Culture medium, method and application for constructing liver cancer or cholangiocarcinoma explant

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a culture medium and method for constructing an ex-plant of hepatocarcinoma or cholangiocarcinoma and application. The culture medium and method for constructing an ex-plant of hepatocarcinoma or cholangiocarcinoma use tumor tissues removed from a hepatocarcinoma patient in surgery, proliferate and culture by using an optimized ex-plant culture medium, and construct a hepatocarcinoma ex-plant model with high activity and complete structure by using an ex-plant culture method, so that the hepatocarcinoma ex-plant model can be used for drug screening and guidance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a culture medium, method, and application for constructing explants for liver cancer or cholangiocarcinoma. Background Technology

[0002] Liver cancer is the third leading cause of cancer-related deaths worldwide, posing a serious threat to human health. Due to its high heterogeneity, aggressiveness, and metastatic potential, liver cancer treatment remains a major clinical challenge. Although multi-kinase inhibitors such as sorafenib and lenvatinib are widely used as first-line treatments for liver cancer, their efficacy is limited, and improvements in overall survival and quality of life are not significant.

[0003] Bile ductal carcinoma is a type of malignant tumor, usually caused by the transformation of cells in the bile ducts (the passageway that transfers bile from the liver to the intestines), accounting for approximately 20% of all primary liver cancers. Patients with bile ductal carcinoma are predominantly male, with a peak incidence between 60 and 70 years of age. Because the cystic duct in the gallbladder merges with the hepatic duct to form the bile duct, the contents of the bile duct and pancreatic duct enter the intestine through a structure called the hepatopancreatic ampulla. Normally, bile ductal carcinoma may originate from intrahepatic or extrahepatic bile duct epithelial cells, but does not include the gallbladder or hepatopancreatic ampulla. Based on its anatomical location in the biliary tree, bile ductal carcinoma can be divided into peripheral or intrahepatic bile ductal carcinoma (ICC) occurring within the liver and extrahepatic bile ductal carcinoma (CCE) occurring in the extrahepatic bile duct. CCE can be further divided into hilar bile duct carcinoma at the confluence of the left or right bile duct (biliary orifice) and distal bile duct carcinoma at the distal end of the bile duct. From a histological perspective, that is, from the characteristics of the constituent cells, bile duct epithelial cell carcinoma has several different variants: in most cases, it is an adenocarcinoma (papillary carcinoma, mucinous adenocarcinoma, clear cell carcinoma, intestinal-type adenocarcinoma, etc.) or carcinoma (squamous cell carcinoma, undifferentiated carcinoma, small cell carcinoma, etc.). Based on the degree of differentiation, that is, the degree of similarity between the tumor cells and the original epithelial cells, the tumor can be classified as well-differentiated, moderately differentiated, and poorly differentiated (or undifferentiated).

[0004] Clinical studies have shown that, although liver cancer cells and bile duct cancer cells both occur in the liver and differ in origin, biological behavior, and treatment, they do share some key similarities, particularly in the development of clinical drug screening models, where they offer potential applications.

[0005] First, there are similarities in their origin and microenvironment. Since both originate in the same organ, the liver, they share a common organ environment, meaning their development is influenced by the liver's unique microenvironment. For example, both benefit from the dual blood supply of the hepatic artery and portal vein, which promotes rapid tumor growth and metastasis. Furthermore, because the liver is an organ with specific immune tolerance functions (due to its constant processing of antigens from the gut), this environment may help both types of cancer cells evade the immune system's surveillance. Moreover, they share the same immune microenvironment, specifically manifested in the sharing of some important risk factors. For instance, chronic liver diseases, including chronic viral hepatitis (HBV and HCV) and cirrhosis, are major shared risk factors for both, although they are more strongly associated with HCC, they are also important risk factors for ICC.

[0006] Secondly, both cancers share certain similarities in molecular and cell biology. Both involve the aberrant activation of many of the same oncogenic signaling pathways, although the specific mutated genes may differ. For example, the MAPK / ERK pathway, related to cell proliferation and survival, is common in both cancers; the PI3K / AKT / mTOR pathway, also closely related to cell growth, metabolism, and survival, is a common target in both; and the VEGF pathway, related to angiogenesis (tumor angiogenesis), is effective for some patients in both highly vascularized tumors. Furthermore, both cancers exhibit widespread epigenetic regulatory abnormalities, such as DNA methylation and histone modifications, which can lead to tumor suppressor gene silencing or oncogene activation. Both also possess high intratumoral heterogeneity, meaning that different subpopulations of cancer cells may exist within the same tumor, leading to treatment resistance and recurrence, posing a significant challenge to treatment.

