Fresh patient specimens obtained from surgical resections or biopsies constitute the primary source of material for the successful establishment of PDTOs (3). When the surgery is not preferred, core needle biopsies can be used for the establishment PDTOs despite providing a smaller amount of cellular material (9). Regarding the gastrointestinal (GI) PDTOs, performing endoscopic biopsies can lead to derivation of epithelial cells from the stomach, colon or the other regions of the GI track which can then be used for PDTO generation (14). In addition, liquid biopsies, relying on capturing circulating tumor cells (CTCs), have recently become a topic of interest as a minimally invasive approach for the generation of PDTOs (15). The efficiency of PDTO establishment and long-term propagation is strongly influenced by the method of specimen collection, tissue type, and the time elapsed between resection and processing. To maximize this efficiency, tissue processing is recommended to take place within the first 2–4-h window of post-specimen collection accompanied with cold, oxygenated medium specific for PDTOs.

To generate a viable single-cell suspension suitable for PDTO establishment, tumor tissues must undergo carefully optimized mechanical and/or enzymatic dissociation (11). Mechanical tissue separation involves the use of scalpels and scissors to separate the cellular parts (16). Enzymatic tissue separation, on the other hand, utilizes collagenase, dispase, trypsin, or DNase to facilitate the disruption of certain extracellular matrix components and therefore releasing individual cells (16). The duration of enzymatic exposure is a critical parameter, as excessive digestion compromises cell viability whereas insufficient digestion reduces PDTO establishment efficiency (17). Depending on the tissue of origin, the tissue dissociation can vary. For example, very fibrotic stroma present in pancreatic tumor tissue requires prolonged enzymatic exposure time while colon tissue with a more diffuse connective tissue structure requires less enzymatic exposure time for more effective digestion (18). Thus, there is no “one-size-fits-all” approach as each tissue type requires a specific optimization to determine the quality of tissue dissociation for the efficiency of PDTO establishment.

Following tissue dissociation, isolated tumor cells are embedded into an extracellular matrix (ECM) that provides both structural support and essential biochemical signals for PDTO self-organization (3). The Matrigel is preferably used ECM derived from Engelbreth–Holm–Swarm mouse sarcoma which is rich of laminin, collagen IV and entactin providing required support and growth for PDTOs (19). This ECM facilitates cell adhesion, proliferation and enables self-organizing for PDTOs (20). Since Matrigel is derived from a murine sarcoma source, it exhibits a batch-to-batch variability, which can significantly affect PDTO morphology, growth rates, and drug response profiles. This variability, in fact, led to the development of synthetic hydrogels as alternatives, offering an opportunity to control mechanical properties and biochemical composition (21). For example, the synthetic hydrogels can be engineered to fine-tune ECM stiffness and present ligands, enabling scientists to study mechanobiology of PDTOs (22). Furthermore, a combination of synthetic hydrogels and natural ECM proteins have emerged, namely hybrid systems, wherein bioactivity and reproducibility can be achieved (23). Therefore, the role of ECM in PDTO generation and maintenance exceeds its role more than as a scaffold and it critically determines the phenotype, lineage specificity and drug response profiles of PDTOs (24). Consequently, a careful selection of ECMs becomes a critical factor for ensuring efficient PDTO establishment and recapitulating patient tissue dynamics.

Defined organoid culture conditions are formulated to mimic the biochemical signaling mechanisms of the tissue niche and sustain tissue-specific stem cell populations (13). Canonical signaling pathways, including Wnt/β-catenin, epidermal growth factor (EGF), and transforming growth factor-beta (TGF-β), are modulated by defined media supplements to regulate stem cell self-renewal, lineage specificity, and cellular differentiation (13). For example, EGF, R-spondin-1, Noggin, Wnt3a, FGF2, FGF10, A83-01 (a TGF-β inhibitor), and nicotinamide contribute epithelial development and preserve structural organization in PDTOs (13). Tissue-specific requirements for media formulations are taken into consideration for each tissue. For example, colorectal cancer-derived PDTOs rely on Wnt3a and R-spondin to maintain their stem cell compartment (9), gastric PDTOs require gastrin for gastric lineage preservation (14), and prostate PDTOs need androgen to maintain luminal differentiation and secretory function (25). Although media formulations have been so far very well established, the issue of batch-to-batch variation in media formulations containing recombinant proteins still remains as a challenge for the reproducibility of PDTO generation across different research laboratories (26).

Patient-derived tumor organoids are typically passaged every 7–21 days, depending on their tissue origin, growth kinetics and differentiation status. PDTOs are passaged via mechanical, including pipette-based agitation, and enzymatic digestion which is then followed by re-embedding into an ECM environment supplemented with fresh growth medium. Effective passaging is crucial to overcome overgrowth and media overconsumption related issues and exhibiting long-term stability in culture to preserve genomic and phenotypic characteristics (1). Nonetheless, PDTOs established from normal tissue may encounter problems associated with cellular senescence and thereby exhibiting short-term culture durations (27). Moreover, in some cases, PDTOs do not maintain their phenotypic stability wherein phenotypic shifts occurs (28). Such phenotypic drift is frequently driven by progressive epigenetic reprogramming induced by prolonged in vitro culture conditions (29). Therefore, it is critical to validate ongoing culture effects in PDTOs to effectively preserve the original tissue representation.

