A drug screening method based on biliary tract cancer organoids

Abstract

The application belongs to the technical field of biological medicine, and provides a drug screening method based on biliary tract cancer organoids. First, biliary tract cancer tumor tissues are used to construct biliary tract cancer tumor organoids with high activity and complete structure through an organoid culture method, a tumor environment is simulated, and a 384-well plate high-throughput drug screening system is used to screen effective drugs from candidate drugs through the biliary tract cancer tumor organoid model. The method uses an organoid model as a means of efficacy evaluation, constructs tumor organoids through intraoperative or biopsy specimens, and applies a small molecule compound library to treat the tumor organoids in vitro, so that the sensitivity of biliary tract cancer cells can be determined simply, quickly and economically, and a basis for individualized treatment is provided. Moreover, the method can quickly screen drugs sensitive to patients, and reduce the great pain of patients caused by multiple chemotherapies.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a drug screening method based on biliary tract cancer organoids. Background Technology

[0002] From 1990 to 2020, existing reports show significant regional differences in the global incidence and mortality rates of biliary tract cancer, primarily concentrated in high-income Asia-Pacific regions and southern Latin America. Biliary tract cancer (BTC) is a type of malignant tumor originating from the digestive system, with a high incidence rate in China. From 1990 to 2020, the number of BTC cases in China showed a continuous increase across all age groups, exhibiting regional heterogeneity with high incidence in the east and high mortality in the north. Obesity is now a newly emerging risk factor. Based on anatomical location, BTC is classified into intrahepatic cholangiocarcinoma (iCCA), extrahepatic cholangiocarcinoma (eCCA), and gallbladder cancer (GBC). The insidious onset, complex etiology, and anatomical heterogeneity of BTC often lead to advanced stages at the time of diagnosis, resulting in low five-year survival rates and poor prognosis. Current treatment for BTC primarily involves surgical resection followed by postoperative radiotherapy and chemotherapy. First-line chemotherapy drugs for advanced cholangiocarcinoma are gemcitabine and cisplatin. While existing treatment regimens, such as combined immune checkpoint inhibitor therapy, have further improved patients' overall survival, the 5-year overall survival rate remains low, the prognosis is poor, and relapse is common, indicating significant limitations in treatment. There is a need to develop new preclinical cell models to predict clinical treatment response.

[0003] In recent years, organoid technology, a newly emerging three-dimensional cell culture technique, has emerged. Organoids are capable of self-renewal and self-organization, and their greatest advantage is their ability to more closely mimic the structure and function of in vivo tissues and their long-term stable passage. Organoids have been applied to simulate various cancers, including breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, and liver cancer, and can relatively stably maintain the structural and genomic characteristics of the tumor organoids and the parent tissue. Studies have reported that tumor organoids, through in vitro drug sensitivity testing, correlate with clinical treatment responses, showing a high degree of consistency in treatment outcomes, providing a valuable preclinical model for the subsequent development and screening of novel drug treatments. Therefore, a new method for drug sensitivity testing of biliary tract cancer organoids is urgently needed to enable the selection of the correct and effective drugs based on the patient's condition during treatment, achieving individualized treatment. Summary of the Invention

[0004] The purpose of this invention is to provide a drug screening method based on biliary tract cancer organoids. This method directly uses biliary tract cancer organoids, takes advantage of the long-term stable passage of tumor organoids, shortens the PDX modeling cycle, significantly reduces costs, and leverages the advantages of simulating the tumor environment to conduct high-throughput drug screening through long-term in vitro amplification.

[0005] The present invention is achieved by the following technical solution: a drug screening method based on biliary tract cancer organoids, which first utilizes biliary tract cancer tumor tissue to construct biliary tract cancer tumor organoids with high activity and complete structure through organoid culture methods to simulate the tumor environment, and then uses the biliary tract cancer tumor organoid model to screen effective drugs through a high-throughput drug screening system of 384-well plates.

[0006] The method for preparing the biliary tract cancer organoids is as follows: biliary tract cancer tumor tissue is washed three times with PBS containing antibiotics and then cut into pieces to 0.3-0.7 mm. 3 The cells were sized and then resuspended in digestive solution for 20-40 minutes. After digestion, FBS was added to a final concentration of 2%-5% to stop the digestion, and the cells were gently mixed by pipetting. The cells were filtered through a 100μm cell filter, centrifuged at 250g for 4 minutes to remove the supernatant, washed three times with basal culture medium, and centrifuged at 250g for 4 minutes to remove the supernatant. 20μL of the cell suspension was used for viable cell counting using AO / PI. The cells were then resuspended in 70% matrix gel, and 30μL of the suspension was dropped into the center of the bottom of a 24-well plate. The plate was placed in a 37℃, 5% CO2 cell culture incubator to solidify for 30 minutes. After the matrix gel solidified, 500μL of complete tumor culture medium was added to adhere the cells. The 24-well plate was then placed in a 37℃, 5% CO2 cell culture incubator and cultured, with the complete culture medium changed every 3 days. The tumor organoids were ready when their diameter exceeded 100μm.

[0007] Furthermore, the biliary tract cancer organoids are subjected to long-term organoid passage culture, or are preserved in cryopreservation solution at -80°C using liquid nitrogen for long-term storage.

[0008] The candidate drugs are gemcitabine, oxaliplatin, cisplatin, irinotecan, 5-fluorouracil (5-FU), and paclitaxel.

[0009] The method for drug screening using the 384-well plate high-throughput drug screening system is as follows: Cultured tumor organoids are digested into small cell clusters and seeded into 384-well plates with black frames and transparent bottoms. 15-20 μL of 2%-3% matrix gel is pre-soiled in the lower layer of each well, and 800-1000 live cells are added to the upper layer of each well, with a total system volume of 50 μL. After the cells are evenly spread, the plates are incubated at 37°C with 5% CO2 and saturated humidity for 24 h. Then, a candidate drug with a final concentration of 1 μM is added in a volume of 10 nL, and the plates are incubated for another 96-120 h. The organoid activity is then detected.

[0010] Furthermore, the organoid activity was detected by adding 20 μL of ATP assay reagent, shaking at room temperature to promote cell lysis, and then performing chemiluminescence detection using a multi-functional microplate reader. Data were analyzed using GraphPad Prism software to calculate cell viability and drug inhibition rate, and a growth inhibition curve was plotted. Cell viability was calculated using the formula: Cell viability = Median value of the blank control group / (Median value of the blank control group - Median value of the candidate drug treatment group), and drugs with cell viability less than 30% were screened.

[0011] Compared with traditional drug sensitivity testing methods, the method described in this invention has the following advantages: The method provides an ATP detection procedure for organoids, making 384-well plate drug screening simple, standardized, and easy to learn. It uses fewer reagents and consumables, reducing costs and requiring fewer cells, thus saving organoid culture time. The culture time, drug administration time, and detection time in organoid drug sensitivity experiments are shorter and faster. This method uses organoid models as a means of efficacy evaluation. Tumor organoids are constructed from intraoperative or biopsy specimens, and then treated in vitro with a small molecule compound library. This allows for a simple, rapid, and economical determination of the sensitivity of biliary tract cancer cells, providing a basis for personalized treatment. Furthermore, it can quickly screen for drugs sensitive to patients, reducing the significant physical suffering caused by multiple chemotherapy sessions. Attached Figure Description

[0012] Figure 1 The growth status of 10 tumor organoids in continuous culture; Figure 2 The growth status of two organoids, PDO26 and PDO37, after continuous culture. Figure 3 The identification results for two organoids, PDO26 and PDO37; Figure 4 The process, images, and drug concentration curves for clinical drug screening of two tumor organoids in 96-well plates are shown. Figure 5 The types of drugs obtained by high-throughput screening of two tumor organoids in 384-well plates; Figure 6 This is a schematic diagram of tumor organoid drug screening. Detailed Implementation

[0013] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0015] Equivalent technologies of the specific embodiments described herein that are readily apparent to those skilled in the art through routine experimentation are included in this application.

[0016] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from regular biochemical reagent stores.

[0017] The main reagents used in this invention are shown in Table 1.

[0018] Table 1: Main Reagents Screening of organoids for biliary tract cancer tumors, illustrated in the diagram. Figure 6 The procedure, as shown, includes the following steps: obtaining biliary tract cancer tissue samples from patients and preprocessing them to obtain tissue blocks; digesting the tissue, coating it with matrix gel, plating it, and adding it to tumor complete culture medium for long-term amplification culture after solidification; after successful organoid culture, expanding the culture, collecting the organoids for fixation, HE and IHC staining, NCG mouse subcutaneous tumor formation, and whole-exome sequencing; detecting the sensitivity of two organoids to six conventional chemotherapy drugs in 96-well plates; and performing high-throughput drug screening on two organoids in 384-well plates, while simultaneously performing correlation analysis with the drug sensitivity results from the 96-well plate screening. The specific methods are as follows.

[0019] I. Methods for constructing organoids from biliary tract cancer tumors, such as Figure 1 As shown, the specific steps include the following: (1) Tissue sample pretreatment: Tumor tissue was transported to the laboratory using tissue preservation solution, washed three times with PBS containing antibiotics, and photographed to record the sample's pre-experimental condition in a sterile cell culture dish. The sample was then minced to approximately 0.5 mm using tissue scissors in a culture dish or centrifuge tube. 3 Left and right sizes.

[0020] (2) Digestion with digestion solution: Transfer the minced tissue to a 15ml centrifuge tube, add 10ml of digestion solution to resuspend, place in a constant temperature shaking incubator at 37℃ and 100rpm, and digest for 20-40min. During digestion, use a pipette to blow 10 times every 10min to ensure thorough digestion. After digestion is complete, add FBS to a final concentration of 2%-5%, gently blow to mix and stop digestion.

[0021] (3) Collection of live cells after filtration: Filter the cells using a 100 μm cell filter, centrifuge at 250 g for 4 min, and discard the supernatant. Wash the cells three times with basal culture medium, centrifuge at 250 g for 4 min, and discard the supernatant. Take 20 μL of the cell suspension and count the live cells using AO / PI.

[0022] (4) Matricella cell encapsulation: Centrifuge the cell suspension after counting at 250g for 4 min, discard the supernatant, and resuspend the cell pellet in 70% Matricella. During the operation, mix on ice to avoid generating air bubbles, and place on ice after mixing. Drop 30 μL of the suspension into the center of the bottom of a 24-well plate, place the culture plate in a 37℃, 5% CO2 cell culture incubator to solidify for 30 min. After the Matricella has solidified, add 500 μL of tumor complete culture medium to the wells to adhere to the cells. Place the 24-well plate in a 37℃, 5% CO2 cell culture incubator and change the complete culture medium every 3 days.

[0023] (5) Long-term culture: When the tumor organoids reach a diameter greater than 100 μm, passage the organoids. Aspirate the supernatant, add 200-500 μL of passage digestion solution, scrape off the matrix gel cell clusters, and thoroughly pipette about 10 times. Mix well and place in a 37°C incubator for 4-8 min, pipetting the cell suspension every 4 min until the biliary carcinoma organoids are digested into small cell clusters. Add 1 ml of basal culture medium to stop digestion, centrifuge at 250 g for 4 min, and discard the supernatant. Wash the cells 3 times with basal culture medium, centrifuge at 250 g for 4 min, and discard the supernatant. Coat the cell pellet with matrix gel, add 30 μL to a 24-well plate, place in an incubator for 30 min, and after solidification, add 500 μL of complete tumor culture medium. If the cells are not cultured and are directly cryopreserved, they will not be plated. After washing the cells, add organoid cryopreservation solution and store at -80°C according to the gradient cryopreservation method. Store in liquid nitrogen for long-term storage.

[0024] (6) Resuscitation Culture: Place the liquid nitrogen-preserved cells in a 37°C water bath and shake rapidly to thaw them quickly. Stop the water bath before the ice melts completely. Transfer the suspension to a 5ml tube, slowly add 3ml of basal culture medium, centrifuge at 250g for 4min, discard the supernatant, wash the cells 3 times, centrifuge at 250g for 4min, and discard the supernatant. Resuspend the cell pellet using matrix gel. Add 30μL of matrix gel cell suspension to a 24-well plate, incubate for 30min, and add 500μL of complete tumor culture medium after solidification.

[0025] (7) Observation and recording: The state of organoids during primary culture, passage and resuscitation was recorded using an inverted microscope.

[0026] The biliary tract cancer tissues used in this invention were all obtained from the First Affiliated Hospital of Shanxi Medical University. Fresh tumor tissues were collected with the informed consent of all patients and without affecting postoperative pathological diagnosis. From the biliary tract cancer tumor tissue samples collected from 32 patients, 10 tumor organoids were successfully constructed. The pathological details of the 10 cases are shown in Table 2.

[0027] Table 2: Pathological findings of 10 cases Tumor organoids were constructed from all 32 obtained biliary tract cancer tumor tissues. Due to factors such as tumor tissue heterogeneity, the viability of the obtained tissue samples, and the operational methods used in organoid construction, the success rate of tumor organoid construction was 31.25%. The growth status of the 10 successfully constructed tumor organoids after continuous culture is as follows: Figure 1 As shown, after 1-2 weeks of continuous culture, the biliary tract cancer organoid model matured, exhibiting solid, regular or irregular cystic structures. Subsequent high-throughput, large-scale drug screening processes require a large number of tumor organoids. However, organoid culture is time-consuming and costly. Therefore, PDO26 and PDO37 organoids, which exhibit rapid growth, high activity, and stable passage, were prioritized. From the 10 constructed tumor organoids, two of the most representative organoids (high malignancy), PDO26 and PDO37, were selected. Continuous observation of their growth status is shown below. Figure 2 As shown, in the experiments involving primary, passaged, and revived tumor organoids (PDO26 and PDO37), the primary cells formed spheroids after more than one week of culture; the cell morphology of the organoids remained unchanged during long-term passage; and the organoids cryopreserved in liquid nitrogen maintained their original growth state after successful revival. These longitudinal observations of the tumor organoids demonstrate that the constructed tumor organoid model can be stably cultured long-term.

[0028] II. Identification of organoids from biliary tract cancer tumors, such as Figure 2 As shown, the specific steps include the following: (1) Xenotransplantation: Two tumor organoids were cultured and expanded to 2x10⁻¹². 6 The right side of the NCG mouse was shaved, and the cells were resuspended in a 1:1 ratio with matrix gel. The suspension was injected subcutaneously into the mouse at a total volume of 250 μL. After one month, a tumor the size of a peanut could be seen.

[0029] (2) Paraffin sections of organoids: Organoid recovery and fixation: When the organoid volume reaches more than 50 μm, remove the supernatant, gently collect the organoid into a 1.5 ml EP tube using ice-cold PBS, let it stand on ice for 10 min, centrifuge at 250 g for 4 min, remove the supernatant, add 1 ml of 4% PFA and fix overnight.

[0030] Dehydration: Gradient ethanol was added to the original tube for dehydration, including 70%, 80%, 90%, 95% phenol red with a concentration of 2% and 100% ethanol, twice for 30 minutes at each concentration. The dehydration process was carried out on a low-speed shaker.

[0031] Transparency and wax impregnation: Treat xylene twice in a fume hood for 30 minutes each time. During this process, melt the paraffin into a liquid state at 65°C in advance. After centrifuging to remove the xylene, use a cut pipette tip to drop the paraffin onto the organoids of phenol red at high temperature. Place the EP tube in a 65°C metal bath overnight.

[0032] Embedding: Using an embedding machine, gently place the paraffin cell clusters into an embedding cassette containing paraffin, sink the cells to the bottom of the metal mold, place the tissue embedding cassette on top, pre-cool on a small ice table, and finally freeze on a freezing table. After solidification, carefully remove the mold.

[0033] Sectioning: Fix the embedded paraffin block onto a microtome to a thickness of 3-5 μm, carefully attach it to a glass slide, and dry it in a 60°C oven. Stain the sections using an automatic staining machine, and mount them with neutral resin. Observe under a microscope and scan using a VS200.

[0034] (3) Paraffin sections and staining of tumor tissue: The tissue corresponding to the organoid was sectioned, stained with hematoxylin and eosin and scanned with VS200 image. The image size corresponds to the scale bar: 100μm.

[0035] (4) IHC staining of organoids and tissues: The baked paraffin sections were stained using an automated immunohistochemistry instrument. Finally, the sections were mounted with neutral resin and scanned with VS200. The image size corresponds to a scale bar of 50 μm.

[0036] (5) Whole exome sequencing: Collect digested organoids and send them to Novogene for DNA sequencing. Use GATK software to generate a copy number map of the whole genome to visually display the copy number gain (amplification) and deletion (deletion) of each region of the chromosome.

[0037] result Figure 3As shown, the two organoids, PDO26 and PDO37, can form tumors in NCG mice. Hematoxylin and eosin staining of organoids and tissue sections confirmed the pathological consistency of cell morphology between the tumor organoids and paired tumor tissues. Immunohistochemical staining of tumor organoids and paired tumor tissues showed good consistency in staining with classic cholangiocarcinoma identification markers (Ki67, EpCAM, CK7, and CK19). Whole-exome sequencing revealed numerous copy number amplifications and deletions in various regions of the chromosomes in both organoids (PDO26 and PDO37). In summary, the above experimental methods confirmed that these two organoids are tumor organoids.

[0038] III. Drug screening of the prepared biliary tract cancer tumor organoids in 96-well plates, such as... Figure 4 As shown, the specific steps include the following: (1) Set up experimental groups (6 gradient concentrations for each drug, 3 replicates each) and blank groups.

[0039] (2) Digest the cultured tumor organoids into small cell clusters and seed them into black 96-well plates. Only the experimental group and the control group are seeded. The volume of matrix gel dropped into each well is 4-5 μL. The number of cells resuspended in each well is 1000-2000. After gelation, add 60 μL of tumor complete culture medium per well and place in an incubator at 37°C and 5% CO2.

[0040] (3) After 48-72 hours of inoculation, the organoid culture plate was removed, and six different conventional chemotherapy drugs to be tested were added. The plate was then cultured at 5% CO2 and 37°C for 96-120 hours. The drug-treated organoids were recorded using a high-content imaging instrument.

[0041] (4) Remove the organoid culture plate, equilibrate the cell culture plate to room temperature for 10 min, and add 30 μL of ATP detection reagent to the blank well, control well, and drug test well of the culture plate. Shake vigorously at room temperature for 5 min to promote full cell lysis, incubate at room temperature for 20 min, and perform chemiluminescence detection using a multifunctional microplate reader with chemiluminescence detection function.

[0042] (5) Use GraphPad Prism to analyze the data, calculate the cell viability rate, and plot the growth inhibition curve.

[0043] The results are as follows Figure 4As shown, two organoids were screened against six conventional chemotherapy drugs: gemcitabine, oxaliplatin, cisplatin, irinotecan, 5-fluorouracil (5-FU), and paclitaxel. Cell morphology was recorded at different concentrations of each drug, and IC50 curves for the corresponding drugs were obtained for the two samples. It was found that both organoids were relatively sensitive to gemcitabine and paclitaxel, with IC50 values ​​in the nM range. This suggests that the drugs screened by the model can be used as the preferred drugs for the treatment of these patients, providing a basis for individualized treatment in clinical practice.

[0044] IV. Drug screening of the prepared biliary tract cancer tumor organoids in 384-well plates, specifically including the following steps: (1) Set up a control group (gemcitabine with 6 gradient concentrations for each drug, 3 replicates each), an experimental group (small molecule drug library), and a blank group.

[0045] (2) Digest the cultured tumor organoids into small cell clusters and seed them into 384-well plates with black frames and transparent bottoms. Only the experimental group and the control group are seeded. 15-20 μL of 2%-3% matrix gel is pre-soiled in the lower layer of each well, and 30-35 μL of cell suspension is added to the upper layer of each well. 800-1000 live cells are added to each well. After the cells are evenly spread, they are placed in an incubator at 37°C and 5% CO2 for culture.

[0046] (3) After 24 hours of inoculation, the organoid culture plate was removed and a small molecule compound library with a final concentration of 1 μM was added using Echo555. Gemcitabine, the control group, was also added. The plate was then cultured for 96-120 hours in a 5% CO2, 37°C incubator.

[0047] (4) Remove the organoid culture plate, equilibrate the cell culture plate to room temperature for 10 min, and add 20 μL of ATP detection reagent to the blank wells, control wells, and drug test wells of the culture plate. Shake vigorously at room temperature for 5 min to promote full cell lysis, incubate at room temperature for 20 min, and perform chemiluminescence detection using a multi-functional microplate reader with chemiluminescence detection function. Analyze the data, calculate the cell viability, and plot the growth inhibition curve.

[0048] The results are as follows Figure 5 As shown, in the two organoids, 48 ​​candidate drugs were screened from 3439 drugs in the drug repositioning library using the 384 high-throughput drug screening method (including gemcitabine, which is relatively sensitive in the 96-well plate conventional chemotherapy drug sensitivity test). The cell viability of both was less than 30%. The drugs were divided into four categories: Category I is clinical chemotherapy drugs for biliary tract cancer, Category II is repurposing of old drugs, Category III is drugs that have been reported in tumor research, and Category IV is drugs in the clinical stage of tumor treatment and FDA approved drugs that have not been reported in biliary tract cancer.

[0049] This invention primarily focuses on the advantages and reliability of high-throughput screening methods. To further demonstrate the accuracy of drug screening, it focuses on the first category: commonly used clinical chemotherapy drugs for biliary tract cancer. Drugs screened include gemcitabine and paclitaxel, which were also tested in 96-well plates. The results showed high sensitivity and IC50 values ​​in the nM range, further confirming the reliability of the 384-well high-throughput screening system. This invention demonstrates a workflow for drug screening of biliary tract cancer tumor organoids in 384-well plates, applicable to high-throughput drug screening of various cancer types, and has identified candidate drugs that can effectively treat patients.

[0050] In summary, the drug screening method for biliary tract cancer tumor organoids using 384-well plates described in this invention utilizes surgically removed tumor tissue from biliary tract cancer patients. Through organoid culture, a biliary tract cancer organoid model with high activity and intact structure is constructed. Using this organoid model, an effective drug screening system using 384-well plates can be employed to screen for drugs that can treat patients. The 384-well drug screening system not only has stability and reliability but also reduces the workload, reagent consumption, and detection cycle associated with cell amplification, while increasing the number of drugs screened and reducing costs.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A drug screening method based on biliary tract cancer organoids, characterized in that: First, using biliary tract cancer tumor tissue, we constructed biliary tract cancer tumor organoids with high activity and complete structure through organoid culture methods to simulate the tumor environment. Then, using the biliary tract cancer tumor organoid model, we screened effective drugs from candidate drugs through a high-throughput drug screening system of 384-well plates.

2. The screening method according to claim 1, characterized in that: The method for preparing the biliary tract cancer organoids is as follows: biliary tract cancer tumor tissue is washed three times with PBS containing antibiotics and then cut into pieces to 0.3-0.7 mm. 3 The cells were sized and then resuspended in digestive solution for 20-40 minutes. After digestion, FBS was added to a final concentration of 2%-5% to terminate the digestion, and the cells were gently mixed by pipetting. The cells were filtered through a 100μm cell filter, centrifuged at 250g for 4 minutes to remove the supernatant, washed three times with basal culture medium, and centrifuged at 250g for 4 minutes to remove the supernatant. The resulting suspension was the primary bile duct cancer cell suspension. 20μL of the cell suspension was used for viable cell counting using AO / PI, and the cell concentration was 2×10⁶ cells / mL. 5 Cells / ml; then the cell suspension was mixed with matrix gel at a ratio of 3:7 and the cells were resuspended. 30 μL of the suspension was dropped into the center of the bottom of a 24-well plate and placed in a 37°C, 5% CO2 cell culture incubator to solidify for 30 min. After the matrix gel solidified, 500 μL of cholangiocarcinoma tumor culture medium was added to the top. The 24-well plate was then placed in a 37°C, 5% CO2 cell culture incubator and cultured for at least 7 days, with the cholangiocarcinoma tumor culture medium being changed every 3 days. The culture was considered complete when the diameter of the tumor organoids exceeded 100 μm.

3. The screening method according to claim 2, characterized in that: The biliary tract cancer organoids are subjected to long-term organoid passage culture, or are stored in cryopreservation solution at -80°C in liquid nitrogen for long-term preservation.

4. The screening method according to claim 2, characterized in that: The candidate drugs are gemcitabine, oxaliplatin, cisplatin, irinotecan, 5-fluorouracil (5-FU), paclitaxel, and a drug repositioning library comprising 3439 small molecules.

5. The screening method according to claim 2, characterized in that: The method for drug screening using the 384-well plate high-throughput drug screening system is as follows: cultured tumor organoids are digested into small cell clusters and seeded into 384-well plates with black frames and transparent bottoms. 15-20 μL of 2%-3% matrix gel is pre-soiled in the lower layer of each well, and 800-1000 live cells are added to the upper layer of each well, with a total system volume of 50 μL. After the cells are evenly spread, the plates are incubated at 37°C with 5% CO2 and saturated humidity for 24 h. Then, a candidate drug with a final concentration of 1 μM is added in a volume of 10 nL, and the plates are incubated for another 96-120 h. The organoid activity is then detected. The screening method according to claim 5 is characterized in that: The organoid activity was detected by adding 20 μL of ATP assay reagent, shaking at room temperature to promote cell lysis, and then performing chemiluminescence detection using a multi-functional microplate reader. Data were analyzed using GraphPadPrism software to calculate cell viability and drug inhibition rate, and growth inhibition curves were plotted. Cell viability was calculated using the formula: Cell viability = Median value of blank control group / (Median value of blank control group - Median value of candidate drug treatment group), and drugs with cell viability less than 30% were screened.

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