[0007] All of these characteristics indicate that liver cancer and bile duct cancer are closely related in many ways.

[0008] In clinical drug development and treatment, effective drug sensitivity screening models are indispensable tools for evaluating drug efficacy and safety. While traditional two-dimensional cell culture models are simple to operate, they struggle to simulate the complex tumor microenvironment in vivo, leading to screening results that often deviate significantly from clinical reality. Animal models, while closer to human physiological states, are time-consuming, costly, and raise ethical concerns. Patient-derived xenograft models, while more accurately replicating the tumor-stromal exchange process during tumor growth and proven to predict clinical outcomes relatively accurately, represent a mature experimental model; however, their long culture periods and high costs limit their role in personalized cancer treatment.

[0009] Patient-derived organoid models fall between cell models and animal models, combining the advantages of both. This is especially relevant since April 10, 2025, when the U.S. Food and Drug Administration (FDA) announced plans to phase out animal testing requirements for monoclonal antibodies and other drugs. The FDA will promote the use of lab-grown human "organoids" and organ-on-a-chip systems, which can mimic human organs (such as the liver, heart, and immune organs) to test drug safety. These experiments can reveal toxic effects that might be easily overlooked in animals, providing a more direct window into human responses. In recent years, explant models have emerged in the field of drug sensitivity screening due to their ability to mimic the in vivo tumor microenvironment. This technology successfully preserves the complex structures and microenvironment of tumors, such as blood vessels, stroma, and immune cells, by obtaining biological materials from patient tumor tissue and culturing them in vitro. This near-realistic microenvironment reproduction significantly improves the accuracy of predicting tumor biological behaviors such as growth, invasion, and metastasis. Furthermore, by constructing explant models of liver cancer or bile duct cancer from different patients, the efficacy differences of drugs in different patients can be further evaluated, providing an important basis for the development of individualized treatment plans.

[0010] In tumor drug development models, tumor explant models and tumor organoid models are the two most crucial in vitro models in modern tumor research. Each has its advantages and disadvantages and is suitable for different research scenarios. Tumor explant models refer to tumor tissue directly obtained from a patient through surgery or biopsy, which is then physically cut into small pieces (usually 1-2 mm). 3 Tumor explant models, which are short-term in vitro cultures, are characterized by their "unaltered" nature, preserving to the maximum extent the complex tumor microenvironment (TME) of the primary tumor, including various types of cancer cells, fibroblasts, immune cells, vascular endothelial cells, and extracellular matrix (ECM). Tumor organoid models, on the other hand, are three-dimensional (3D) cell aggregates formed by the self-renewal, proliferation, and differentiation of tumor stem cells (CSCs) or stem cells under specific culture conditions (such as in Matrigel with the addition of specific growth factors), after tumor tissue is digested into single cells or small cell clusters. Their core characteristic is "artificial reconstruction," where self-renewing tumor cells are screened and enriched in vitro, resulting in simplified but highly organized micro-tumor structures. Therefore, in this field, tumor explant models and tumor organoid models are two parallel but completely different models for drug development, each with its own advantages and disadvantages, leading to different clinical applications. The specific differences between tumor explant models and tumor organoid models are shown in Table 1 below.

[0011] Table 1

[0012] Feature Dimension Tumor explant model Tumor organoid models Sources and preparation Physical cutting: Directly cutting small pieces of tissue. Enzymatic digestion + screening: After digesting the tissue, tumor stem cells are cultured. Cellular composition Highly complex and heterogeneous: • Multiple tumor cell subsets • Immune cells (T cells, B cells, macrophages, etc.) • Fibroblasts • Vascular endothelial cells • Extracellular matrix (ECM) Relatively simple and singular: • Mainly composed of tumor cells of epithelial origin • Usually lacks most stromal cells and immune cells (which need to be added later through co-culture techniques). Organizational structure It perfectly preserves the tissue structure, spatial heterogeneity, and authentic cell-to-cell interactions of the primary tumor. It self-assembles and possesses some of the histological features of the primary tumor (such as glandular lumens and cell polarity), but the structure is reconstructed, simplified, and does not represent true cell-to-cell interactions. cultivation cycle Short-term (usually 1-2 weeks) • Difficult to passage for long periods • Prone to overgrowth of normal fibroblasts Long modeling and culture periods; cryopreservation thawing success rate is only 15%; easy to establish a biobank. