Cholangiocarcinoma (CCA) is a highly heterogeneous epithelial carcinoma with variable degrees of differentiation (1,2). Based on anatomical location, CCA is classified into intrahepatic CCA and extrahepatic cholangiocarcinoma (eCCA). According to the location relative to the cystic duct, eCCA is further subdivided into perihilar cholangiocarcinoma and distal cholangiocarcinoma (3,4). eCCA accounts for 20–30% of all biliary tract cancers (5). Established risk factors mainly include non-alcoholic fatty liver disease, liver cirrhosis and primary sclerosing cholangitis (6). Over recent decades, the incidence of eCCA has increased steadily. However, the prognosis remains poor, with a 5-year survival rate of ~11% (7,8). Surgery is still the only curative treatment for non-metastatic eCCA. For patients with advanced disease, the primary treatment strategy is chemotherapy combined with immunotherapy, although the therapeutic efficacy remains limited. Therefore, investigation of the pathogenesis of eCCA and development of more effective therapies are of great importance.

Unlike tumor cell lines, organoids are three-dimensional (3D) structures derived from pluripotent stem cells or isolated organ progenitor cells. These structures can self-organize into ‘mini-organs’ that recapitulate the architecture of native tissues (9) and reproduce the pathophysiological characteristics of the parental tumors (10). Organoids closely mimic the structure and function of primary tissues while retaining high genetic fidelity and donor-specific therapeutic responses (11). Furthermore, the establishment of organoids requires substantially less time and tissue than the generation of patient-derived xenograft models (12). Importantly, compared to animal-based models, organoid systems offer a significant ethical advantage by reducing or replacing the need for animal experimentation, thereby addressing animal welfare concerns. While the use of human-derived cells for in vitro culture involves its own distinct ethical considerations, it bypasses many of the complex ethical implications inherent in animal testing.

This study aimed to explore the feasibility of establishing eCCA organoids using suspension culture, with the goal of providing a potentially more economical and rapid alternative construction platform for basic research on this malignancy.

The remaining organoids in the centrifuge tube were centrifuged at 340 × g for 5 min at 4°C. According to the pellet size, 1–2 ml of cryopreservation medium was added and the pellet was gently resuspended by pipetting. A commercial cell-freezing medium was also acceptable. The suspension was then transferred to a 2-ml cryovial and stored in liquid nitrogen for long-term preservation.

For organoid recovery, the cryovial was rapidly thawed in a 37°C water bath. The organoid suspension was then immediately transferred to a 15-ml centrifuge tube using a 1,000-µl pipette tip. Next, 5 ml of complete organoid culture medium was added and the suspension was gently mixed by pipetting. After centrifugation at 340 × g for 5 min at 4°C, the supernatant was discarded. The pellet was then resuspended in 12 ml of complete organoid culture medium, gently mixed again and evenly seeded into a 12-well ultra-low-attachment plate. Organoid culture was continued as described above.

After centrifugation of the organoid suspension at 340 × g at 4°C for 5 min, 5 ml of 4% paraformaldehyde fixative was added to the centrifuge tube and fixation was carried out at 37°C for 24 h. The samples were then dehydrated through a graded ethanol series (50, 70, 80, 95 and 100% ×2), with each step lasting 30 min. Subsequently, the fixed organoid blocks were immersed in a xylene-ethanol mixture (1:1) for 30 min, followed by two incubations in pure xylene for 1 h each. The organoid blocks were then embedded in paraffin and the paraffin blocks were sectioned into slices ~5 µm thick using a microtome. The sections were mounted on glass slides, air-dried and stored for subsequent use.

Sections from paraffin-embedded organoid blocks and primary tumor tissue blocks were mounted on glass slides and placed on staining racks. Deparaffinization was performed in xylene (3×2 min), followed by rehydration through a graded ethanol series consisting of 100% ethanol (3×2 min), 95% ethanol (2 min) and 70% ethanol (2 min). The slides were then rinsed under running tap water at room temperature for at least 5 min.

Staining was performed with eosin solution for 2 min, followed by differentiation in 95% ethanol with ~20 dips. The sections were then dehydrated sequentially in 95% ethanol for 2 min and 100% ethanol (2×2 min). Clearing was performed in xylene (3×2 min). Finally, the sections were mounted with neutral balsam and covered with coverslips. Slides that passed microscopic quality control were subjected to image acquisition and analysis.

