. Their findings suggest that HIOs may serve as viable in vitro models for coronavirus research.

Enterovirus

Zhao et al. improved the villus differentiation of HIOs by optimizing the differentiation medium, enabling better simulation of human intestinal epithelium. They utilized the optimized HIOs model to culture and compare the replication capacities of two enteroviruses, Enterovirus A71 (EV-A71) and Coxsackievirus A16 (CVA16), finding that EV-A71 exhibited higher replication rates in HIOs compared to CVA16, which triggered more intense cellular responses [72].

Advantages, limitations, and optimization of HIOs

Advantages and disadvantages of HIOs

A fundamental advantage of organoids lies in their ability to be cryopreserved, rapidly and indefinitely proliferated, with significant advantages in terms of cost, modeling success rate, and high-throughput analysis [73]. Compared to traditional cell models, HIOs derived from stem cells possess a complex cellular environment, enabling better simulation of tissue development processes and recapitulation of interactions between the organism and pathogens. In contrast to in vivo models, HIOs, developed from hESCs or hPSCs—and even from hPSCs of patients carrying specific mutations—can address the challenge of interspecies differences in pathogen infections, making them potentially more suitable for studying human-related diseases and formulating personalized medical plans [4].

Despite these advantages, current organoid culture techniques produce models containing only epithelial cells, lacking vascular systems, immune cells, and neurons, thus presenting certain limitations. In recent years, with the emergence and development of various emerging technologies such as co-culture of organoids with immune cells, optimization of viral infection pathways, and CRISPR/Cas9 gene editing, organoid technology will gradually address these shortcomings. This will facilitate the construction of research models that are more accurate and closer to in vivo intestinal tissues, which, together with 2D cell models and animal models, will better contribute to advancements in human medicine (Table 2) (Fig. 3).

Table 2.

The advantages and disadvantages of HIOs models, cell models, and animal models

	Advantages	Disadvantages
HIOs models	High physiological similarity to in vivo tissues, with relatively diverse functions.	Only simulate epithelial tissues, with insufficient complexity.
	Minimal ethical controversies, reduced reliance on experimental animals, and lower costs.	Challenges exist in standardizing culture conditions.
Cell models	Simple and convenient to operate, suitable for short-term experiments.	Low physiological relevance to in vivo cells.
		Relatively single cell type and limited functions.
Animal models	Can simulate in vivo physiological environments and multi-organ interactions.	Exist differences between species and individual variations, making it difficult to establish specific models.
	Served as the “gold standard” for preclinical drug research and the basis of translational medicine.	High ethical controversies, high costs, and long cycles.

Fig. 3.

The advantages and disadvantages, application optimization of HIOs

Current optimization methods for HIOs

Organoid-immune cell co-culture systems

Intestinal epithelium and mucosal immune system together form the cornerstone of the intestinal immune system, ensuring effective defense against pathogens and appropriate tolerance to harmless antigens. This not only maintains intestinal health but also safeguards immune homeostasis of the entire organism. Conventionally cultured HIOs typically lack immune cells, failing to simulate the infection microenvironment. However, this limitation has been gradually addressed with the development of organoid-immune cell co-culture technologies.

Intraepithelial lymphocytes (IELs) in the intestine play a crucial role in regulating local immune responses. Although their functions have been studied in various animal experiments, the lack of suitable culture systems has hindered in vitro research on their interactions with intestinal epithelial cells. In 2016, Nozaki et al. found that HIOs can support the in vitro maintenance of IELs in the presence of IL-2, IL-7, and IL-15. They developed a novel IEL-HIO co-culture system, which can be used to investigate the pathogenesis of intestinal diseases and immune response processes, providing a theoretical basis for developing new therapeutic approaches [74].

In 2017, Noel et al. developed and characterized the first primary human macrophage-HIO co-culture model, demonstrated its suitability for studying intestinal physiology and host response to intestinal pathogens [75]. Tominaga et al. obtained HIOs with functional tissue-resident macrophages from hPSCs, where both HIOs and macrophages were derived from directed differentiation of hPSCs and combined in vitro. The results showed that macrophages can be incorporated into developing HIOs and maintained for a certain number of weeks. They also found that these co-cultured macrophages exhibited transcriptional profiles similar to those in human fetal intestines, indicating there are obtaining the characteristics of tissue-resident macrophage. Thus, this new organoid system could be used to study molecular mechanisms involved in inflammatory bowel disease [76].

In 2023, Bouffi et al. generated HIOs containing immune cells by transplanting HIOs under the renal capsule of mice with a humanized immune system. They found that human immune cells could temporarily migrate to the mucosa and form cell aggregates resembling human intestinal lymphoid follicles. This in vivo HIOs system with human immune cells can provide a new model for the study of immune system related intestinal diseases [77].

The lack of tissue-specific immune compartments in HIOs limits their application in key aspects of intestinal pathophysiology research. In 2024, the Recaldin team co-cultured epithelial organoids with tissue-resident memory T (TRM) cells in the intestine that have memory function but do not participate in recycling, successfully constructing human intestinal immuno-organoids (IIOs) containing tissue-resident and autologous immune compartments. IIOs can be used to study tissue-resident immune responses in the context of tumorigenesis, infectious diseases, and autoimmune diseases [78].

Simultaneous differentiation of multiple cell lineages in HIOs

Given the absence of vascular, neural, and muscle tissues in HIOs, the missing cell types are usually obtained via co-culture, transplantation into mice, and modification of the growth factors required during the differentiation process. However, the co-culture method cannot induce the differentiation of multiple cell lineages within a single organoid at the same time. Childs et al. found that Epiregulin (EREG), a niche factor of intestinal stem cells, can promote the generation of induced pluripotent stem cell (iPSC)-derived HIOs containing epithelium, stroma, neurons, endothelial cells, and organized smooth muscle, without the need for co-culture. After transplantation into mice, HIOs can develop more mature enteric nervous system functions and functional blood vessels, with normal neuromuscular unit functions [79].

Reversal of intestinal epithelial polarity to optimize viral infection pathways

Normal intestinal function depends on the establishment of polarity, which requires the formation of apical junction complexes and appropriate compartmentalization of apical and basal lateral proteins. Since organoids are sphere-like structures with the apical surface of the epithelium enclosed within the sphere, studying interactions between the epithelium and luminal contents using HIOs poses challenges in accessing the apical surface of the epithelium for pathogens. To address this issue, some research teams have employed microinjection techniques and organoid monolayer culture technologies [80, 81]. Organoid single-layer culture technology has been applied in the cultivation of HuNoV [45]. In addition, CO JY et al. developed a method to reverse HIOs polarity: by removing extracellular matrix proteins, polarity is inverted, and the apical surface is everted to face the medium. This model preserves the functions of various intestinal epithelial cells and can be used for research on host pathogen interactions, barrier integrity, intestinal epithelial absorption function, and more [48]. The reversal of intestinal epithelial polarity may also provide a new way to solve the problem of accessing the apical side of the intestinal epithelium.

Conclusion

In summary, this article reviews the cultivation, functional characterization, and optimization of HIOs, as well as the historical and recent advances in their application to the study of acute gastroenteritis viruses. Since their successful establishment by Hans Clevers in 2009, HIOs have ushered in a new era of organoid research, offering a physiologically relevant platform that closely mimics in vivo conditions and authentically recapitulates both physiological and pathological processes. As such, HIOs hold significant promise for disease modeling, drug screening, gene therapy, and preclinical development of vaccines and personalized treatments. Furthermore, they have advanced our understanding of pathogenetic mechanisms and host–pathogen interactions, providing innovative models for studying specific infections. Despite these achievements, several challenges remain before HIOs can be widely adopted. Limitations in long-term stable culture, scalable expansion, and the absence of key physiological components—such as vascularization, innervation, and mechanical stimuli—hinder their ability to fully emulate intestinal functions in vivo. Therefore, to fully realize the potential of HIOs in drug development, pathogen research, and clinical translation, further technical improvements and conceptual breakthroughs are essential to pave the way for novel insights into human intestinal health and disease.

Abbreviations

RV

Rotavirus

HuNoV

Human norovirus

HAstV

Human astrovirus

HAdV

Human adenovirus

HuSaV

Human sapovirus

3D

Three-dimensional

HIOs

Human intestinal organoids

hESCs

Human embryonic stem cells

hPSCs

Human pluripotent stem cells

EGF

Epidermal growth factor

2D

Two-dimensional

IBD

Inflammatory bowel disease

DHODH

Dihydroorotate dehydrogenase

BQR

Brequinar

LFM

Leflunomide

DGAT1

Diacylglycerol O-acyltransferase 1

MNV

Murine norovirus

VLP

Virus-like particle

HBGAs

Histo blood group antigens

FUT2

Fucosyltransferase 2

2′FL

2′-Fucosyl lactose

EV-A71

Enterovirus A71

CVA16

Coxsackievirus A16

IELs

Intraepithelial lymphocytes

TRM

Tissue-resident memory T

IIOs

Intestinal immuno-organoids

EREG

Epiregulin

iPSC

Induced pluripotent stem cell

CRISPR-HOT

CRISPR-Cas9-mediated homology-independent organoid transgenesis

NHEJ

Non homologous end joining

Author contributions

Conceptualization, KYZ; data curation, KYZ; writing—original draft preparation, KYZ and YZ; writing—review and editing, YZ and HJL; funding acquisition, YZ and HJL. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by funding from Major Science and Technology Special Project of Yunnan Province (Biomedicine) (202402AA310020, 202202AA100006), Yunnan Fundamental Research Projects-general program (202201AT070236), Yunnan Talent Support Program for Industrial Technology Leading Talents (YNWR-CYJS-2019-017) and Yunnan Talent Support Program for Top Young Talents (YNWR-QNBJ-2018-390).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.