The seed cell sources for digestive system organoids can be categorized into somatic cells and stem cells. Somatic cells primarily consist of epithelial cells from various parts of the digestive system, such as the intestine and liver. Among stem cells, ASCs and PSCs are extensively utilized.

Somatic cells possess a degree of stemness, allowing them to maintain their original tissue characteristics in vitro over extended periods with good genetic stability, making them suitable as seed cells for digestive system organoids (Fujii and Sato, 2021). In 2023, Hermans et al. successfully established stable tooth organoids using molar and incisor teeth from mice (Hermans et al., 2023). These organoids expressed dental epithelial stem cell markers and demonstrated the ability to differentiate into ameloblasts in vitro, providing a novel platform for studying tooth biology and development. Cancer cells can also serve as seed cells for establishing digestive system tumor organoids. Kasagi et al. successfully cultured esophageal organoids using the human esophageal cell line EPC2-hTERT, which replicated the natural differentiation process of esophageal epithelium (Kasagi et al., 2018). This study further revealed that Notch signaling promotes esophageal epithelial differentiation, while inhibiting this pathway impairs epithelial differentiation. Xu et al. obtained colorectal cancer tissue samples through surgical resection or endoscopic biopsy, washed them thoroughly, enzymatically digested them to form single tumor cells, embedded them in matrix gel, and cultured them for 7–10 days to generate colorectal organoids (Xu et al., 2018).

Adult stem cells are non-specialized cells located in developed tissues, exhibiting stem cell capabilities and existing within different tissues and organs throughout the body. As an example, Lgr5⁺ stem cells identified in the small intestine and colon can be employed to create organoids that replicate the structural and functional characteristics of natural tissue (Parente et al., 2024). By leveraging the intrinsic self-organization properties of intestinal epithelial stem cells (ISCs) and employing air-liquid interface culture in a minimally defined medium, Kwon et al. successfully induced ISCs to differentiate into intestinal epithelial organoids characterized by cellular diversity, villous structures, and barrier integrity, thereby providing a valuable tool for regenerative medicine and disease modeling (Kwon et al., 2024). Schumacher et al. developed gastric organoids containing diverse gastric epithelial cells, including chief and parietal cells, through co-culture of immortalized gastric mesenchymal cells with gastric epithelial stem cells, facilitating studies on damage repair and other functions of gastric epithelial cells (Schumacher et al., 2015). Basak et al. demonstrated that silencing Lgr5⁺ stem cells in vitro could be achieved through the inhibition of either the epidermal growth factor receptor (EGFR) or the mitogen-activated protein kinase (MAPK) signaling pathways, which subsequently promoted organoid development favoring enteroendocrine cell differentiation (Basak et al., 2017). Additionally, they found that concurrent suppression of Wnt, Notch, and MAPK signaling pathways facilitated the transformation of these organoids into various types of intestinal secretory cells (Basak et al., 2017). In a follow-up investigation, Fujii et al. refined the culture conditions for small intestinal organoids, showing that insulin-like growth factor 1 and fibroblast growth factor (FGF) 2 considerably boosted the clonogenic potential of human small intestinal stem cells, supporting both the self-renewal and multi-lineage differentiation capabilities of intestinal organoids (Fujii et al., 2018).

Pluripotent stem cells, such as human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs), can be directed to differentiate into specific gastrointestinal epithelial cell types and ultimately form gastrointestinal organoids. hiPSCs have been utilized to generate a variety of gastrointestinal organoids, including those of the stomach, small intestine, and colon (Wang et al., 2022). Zhang et al. combined PSCs with retinoic acid and fibroblast growth factor (FGF) 10 in co-culture to establish salivary gland organoids. This method provides a robust model for studying salivary gland development in vitro and developing novel cell therapies (Zhang et al., 2022). Zhang et al. exposed RUES2-derived embryonic stem cells to a range of growth factors, such as activin A, FGF2, BMP4, and the Rho kinase inhibitor Y-27632. This exposure facilitated their differentiation into anterior foregut progenitors by suppressing the BMP, transforming growth factor (TGF)-β1, and Wnt signaling pathways. The resulting esophageal progenitor cells were subsequently cultivated into esophageal organoids (Zhang et al., 2018). Furthermore, directed differentiation can also be achieved through cell co-culture systems. For instance, co-culturing hepatocytes with mesenchymal stem cells or stellate cells can produce liver-like tissues (Afonso et al., 2024; Christine Verawaty Sibuea, 2020), while combining mesenchymal stem cells, iPSCs, and endothelial cells in co-culture can create vascularized liver organoids, which is advantageous for constructing larger organoids (Abbasalizadeh et al., 2023). In addition, to further replicate the complexity of the intestinal microenvironment, researchers have established a range of gastrointestinal organoid co-culture systems, including those involving immune cells, mesenchymal cells, or gut microbiota (Al-Qadami et al., 2025; Flood et al., 2024). These co-culture systems enable the simulation of intricate cell-cell and host-microbe interactions within the gut, thereby offering novel insights into the investigation of inflammatory bowel diseases and infectious diseases.

The construction technology for digestive system organoids can be categorized into traditional and novel methodologies. Traditional methodologies typically encompass embedding culture, rotary culture, hanging drop culture, magnetic levitation culture, and ultra-low attachment culture techniques, with embedding culture being the most prevalent. Novel construction technologies primarily consist of organ-on-a-chip, 3D printing, and 4D printing techniques (as illustrated in Figure 2).

The modeling of disease organoids requires a relatively short period, allowing for more intuitive tracking and investigation of tissue and cellular responses and changes. This approach holds significant promise in disease modeling and mechanism research. A few countries have established organoid biobanks for cancer, confirming the feasibility of using organoids as experimental models for targeted therapy. In an oral squamous cell carcinoma (OSCC) organoid model, Zhao et al. demonstrated that co-culturing cancer-associated fibroblasts (CAFs) with CD44-expressing cancer stem cells (hereafter referred to as CD44⁺ cells) resulted in the formation of OSCC organoids (Zhao et al., 2021). They observed increased expression levels of CD44 and OCT-4 in these organoids through immunofluorescence and Western blot analyses, indicating that CAFs enhance the organoid-forming capability of CD44⁺ cells. In 2023, researchers further discovered that CAFs in OSCC organoids express nicotinamide N-methyltransferase, which reduces the enrichment of H3K27me3 at the promoter region of the lysyl oxidase gene. This reduction leads to increased deposition of type I collagen, thereby promoting the growth and development of OSCC (Zhao et al., 2023).

Zhao et al. identified a therapeutic target for OSCC (Zhao et al., 2019b). By silencing monocarboxylate transporter 1 (MCT1), the levels of lactate, which is associated with tumor prognosis, were reduced, and the proliferative capacity of cancer cells was diminished. Therefore, inhibiting MCT1 can serve as a potential therapeutic target for OSCC treatment. Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) binds to CEACAM1 on natural killer cells and Tim3 on T cells, thereby suppressing the body’s anti-tumor immune response. Blocking CEACAM1 using targeted antibodies or small molecules may restore the body’s anti-tumor immunity and represents a promising new immunotherapy approach for head and neck squamous cell carcinoma (HNSCC) (Tsang et al., 2022). Considering the individual variability of tumors, Driehuis et al. established HNSCC organoids from 31 patients in vitro and observed diverse responses to cisplatin, carboplatin, cetuximab, and radiotherapy (Driehuis et al., 2020a). The in vitro responses mirrored the clinical outcomes of patients, highlighting the potential of tumor-derived organoids to guide personalized therapies.

As an effective tool for modeling the structure and function of the esophagus, esophageal organoids have gained widespread application in recent years, particularly in the study of esophageal inflammation and esophageal cancer. Compared with PSCs-derived organoids, tissue-derived esophageal organoids are more straightforward to construct and better preserve certain characteristics of the original tissue. Consequently, tissue-derived esophageal organoids play a pivotal role in the study of esophageal disease pathogenesis and their applications in regenerative medicine (Cabeza-Segura et al., 2023). Nakagawa et al. developed an organoid model of eosinophilic esophagitis using patient-derived esophageal tissues (Nakagawa et al., 2020). Research has demonstrated that eosinophilic esophagitis induces basal cell proliferation, and exogenous recombinant cytokines such as IL-13 can prompt organoids to replicate the inflammatory response characteristic of this condition. This study underscores the potential of the eosinophilic esophagitis organoid model to simulate disease pathogenesis through induced inflammatory responses, thereby facilitating the identification and development of potential therapeutic strategies. Advances in tumor-derived organoid culture techniques have led to the successful establishment of several esophageal cancer models. Organoids derived from tumor tissues exhibit high similarity to primary tumors and preserve their heterogeneity, providing a platform for personalized treatment options for cancer patients. Esophageal squamous cell carcinoma (ESCC), which is the primary subtype of esophageal cancer in Asia, represents 40% of worldwide esophageal cancer cases (Thrift, 2021). Kijima et al. developed a technique for cultivating ESCC organoids derived from patients. These organoids can be efficiently produced from single-cell suspensions embedded in a basement membrane matrix within 2 weeks, with a success rate of around 60%. They also investigated the ex vivo response of these organoids to 5-fluorouracil, revealing that cancer cells with high CD44 expression may contribute to tumor resistance (Kijima et al., 2019).

Barrett’s esophagus (BE) is recognized as a precancerous lesion associated with esophageal adenocarcinoma (EAC), a type of cancer with a poor prognosis and rapidly increasing incidence in Western countries (Peters et al., 2019). In 2011, Sato et al. pioneered the generation of an esophageal epithelial organoid using biopsy tissue from BE, marking the inception of organoid-based research for this condition (Sato et al., 2011). The cellular origin of esophageal tumors remains a subject of debate, and existing studies have not conclusively determined whether esophageal adenocarcinoma (EAC) develops from BE, as approximately half of EAC patients do not exhibit BE metaplasia at diagnosis. In 2021, Nowicki-Osuch and colleagues leveraged esophageal epithelial organoids to show that BE emerges from the gastric cardia and is propelled by c-MYC and hepatocyte nuclear factor 4 alpha (HNF4α). This discovery suggests that EAC develops via BE-like epithelial metaplasia, filling a crucial gap in prior research and highlighting the significance of esophageal organoids in modeling the esophagus (Nowicki-Osuch et al., 2021). Kunze and collaborators explored the connection between Notch signaling and goblet cells in BE, demonstrating that activation of the Notch pathway results in decreased goblet cell density in BE, which is closely linked to the activation of nuclear factor kappa-B (Kunze et al., 2020). Considering the pivotal role of Notch signaling in tumor formation, these insights offer meaningful contributions to future EAC prevention strategies. The combination of gene editing with organoid technology has expanded the utility of organoids in elucidating disease mechanisms. Liu and associates utilized CRISPR/Cas9 technology to examine the function of the Wnt signaling pathway in tumor transformation associated with BE (Liu et al., 2018). Their results indicated that activating the Wnt signaling pathway enhances proliferation and replication capabilities while reducing apoptosis in BE organoids compared to their wild-type counterparts. At present, esophageal organoids are primarily applied in esophageal cancer research, with limited exploration in other diseases. The unclear cellular origin of esophageal tumors has led most studies to focus on elucidating the mechanisms of tumorigenesis, which may explain the restricted use of esophageal organoids in researching other conditions. A large number of studies have shown the substantial importance of esophageal organoids in simulating tumor progression and performing tumor drug testing. Looking ahead, esophageal organoids hold promise for expanding into the study of other esophageal diseases.

Rodents and gastric cancer cell lines are frequently utilized models for investigating Helicobacter pylori infection; however, both models possess inherent limitations. Mouse models typically exhibit only mild inflammation and do not progress to gastric ulcers or gastric cancer (Idowu et al., 2022). Gastric cancer cell lines often harbor mutated oncogenes and lack the capacity for self-renewal (Idowu et al., 2022). In contrast, gastric organoids can faithfully replicate the structural complexity of the stomach, thereby playing a crucial role in elucidating H. pylori infection and gastric cancer pathogenesis.

McCracken et al. directly microinjected H. pylori into the epithelial lumen of organoids, observing the resultant pathophysiological responses (McCracken et al., 2014). This study demonstrated that cytotoxin-associated gene A could invade organoid epithelial cells and interact with the c-Met receptor, underscoring its significance in H. pylori infection. Gastrointestinal pancreatic neuroendocrine neoplasms (GEP-NEN) represent a rare disease, characterized by limited clinical samples, which has historically hindered research progress. Organoids offer a promising solution to this challenge. Kawasaki et al. established a library of 25 GEP-NEN organoids derived from patient gastric tissues and conducted comprehensive analyses, including whole-genome sequencing (Kawasaki et al., 2020). Their findings revealed frequent RB1 mutations and extensive chromosomal aberrations, which closely resemble the genetic alterations observed in adenocarcinoma organoids. Additionally, CRISPR-Cas9 technology was employed to knockout TP53 and RB1 genes in normal gastric organoids, generating a model that accurately reflects the genetic profile of GEP-NEN for mechanistic studies (Kawasaki et al., 2020). Collectively, gastric organoids provide a more effective platform for studying gastric diseases and will likely become an indispensable tool in this field.

Small intestinal organoids are capable of self-assembling into micro-organs with intricate three-dimensional architectures, encompassing a diverse range of intrinsic intestinal cell types, including intestinal epithelial cells, goblet cells, and Paneth cells (Ghorbaninejad et al., 2023). This high level of structural and functional fidelity allows small intestinal organoids to more accurately recapitulate the in vivo physiological state of the intestine, thereby providing a robust model for elucidating the mechanisms underlying intestinal diseases.

The small intestinal organoid system, established as the earliest organoid model, has been employed to study a range of diseases, such as cystic fibrosis and infections caused by bacteria and viruses. Cystic fibrosis is a rare genetic condition marked by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel within epithelial cells (Yin et al., 2019). Reproducing the varied phenotypes of CFTR mutants poses significant challenges for traditional cell lines and animal models, and there is still a lack of effective clinical therapies. As a result, organoids have become an essential tool for researching these disorders.