Unlike pluripotent stem cell-derived organoid systems, where karyotype anaylysis is a primary quality control step, validation of PDTOs is primarily based on histopathological, genomic, transcriptomic, and functional concordance with the parental tumor tissue (3). It is essential to demonstrate that PDTOs accurately recapitulate the molecular, histopathological and functional characteristics of their parental tumors. To verify that PDTOs resembles the donor tissue, a comprehensive analysis including morphological, molecular and functional validations are performed (3). For example, the glandular architecture and polarity of PDTOs are compared and validated across the donor tissue sections using histopathology analysis (30). Immunohistochemistry is used to confirm lineage-specific biomarkers including CK20 and CDX2 in colorectal cancer PDTOs (9). To validate the genomic resemblance between PDTOs and the donor tissue, whole-genome, whole-exome, targeted panel sequencing or long read sequencing can be employed (31, 32). This approach is based on the assessment of the stability of somatic mutations and mutational burden (9). Another critical molecular validation is that transcriptional profiling through RNA sequencing to verify stable gene expression and signaling activities across the PDTO and the donor tissue (33). Lastly, functional assays assess whether PDTOs exhibits drug sensitivity for oncological PDTO applications (18, 34, 35). Collectively, these validation methods provide a comprehensive approach for assessing the reliability of a PDTO model, and thereby offering a valuable resource for both basic research and translational applications in precision medicine.

Biobanking efforts for PDTOs have emerged as critical infrastructure for functional precision oncology by enabling systematic generation, storage, molecular annotation, and large-scale drug testing of patient-specific tumor models. In precision medicine workflow, PDTO biobanks are not only repositories for long-term storage but are increasingly established as functional biobanks that integrate clinical metadata, multi-omics profiling, and high-throughput drug screening pipelines. These efforts also enable researchers to perform independent PDTO culturing, validating the reproductivity of culture conditions (36). PDTOs in the culture are harvested for their subsequent freezing which includes a suspension in a freezing media containing 10% dimethyl sulfoxide (DMSO) (11). After PDTOs are mixed with the freezing solution, the samples are subjected to controlled rate of decrease in the freezing before long-term storage in the liquid nitrogen tank (11). Efficient cryopreservation of PDTOs enables the preservation of organoids for their functionality in the biobanks when they are shared and used for additional experiments by other researchers. Importantly, in functional PDTO biobanks, thawed organoids are routinely re-expanded for systematic testing of standard-of-care therapies as well as investigational and targeted compounds, enabling real-time assessment of patient-specific drug sensitive. The resulting functional drug response profiles can be integrated with molecular signatures and, in selected clinical settings, used to support therapeutic decision-making. International efforts for PDTO biobanking such as the Human Cancer Models Initiative and the European Organoid Resource offer established standardization in biobanking, facilitating quality control, electronic health records for clinical, histological and genomic data (37). These coordinated platforms provide not only physical access to well-annotated PDTO models but also integrated clinical, histopathological, and genomic datasets, enabling reproducible drug testing across laboratories and accelerating translational research. As a result, modern PDTO biobanks now function as living resources that bridge basic research, drug discovery, and personalized oncology by continuously linking patient material with functional therapeutic testing and clinical outcome data.

Patient-derived tumor organoids pre-present a cornerstone of contemporary cancer modeling by preserving tumor architecture, epi(genetic) heterogeneity, and therapy response profiles (43). For example, the functional consequences of recurrent driver mutations, such as APC, KRAS, TP53 in colorectal cancer have been systematically investigated using PDTO platforms (44). Moreover, the relationships between oncogenic signaling and tumor growth dynamics have been studied using PDTOs in a genetically or pharmacologically altered conditions (45). Similarly, PDTOs originating from pancreatic ductal adenocarcinoma (PDAC) showed significant potential, reflecting the chemoresistance seen in patients and indicating as a promising predictive model for effective treatment combinations (46). In addition, triple-negative breast cancer PDTOs helped to test number of different customized therapy options (47). Taken together, PDTOs used in modeling of cancer contributes to the functional analysis of tumor biology within a patient-specific framework and serve as a platform for testing treatment modalities.

The tumor microenvironment (TME) plays a critical role in regulating tumor growth, immune evasion, metastasis, and therapeutic resistance (48). While traditional PDTO cultures were established using epithelial tumor cells, selected number of recent research have shown that incorporation of stromal and immune components significantly boosts the physiological relevance of PDTOs (49). Co-culture systems combining cancer-associated fibroblasts (CAFs) with PDTOs have been shown to modulate tumor proliferation, ECM remodeling, and drug sensitivity (50). Similarly, PDTO-immune cell co-culture platforms enable functional interrogation of immune checkpoint blockade, tumor immune presentation, and mechanisms of immune evasion (48). In addition, incorporation of endothelial cells into PDTO cultures enables angiogenic signaling and provides nutrient diffusion and drug delivery observed in in vivo (51). Collectively, these advances demonstrate that accurate modeling of the TME is essential for reproducing patient-specific therapeutic responses and resistance mechanisms. The integration of stromal, immune, and vascular components into PDTO systems is therefore increasingly recognized as a prerequisite for effectively translating PDTO-based discoveries into clinical oncology.

Patient-derived tumor organoids have proven to be critical preclinical model for drug screening and precision oncology (38). Hundreds of therapeutic compounds, either as monotherapies or rational combinations, can be systematically evaluated using high-throughput PDTO-based drug screening platforms (52). Since PDTOs preserve all major characteristics of the donor tissue, HTS using PDTOs facilitate more physiologically relevant platform than conventional two-dimensional cell lines and has demonstrated strong predictive accuracy in clinical research. For example, a pioneering study by Vlachogiannis et al. reported that there was over 80% concordance between the drug response in PDTOs and metastatic GI patients, demonstrating their efficacy as a functional indicator for treatment selection (9). When combined with comprehensive genomic profiling, high-throughput drug screening data derived from PDTOs can be integrated with molecular signatures sensitivity or resistance to guide rational therapeutic selection. Moreover, PDTOs can offer adaptive therapy testing, wherein the consequences a sequential drug testing is monitored for the evolution of resistance (53). Collectively, PDTOs are considered as “living patient avatars” potentially helping to connect molecular diagnoses with individualized treatment plans, so contributing to the improvement of therapeutic accuracy (54).

Besides drug screening, PDTOs have been instrumental for the identification and functional validation of predictive biomarkers of drug sensitivity and resistance, which is a key factor for precision oncology. For example, KRAS and NRAS mutations have been associated with intrinsic resistance to EGFR-targeted therapies such as cetuximab and panitumumab in colorectal cancer PDTOs (9). On the other hand, BRAF V600E mutations have been shown to correlate with reduced response to standard chemotherapy but increased sensitivity to combined BRAF-MEK inhibition in such models (44). In addition, PIK3CA mutations and PTEN loss detected in PDTOs have been linked to an increase in the response to PI3K-AKT-mTOR signaling pathway inhibitors (47). In pancreatic ductal adenocarcinoma PDTOs, BRCA1, BRCA1 and PALP2 mutations leading to alterations in DNA damage repair machinery have emerged as critical biomarkers of sensitivity to platinum-based chemotherapy and PARP inhibitors (18). Conversely, distinct transcriptional subtypes identified from RNA sequencing of PDTOs have been associated with differential sensitivity to gemcitabine, FOLFIRINOX, and MEK inhibitors (18). In breast cancer PDTOs, ER, PR, and HER2 expression levels maintain their predictive power for endocrine therapy and HER-directed treatments (35). Likewise, in prostate cancer PDTOs, androgen receptor (AR) amplification and splice variants, such as AR-V7, have been linked to resistance to androgen signaling inhibitors (25). Taken together, these examples, demonstrate that PDTO models not only recapitulate patient-specific drug responses but also provide a powerful system for the functional validation of genomic, transcriptomic, and phenotypic biomarkers of drug sensitivity and resistance with an ultimate aim to strength their role in biomarker-driven precision oncology.

Organoid systems are considered highly disease-relevant models since they reproduce human epithelial biology, including host-pathogen interactions, under controlled experimental conditions (40). In virology, the reproduction of human norovirus has been demonstrated, for the first time, in human intestinal organoids. Specifically, in this study, viral entry, replication and host organoid response was uncovered (55). Furthermore, airway epithelial-derived organoids have been utilized to investigate respiratory diseases, specifically in relation to the role of the ACE2 receptor and proteolytic cleavage mediated by SARS-CoV-2 (56), which has also been documented, in a separate study, as a critical factor in GI cancers (57, 58). Lastly, gastric organoid models have been developed for simulating Helicobacter pylori infection wherein, as a result, epithelial cell damage, cytoskeletal reorganization, and activation of proinflammatory signaling pathways have been studied (59). These studies show the power of organoids in studying complex host–microbe interactions that traditional culture systems or animal models were unable to facilitate.

Patient-derived organoids (PDOs) have been broadly considered as supporting scaffolds for tissue regeneration and functional restoration in regenerative medicine (42). For example, wounded mice were examined after healthy liver PDOs transplantation and as a result, partially restored liver function was observed, demonstrating the promise of organoid-based hepatocyte replacement therapy (60). Furthermore, corneal epithelial-derived PDOs were into animal models with corneal injury and as a consequence, transparency was restored, indicating a promising vision-restoration therapy (61). Moreover, PDOs from intestinal tissue transplanted into an animal model of ulcerative colitis resulted in mucosal healing with restoration of epithelial barriers, indicating the role of PDO-based therapies for chronic inflammatory diseases (62). Despite these promising studies, there are still challenges associated with PDO-based approaches in regenerative medicine including the organoid production rate, immunological compatibility, and integration into host tissues (50).