Cryopreserved eCCA tumor tissues stored at −80°C were fixed in 4% paraformaldehyde and embedded in paraffin to prepare 5 µm-thick sections, following the protocol previously described for organoids. To characterize the tissue origin and proliferative activity, both organoid sections and tumor tissue sections were subjected to standard chromogenic immunohistochemistry. After deparaffinization and rehydration through a graded ethanol series, antigen retrieval was performed in citrate buffer using a microwave oven, and endogenous peroxidase activity was blocked with 3% H₂O₂. The sections were then incubated overnight at 4°C with primary antibodies against CK7, CK19 and Ki-67, each at a dilution of 1:100. On the following day, a horseradish peroxidase-conjugated universal secondary antibody (ready-to-use) was applied for 30 min at room temperature, followed by visualization with 3,3′-diaminobenzidine. Positive signals were identified as brownish-yellow precipitates under a light microscope. Finally, the slides were counterstained with hematoxylin and mounted. Image acquisition and analysis were performed using optical microscopy. For Ki-67 quantification, two pathologists independently and blindly evaluated five randomly selected high-power fields per slide using ImageJ software (version 1.54f; National Institutes of Health). The mean percentage of positive nuclei relative to the total cell count was then calculated to determine the final labeling index.

Organoids were collected into a 15-ml centrifuge tube using a Pasteur pipette, centrifuged at 97 × g for 5 min at room temperature and the supernatant was discarded. The organoid pellet was then digested with trypsin to obtain a single-cell suspension. After cell counting with a hemocytometer, 100 µl of cell suspension adjusted to a cell density of 1.2×10⁵ cells/ml was evenly added into each well of a 96-well plate (NEST Biotechnology Co., Ltd). After 24 h, four antitumor drugs, gemcitabine, oxaliplatin, 5-FU and albumin-bound paclitaxel, were diluted in complete medium (RPMI-1640 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin) to the following concentrations. These concentration ranges were established based on our preliminary dose-finding experiments. Gemcitabine (600, 150, 30, 6, 1.5, 0.3, 0.06, 0.015, 0.003, 0.0006, 0.00015, 0.00003, 0.000006 and 0.0000012 µmol/l), oxaliplatin (400, 200, 100, 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078 and 0.039 µmol/l), 5-FU (7,680, 1,920, 384, 96, 24, 6, 1.5, 0.3, 0.06, 0.015, 0.003, 0.0006, 0.00015 and 0.00003 µmol/l) and albumin-bound paclitaxel (40, 20, 10, 5, 2.5, 0.5, 0.1, 0.025, 0.005, 0.001, 0.00025, 0.00005, 0.00001 and 0.0000025 µmol/l). After removal of the spent medium, 170 µl of the corresponding drug solution was added to each well. The plate included a control group and drug treatment groups. The control group received 170 µl of complete medium, whereas the treatment groups received an equal volume of the corresponding drug solution. The control group and each drug concentration group were set up in quadruplicate. After 72 h of culture, the medium was removed and 100 µl of Cell Counting Kit (CCK)-8 (APExBIO Technology LLC) diluted in RPMI-1640 at a 1:9 ratio was added to each well. Following incubation at 37°C for 3 h, the absorbance at 450 nm was measured using a microplate reader (Synergy H1; Biotek China). The experiment was independently performed in quadruplicate. Drug dose-response curves were generated and the half-maximal inhibitory concentration (IC₅₀) values were calculated using GraphPad Prism software, version 9.5.1 (Dotmatics). IC₅₀ values were determined by a nonlinear regression using the following four-parameter logistic equation:

where X is the logarithm of the drug concentration, Y is the normalized cell viability and HillSlope represents the slope of the dose-response curve. In the four-parameter logistic equation, Top represents the maximum response (cell viability at the lowest drug concentration, typically approaching 100% viability) and Bottom represents the minimum response (cell viability at the highest drug concentration, where inhibition reaches a plateau).

In the present study, a human eCCA organoid culture system was successfully established using suspension culture and its potential for clinical drug screening was preliminarily validated. These findings provide a novel research model and suggest a new direction for personalized therapy in this highly aggressive malignancy with a poor prognosis. Although the conventional Matrigel-based method remains the standard approach, batch-to-batch variability associated with its animal-derived origin may compromise reproducibility. In theory, the matrix-free approach used here may offer advantages in scalability and cost-effectiveness (14). It should be clarified that while the present study is not the first to establish CCA organoids, as Matrigel-based models for both intrahepatic and extrahepatic CCA have been described previously (15), it represents a novel implementation of a matrix-free suspension culture system specifically optimized for human eCCA. Currently, organoid technology is a rapidly advancing field and has been successfully established and utilized as ‘patient surrogates’ for various malignancies beyond eCCA, including gastric cancer (13), primary liver cancer (16) and lung cancer (17), demonstrating its robust predictive value for clinical drug responses.

In the present study, H&E staining confirmed that suspension-cultured CCA organoids retained the histological architecture of the primary tumors, as well as the characteristic strong cytoplasmic expression of biliary markers (CK7 and CK19). This high degree of resemblance may underlie the potential clinical relevance of the experimental findings and supports the use of these organoids as a promising alternative platform for drug screening. Previous work by our research group successfully established gastric cancer organoids using suspension culture, and these organoids closely resembled the original tissues and served as effective models for personalized drug screening (13). In addition, the matrix-free suspension culture strategy adopted in the present study was based on previous work on hepatic ductal organoids, in which efficient organoid expansion was achieved using stirred-tank bioreactors (18). Furthermore, a recent study on polyisocyanopeptide-based hydrogel functionalized with invasin hydrogel have shown that it can support the long-term three-dimensional expansion of various epithelial organoids, with efficacy comparable to that of Matrigel (19). Collectively, these studies help reduce reliance on complex natural matrices and promote the development of chemically defined and scalable organoid culture systems.

In the context of precision medicine, drug sensitivity testing using eCCA organoids show considerable potential for advancing personalized therapy. Organoids derived from different patients exhibit markedly different responses to standard chemotherapy regimens, such as gemcitabine/cisplatin, and these differences are closely associated with actual clinical outcomes (16). This suggests that prospective drug screening using patient-derived organoids before treatment initiation may help identify effective therapeutic strategies while avoiding the toxicity and treatment delays associated with ineffective therapies (16,17). This approach is particularly valuable for patients with eCCA, who usually face limited treatment options and poor outcomes. Conventional two-dimensional cell lines are often limited by low culture success rates. In addition, they are prone to genetic drift and loss of heterogeneity during in vitro passaging, which markedly restricts their predictive value for clinical drug responses (20). Multiple prospective clinical studies have reported a high degree of concordance between organoid drug sensitivity and patient outcomes, supporting the role of organoids as promising in vitro models for personalized medicine. Consistent with these findings, the present study showed that the drug-sensitivity profile of Organoid-P1 was in agreement with the clinical response of patient 1. Notably, Organoid-P1 exhibited marked resistance to oxaliplatin (IC₅₀=331.8±73.1 µmol/l), which is consistent with the clinical selection of the GS regimen (gemcitabine and S-1) rather than an oxaliplatin-based regimen for patient 1. This resistance is also consistent with previous findings showing that a subset of patients with eCCA derives limited benefit from platinum-based therapies, further supporting the utility of organoids in identifying potentially ineffective treatments (21,22). However, despite the high biological fidelity of this model, this single clinical correlation should be interpreted cautiously as preliminary and hypothesis-generating. Validation in larger prospective cohorts is still required to determine the definitive predictive value of this platform in eCCA.

However, the translation of organoid-based drug sensitivity testing into routine clinical practice still faces several challenges. A major limitation of the current model is its simplified composition, as it consists predominantly of tumor epithelial cells and lacks critical components of the tumor microenvironment, such as immune cells and fibroblasts (23). Although Patient 1 showed no recurrence after receiving a regimen that included the programmed cell death-1 inhibitor sintilimab, the current organoid model, composed solely of tumor epithelial cells, cannot predict or evaluate responses to immunotherapy. Therefore, the favorable clinical outcome observed in this patient may mainly reflect sensitivity to the chemotherapy components, gemcitabine and S-1, rather than immune-mediated effects. Future studies should address this limitation by establishing co-culture systems of organoids with autologous immune cells from patients (24,25) thereby enabling evaluation of the efficacy of immune checkpoint inhibitors (17,26) and facilitating the development of more predictive ‘all-in-one’ drug sensitivity models. In parallel, the standardization and automation of culture protocols, together with cost reduction, will be essential for large-scale clinical application.

With regard to technical feasibility, the successful establishment rate in this study was 60% (3/5), which reflects the inherent difficulty of organoid culture in biliary tract cancer. The failures in the remaining two cases may be attributable to both biological and technical factors. First, marked intratumoral heterogeneity and differences in the proportion of cancer stem cells may lead to variation in malignant proliferative capacity among samples. Second, the advanced age of the two patients in the failed cases (66 and 68 years) compared to the successful cases (58, 58 and 61 years) may have been associated with reduced cellular proliferative potential. In addition, both failed cases were moderately differentiated tumors, and their relatively lower degree of malignancy and slower proliferative rate, compared to poorly differentiated tissues, may have led to poor adaptation to the matrix-free suspension environment, subsequently promoting apoptosis. From a technical perspective, the high stromal content of these specimens required prolonged enzymatic digestion, which may have reduced primary cell yield and increased cellular damage. Finally, the anatomical proximity of the bile duct to the intestinal lumen increases the risk of microbial contamination. This issue was encountered during the culture process and remains a major obstacle to successful organoid establishment. In future work, the organoid biobank will be expanded by incorporating a broader range of sample types. In addition, the culture process should prioritize the maintenance of cellular viability rather than prolonged culture duration, and the methodology should be further standardized accordingly.

In summary, this study successfully established eCCA organoid models using suspension culture and preliminarily demonstrated their potential for exploring personalized treatment strategies. With continued technical optimization and further clinical validation, suspension-cultured organoids may become a valuable tool for guiding precision therapy in patients with eCCA.