Dekkers et al. introduced a novel method termed “forskolin-induced swelling (FIS)” for the functional assessment of cystic fibrosis using small intestinal organoids (Dekkers et al., 2013). This study demonstrated that forskolin activates CFTR in organoids, resulting in observable swelling. The extent of this swelling is diminished in samples lacking functional CFTR or harboring CFTR mutations. FIS has established a robust research model for drug screening in cystic fibrosis and offers potential for personalized therapeutic approaches. Small intestinal organoids exhibit characteristics closely resembling those of human intestinal epithelium, making them an ideal platform for investigating the pathogenesis and treatment of infectious diseases. Norovirus, an enterovirus responsible for acute gastroenteritis, lacks an effective antiviral drug or vaccine due to the absence of a suitable in vitro culture system (Flynn et al., 2024). While traditional laboratory methods for detecting norovirus RNA are highly sensitive, they cannot differentiate between infectious and non-infectious viral particles. Chan et al. successfully cultured norovirus in intestinal organoids and utilized real-time reverse transcription PCR to determine the threshold of norovirus replication (Chan et al., 2019). They found that when the C t value was ≤30, the virus replicated efficiently within organoids, providing a valuable tool for assessing viral infectivity in clinical settings. Additionally, rotavirus, Shigella, and Escherichia coli, which are major pathogens causing diarrhea, have also been studied using organoid models.

Finkbeiner et al. demonstrated that small intestinal organoids are susceptible to infection by both experimental rotavirus (simian SA11) and clinical rotavirus isolates (Finkbeiner et al., 2012). Furthermore, the study revealed that iPSC-derived small intestinal organoids support pathogen replication, indicating their potential for culturing intestinal pathogens that are challenging or impossible to grow using traditional models. Pradhan and colleagues developed a model using Shiga toxin-infected small intestinal organoids to examine how small intestinal tissues respond biologically to Shiga toxin exposure (Pradhan et al., 2020). Their study revealed that Shiga toxin triggers necrosis and apoptosis in both intestinal epithelial and stromal cells. Additionally, preserving the integrity of the intestinal epithelial barrier strengthens the organoids’ resilience against Shiga toxin infection. Barron et al. used non-pathogenic E. coli ECOR2 to microinject small intestinal organoids and discovered that deletion of the RpoS gene reduces ECOR2’s ability to colonize these organoids (Barron et al., 2020). Serra et al. identified yes-associated protein 1 (Yap1) as a signaling factor that detects organoid integrity; upon organoid disintegration, Yap1 activation drives tissue repair, which subsequently induces specific Yap1 activation in local cell clusters (Serra et al., 2019). Yap1 also promotes delta-like canonical Notch ligand 1 expression and Paneth cell formation in vivo. The Wnt signaling pathway plays a crucial role in organoid culture. Miao et al. engineered a modified Wnt molecule that forms heterodimers with Wnt Frizzled receptors (Fzd) and LDL receptor-related protein 6 (Miao et al., 2020). Administration of Fzd-specific Wnt agonists enhances the proliferation of adult intestinal crypt cells and improves the long-term proliferation and maintenance of organoids. In summary, organoids hold significant potential for modeling diseases and investigating disease mechanisms in the small intestine. In addition, the enteric nervous system (ENS) plays a crucial role in the regulation of intestinal function. The co-culture of enteric nerves and intestinal organoids can mimic the interaction between enteric nerves and the epithelium, thereby offering a novel model for investigating ENS function and associated diseases (Özkan et al., 2024). A sophisticated 3D culture technique was developed to enable the co-culture of small intestinal organoids with myenteric and submucosal neurons. Through the refinement of isolation methods, intestinal organoids containing both intestinal neurons and glial cells from the two nerve plexuses were successfully established, providing a unique platform for studying the regulatory mechanisms of the enteric nervous system.

In terms of organ development, homeostasis maintenance, and pathogenesis, organoid models are more accurate than animal models in providing basic information similar to that of the human body. So far, researchers have successfully constructed different kinds of liver disease models.

Biliary organoids provide a crucial platform for studying diseases like biliary atresia, biliary tract cancer, and primary sclerosing cholangitis, enabling a deeper understanding of the underlying disease mechanisms. Chen et al. were the first to develop a method for cultivating biliary organoids using gel embedding. These organoids were then co-cultured with rotavirus, allowing for the successful creation of a biliary atresia (BA) disease model (Chen et al., 2020). Their findings demonstrated that rotavirus causes damage to biliary tract cells through interactions with host cells, which contributes to the onset of BA. Additionally, they suggested that suppressing rotavirus replication and providing antibodies targeting the VP7 protein of rotavirus might serve as promising treatment approaches for BA. Maier et al. reported a protocol for the establishment of cholangiocarcinoma organoids in stable culture conditions. They mechanically dissociated cholangiocarcinoma tissues and enzymatically digested them with tissue-specific enzymes for 2 h, followed by filtration through a 40–100 μm cell strainer and differential centrifugation at 200 g for 3 min. The isolated cells and cell aggregates were subsequently co-seeded in a matrix gel supplemented with ROCK inhibitor, forskolin, insulin, transferrin, and selenite to form stable cholangiocarcinoma organoids (Maier et al., 2021). Du et al. utilized organ-chip technology to create a vascularized bile duct chip-based organoid model derived from PSC) (Du et al., 2023). The expression patterns of critical markers, including bile duct cell indicators, polarity proteins, collagen IV, laminin, bile salt transporters, secretin receptors, and tight junction proteins (such as zonula occludens-1), closely matched those found in primary bile duct cells obtained from PSC patients. This sophisticated disease model provides substantial benefits for exploring the physiological and pathological processes associated with PSC. In recent years, Jalan-Sakrikar et al. successfully reprogrammed fibroblasts from PSC patients into iPSCs and cultivated them under three-dimensional conditions to establish PSC organoids (Jalan-Sakrikar et al., 2022).

Existing biliary tract models, including two-dimensional cell cultures, are inadequate for replicating the complex structure of the biliary system. Moreover, these models present challenges in precisely controlling the dimensions of the biliary tract and the positioning of cells. Consequently, there is a critical need for an advanced in vitro biliary tract model to facilitate comprehensive studies of biliary physiology and pathology. Organoids, which are distinguished by their distinctive spatial structure and cell-specific properties, hold promise for tissue regeneration and the recovery of many original organ functions. This feature renders them a perfect model for exploring the physiological and pathological processes of the biliary tract. Jalan-Sakrikar et al. successfully reprogrammed fibroblasts derived from PSC patients into hiPSCs and then generated biliary organoids through a three-dimensional culture method (Jalan-Sakrikar et al., 2022). Through electron microscopy, they observed that these organoids were diminutive, lacked a central lumen, and exhibited accelerated aging. Additionally, they noted increased secretion of fibronectin, interleukin-6, and C-C motif chemokine ligand 2, which highlighted the disease-specific characteristics of PSC. Amarachintha et al. generated bile duct atresia cystic organoids (BACOs) by culturing liver tissue from infants with biliary atresia in a three-dimensional environment (Amarachintha et al., 2022). Transmission electron microscopy showed a limited number of ciliated cells with abnormal lateral cilia development, which may be associated with decreased levels of F-actin, β-catenin, and ezrin secretion. In a separate experiment, it was observed that BACOs had reduced expression of the tight junction protein zonula occludens 1 in biliary epithelial cells, resulting in impaired barrier function and elevated permeability. Additionally, stimulation of the EGF/FGF signaling pathway in biliary epithelial cells promoted epithelial differentiation and enhanced the integrity of the biliary epithelial barrier (Amarachintha et al., 2022). Verstegen et al. developed a cystic fibrosis model using organoids that exhibited normal chloride channel and MDR1 transporter activity but lacked functional CFTR channel activity (Verstegen et al., 2020).These studies highlight the crucial role of biliary organoids as a platform for visualizing and studying metabolic and regulatory processes within the biliary system.

Advancements in pancreatic organoid technology have enabled the development of three-dimensional models that accurately replicate the heterogeneity, structure, and function of native pancreatic tissue, which is crucial for modeling pancreatic diseases (Liu et al., 2023). Pancreatic organoids can emulate a diverse array of pancreatic cell types, including mature ductal cells and acinar cells. These 3D models facilitate a more profound understanding of drug mechanisms of action, offer faster and more cost-effective assessments, reduce reliance on animal models, and enhance the prediction of patient responses.

Wang et al. utilized salivary gland organoids to investigate the mechanism of progenitor cells in response to β-blockers for treating salivary insufficiency (Wang et al., 2021). Their findings revealed that β-blockers induce a reduction in Notch signaling within intercalated duct cells, thereby impeding the proliferation and differentiation of these cells into acinar cells, leading to persistent hypopsialsecretion in patients on β-blocker therapy. Tanaka et al. refined the spheroid culture method for tumor cells, demonstrating that regardless of the status of the tumor suppressor gene TP53 or human papillomavirus, organoids resembling original head and neck tumors can be formed (Tanaka et al., 2018). This model allows for predicting in vivo drug sensitivity of tumor cells, indicating its potential for drug screening and toxicity simulation. Belair et al. identified that tributyltin oxide, all-trans retinoic acid, valproic acid, theophylline, and triamcinolone acetonide interfered with palatal fusion among 12 putative teratogens (Belair et al., 2018). Tigani et al. discovered that triethylene glycol dimethacrylate, a component in dental restorations, exhibits toxic effects on gingival and dental pulp tissues, inhibiting cell migration and aggregation, potentially suppressing the expression of adhesion receptors necessary for cell-ECM connections, and altering cellular structure and morphology (Tigani et al., 2019). Driehuis et al. utilized mouse tongue epithelial organoids to demonstrate that acyclovir can inhibit herpes simplex virus 1 proliferation (Driehuis et al., 2020a). Leucovorin serves as an antidote for methotrexate toxicity, mitigating chemotherapy-induced damage, including oral mucositis, when administered within 72 h post-methotrexate treatment (Driehuis et al., 2020b).

Gastric cancer is a complex disease characterized by diverse histological features and molecular subtypes. To elucidate the mechanisms underlying its development, it is essential to investigate the specific expression patterns of these features in appropriate models.

Nanki et al. utilized CRISPR/Cas9 technology to generate gastric cancer organoids harboring multiple mutations. They also established a biobank comprising 37 patient-derived gastric cancer organoids, thereby constructing a comprehensive resource for studying genetic and histopathological changes (Nanki et al., 2018). This biobank facilitates modeling, drug screening, and personalized treatment strategies for gastric cancer. Yan et al. established an additional biobank consisting of gastric cancer organoids derived from 34 patients. This collection included almost all recognized molecular subtypes and mutation profiles, following a meticulous sample selection process (Yan et al., 2018). They conducted extensive whole-exome and transcriptome analyses, providing detailed genomic data on tumors. Additionally, they performed large-scale drug screenings, revealing significant sensitivity of tumor organoids to napabucasin, abemaciclib, and ataxia telangiectasia and Rad3-related inhibitors such as VE-822. Chemotherapy remains a primary treatment modality for gastric cancer; however, challenges like drug resistance and adverse reactions persist. Ouyang et al. developed a selective inhibitor of signal transducer and activator of transcription 3 (STAT3), W1131, which was tested in gastric cancer organoids (Ouyang et al., 2022). It was found that W1131 could reduce tumor cell resistance to 5-fluorouracil by inhibiting STAT3 activity. Zou et al. investigated nano-formulations with fewer adverse effects, comparing the efficacy of two paclitaxel nano-formulations in patient-derived gastric cancer organoids (Zou et al., 2022). They observed that both nanoparticles demonstrated anti-tumor effects, but liposomal paclitaxel exhibited superior cytotoxicity compared to albumin-bound paclitaxel. This study highlights the potential of PDOs as an effective platform for evaluating nanomedicine drugs, suggesting that more such agents may be tested using organoid models in the future. In addition, the recent adoption of conditioned medium as an alternative culture method for recombinant hepatocyte growth factors has substantially decreased the cost of culturing human gastrointestinal tract (GIT) organoids. This advancement facilitates large-scale cultivation of GIT organoids and compound screening. Despite existing challenges in GIT organoid development, such as their inability to form paired structures, limited cell type diversity, and reliance on single drug exposure patterns, these organoids hold significant potential for drug screening (Zhou et al., 2024a). The utilization of GIT organoids in this context is anticipated to enhance the precision of medical treatments for patients with gastrointestinal diseases.

Human intestinal cell lines, such as Caco-2, have traditionally served as foundational platforms for drug development (Bein et al., 2018). The emergence of small intestinal organoids has introduced a novel and advanced research platform for drug screening. Vijftigschild et al. utilized the FIS model to screen small molecule compounds regulated by G protein-coupled receptors, identifying β2-adrenergic receptor agonists as potent inducers of CFTR function (Vijftigschild et al., 2016). This research highlights the promise of small intestinal organoids as a reliable preclinical model for designing and assessing effective treatments for cystic fibrosis. Yin et al. utilized human small intestinal organoids to identify potential antiviral compounds against rotavirus infection, revealing that cyclosporine A and mycophenolic acid significantly hindered rotavirus replication. These findings validated the practicality of employing small intestinal organoids in drug investigations targeting intestinal infections (Yin et al., 2018). Overall, small intestinal organoids better replicate the structural and functional attributes of human small intestine tissues, offering an advanced system for analyzing drug effects in humans. As such, small intestinal organoids are expected to play a crucial role in upcoming drug screening initiatives.

Mutations in the Wnt signaling pathway are observed in approximately 90% of colorectal cancers. Although numerous targeted therapies aimed at this pathway have been proposed, a subset of patients do not derive clinical benefit from these treatments. Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations are prevalent in colorectal cancer. Verissimo et al. employed patient-derived organoids to evaluate therapeutic agents aimed at the EGFR-RAS-ERK signaling pathway. Their findings revealed that afatinib, an inhibitor of the epidermal growth factor receptor and human epidermal growth factor receptor (HER) 2, demonstrated efficacy against organoids with wild-type KRAS but was ineffective against those harboring KRAS G12V mutations. Additionally, the mitogen-activated protein kinase kinase (MEK) inhibitor selumetinib exhibited no therapeutic benefit as a standalone treatment. However, when combined with afatinib, MEK inhibitors were observed to increase the sensitivity of RAS-mutated tumors to HER2 inhibition (Verissimo et al., 2016). Moreover, simultaneous targeting of both the PI3K-AKT and EGFR-RAS-ERK pathways through the inhibition of phosphatidylinositol 3-kinase (PI3K) or serine/threonine protein kinase AKT, in conjunction with anti-EGFR therapy, did not improve treatment outcomes in KRAS-mutated tumors (Verissimo et al., 2016). As a result, the use of MEK inhibitors in conjunction with PI3K, AKT, or mammalian target of rapamycin (mTOR) inhibitors has not led to desirable outcomes in the treatment of KRAS-mutated colorectal cancer in clinical settings (Atanasova et al., 2023). The issue of drug resistance continues to be a major clinical obstacle, affecting roughly 40% of patients with KRAS-mutated colorectal cancer.

Knight et al. developed a colorectal cancer organoid harboring a KRAS mutation (Knight et al., 2021). They discovered that the combined inhibition of mitogen-activated protein kinase-interacting kinase (MNK) and mechanistic target of rapamycin complex 1 (mTORC1) enhanced the sensitivity of the organoids to rapamycin. The primary mechanism involves reducing the phosphorylation of eukaryotic translation initiation factor 4E and decreasing c-MYC expression, which may potentially inhibit tumor recurrence and metastasis in patients with KRAS mutations in clinical settings (Knight et al., 2021). Ringel et al. conducted CRISPR screening by integrating single guide RNAs from both wild-type and APC mutant human intestinal organoids (Ringel et al., 2020). By optimizing experimental conditions, they achieved a genome-wide CRISPR screen of organoids to identify tumor suppressor genes mediating TGF-β resistance, providing novel insights for drug development. Chimeric antigen receptor T-cell immunotherapy (CAR-T) has demonstrated promising therapeutic efficacy in leukemia. Consequently, Schnalzger et al. established PDOs of colon cancer to evaluate the cytotoxic effects of chimeric antigen receptors on solid tumors (Schnalzger et al., 2019). They found that engineered EGFRvIII-CAR NK-92 cells specifically targeted organoids transfected with the neoantigen EGFRvIII without exhibiting cytotoxicity towards normal organoids, suggesting that CAR-T technology may offer improved treatment options for colorectal cancer and other solid tumors. Ding et al. utilized droplet emulsification microfluidic technology to rapidly generate thousands of micro-organospheres (MOSs) from small colorectal cancer tissue samples. In this study, a total of eight MOSs derived from metastatic colorectal cancer patients were established. Four of these MOSs exhibited sensitivity to the drug, while the remaining four demonstrated resistance. Based on the drug sensitivity results, clinical treatment strategies were guided, and the drug responses of the sensitive and resistant MOSs were found to be consistent. Additionally, tumor stromal cells and immune cells, among other components of the tumor microenvironment, were detected within the MOSs. In vitro experiments showed that added T cells could infiltrate the MOSs and elicit a cytotoxic response to immunotherapy, thereby enhancing the killing effect on MOSs. This approach provides a platform for clinical trials to evaluate immuno-oncology therapies, including PD-1 blockade in patient tumors, bispecific antibodies, and T-cell therapies (Ding et al., 2022).

Compared to traditional cell lines and xenograft models, organoid models exhibit superior performance in terms of construction success rate, culture duration, and preservation of disease characteristics. This makes them an invaluable tool for drug screening and adverse reaction research (Chen et al., 2024).

Li et al. utilized liver cancer organoids to screen 129 anticancer drugs, revealing that sorafenib, gemcitabine, and other antitumor agents demonstrated heterogeneous efficacy among liver cancer patients. They also identified pramikacin and idarubicin as potentially beneficial treatments for liver cancer (Li et al., 2019). Wang et al. employed organoids to investigate the mechanisms underlying sorafenib resistance (Wang et al., 2020). Kim et al. developed a MASH-related HCC mouse organoid model to evaluate drug responses, particularly Lenvatinib resistance (Kim et al., 2024b). Their findings indicated that while Multi-biotics (a soymilk fermented with lactic acid bacteria) did not directly inhibit tumor growth, it enhanced the efficacy of Lenvatinib, thereby indirectly suppressing tumor progression. Transcriptomic analysis revealed key pathways associated with KRAS signaling, inflammation, and epithelial-mesenchymal transition, identifying genes such as Itga7, Col7a1, and Slpi as potential targets to overcome Lenvatinib resistance. These insights provide valuable information on MASH-related HCC progression and drug resistance. Blukacz et al. demonstrated that inhibiting ABCB1, a drug efflux pump within the ABC transporter superfamily, increased adriamycin sensitivity in drug-resistant hepatocellular carcinoma organoids. They proposed combining adriamycin with ABCB1 inhibitors to enhance adriamycin efficacy and improve the response to transarterial chemoembolization (Blukacz et al., 2024). Collectively, these studies highlight the utility of organoids for in vitro drug sensitivity testing and the study of drug side effects.

Organoids are capable of accurately replicating the drug sensitivity and resistance characteristics seen in solid tissues. Additionally, they provide benefits like a brief preparation period and reliable passaging, which makes them highly useful for high-throughput drug screening (Yang and Yu, 2023).

Yuan et al. established a gallbladder cancer (GBC) organoid model through the co-culture of bile duct epithelial cells extracted from GBC with Matrigel (Yuan et al., 2022). Utilizing this model, they assessed the treatment potential of the dual PI3K/HDAC inhibitor CUDC-907 on GBC. Their results demonstrated that CUDC-907 effectively suppressed the growth of multiple GBC organoids and showed reduced toxicity to normal gallbladder organoids compared to other anticancer drugs in a double-controlled trial. These outcomes highlight the value of biliary organoids as tools for drug evaluation. Ren et al. managed to create cholangiocarcinoma organoids by isolating bile duct epithelial cells from cholangiocarcinoma tissues and culturing them together with Matrigel, achieving a high success rate (Ren et al., 2023). They further examined the therapeutic effects of seven frequently used chemotherapeutic agents—gemcitabine, cisplatin, capecitabine/5-fluorouracil, SN-38 (the active metabolite of irinotecan), oxaliplatin, mitomycin C, and paclitaxel—on these organoids. The results were then compared with follow-up data from cholangiocarcinoma patients and therapeutic outcomes from cholangiocarcinoma mouse models. The alignment across these three sets of findings further confirms the reliability and efficiency of biliary organoids as a platform for drug screening.

In recent years, the advent of targeted therapies, including immunotherapeutic agents, has significantly improved patient outcomes. However, a subset of patients remains unresponsive to current treatments due to tumor heterogeneity. This heterogeneity underscores the critical relationship between individual patient variability and drug efficacy, leading to diverse responses even among different cancer cells within the same tumor. Pancreatic cancer is often diagnosed at an advanced stage, with only 10%–15% of cases being amenable to surgical intervention. For patients with unresectable pancreatic cancer, organoid models offer a reliable platform for precision drug screening. High-throughput drug screening using organoids derived from pancreatic cancer tissues can facilitate the identification of effective therapeutic agents (Li et al., 2022).

Tiriac et al. utilized 156 patient-derived organoids to establish a platform for evaluating single-agent chemotherapy and targeted therapies, demonstrating that the therapeutic responses of pancreatic cancer organoids correlated with clinical outcomes in patients (Tiriac et al., 2018). Huang et al. developed a three-dimensional cell culture technique to expand and maintain primary pancreatic cancer organoids from patient tissues, enabling drug sensitivity testing (Huang et al., 2015). The researchers treated tumor organoids from five patients with gemcitabine and an epigenetic inhibitor, revealing differential drug sensitivity that correlated positively with resistance biomarkers. This study confirmed that pancreatic cancer organoids retained the sensitivity of patient tissues to novel agents in vitro. Hirt et al. selected 31 patient-derived pancreatic cancer organoids representing common genetic mutations and conducted high-throughput drug screening using an FDA-approved library of 1,172 compounds, including anti-tumor, cardiovascular, neurological, and anti-inflammatory drugs. Through automated drug administration, emetine and ouabain were identified as potential effective treatments, validated by in vitro and in vivo experiments. These compounds were found to induce tumor cell death by disrupting the hypoxic tolerance of pancreatic cancer organoids (Hirt et al., 2022). Watanabe et al. constructed a PDOX organoid model to screen for gemcitabine-sensitive and -resistant pancreatic cancer organoids. High-throughput screening of 375 kinase inhibitors was performed, and effective drugs were selected based on their efficacy and toxicity profiles (Watanabe et al., 2022). Zhou et al. demonstrated that the CLDN18.2/CD3 bispecific T cell engager (BiTE) effectively inhibited tumor growth in the early stages using a patient-derived organoid xenograft (PDOX) model. However, its efficacy markedly diminished in later stages. Notably, the combination of vilanterol and STING agonists synergistically enhanced BiTE efficacy by inhibiting CD64-positive CAFs and promoting the proliferation of stem-like CD8 T cells, thereby sustaining antitumor activity. Consequently, they proposed that the combination of vilanterol and STING agonists sensitizes PDAC to CLDN18.2-targeted BiTE therapy, enhancing its efficacy as a promising new strategy (Zhou et al., 2024b).In summary, these studies have validated the utility of organoid technology for investigating tumor heterogeneity, paving the way for establishing a living biobank of multiple patients’ tumor tissues to study individual pathogenic mechanisms, which is crucial for targeted research and personalized drug testing (Magré et al., 2023). The application of pancreatic organoids is shown in Figure 3.

As an emerging in vitro model, digestive system organoids have demonstrated significant potential in the field of drug screening. Oral organoids can serve as a disease model for oral cancer to investigate the anti-tumor effects of drugs. Through the establishment of gastric cancer organoids, metastatic colorectal cancer and pancreatic cancer cell line biobanks, large-scale drug screening has been performed, identifying compounds with notable sensitivity to organoid models. This provides valuable guidance for clinical drug selection and anti-cancer drug development. Drug-induced liver injury (DILI) represents one of the primary causes of clinical trial failure and high attrition rates in drug development. High-throughput generation of liver organoids can markedly accelerate the drug screening process and facilitate the discovery of novel therapeutics. The development of drug screening platforms based on microfluidic technology, in combination with pancreatic ductal adenocarcinoma (PDAC)-derived organoids, enables high-throughput drug screening and expedites drug discovery. However, organoid models still exhibit limitations in recapitulating the complexity of the in vivo microenvironment, such as the absence of key components like immune cells and the nervous system. Variability in organoids constructed across different laboratories may affect the reproducibility of drug screening results, highlighting the need for further research in this area.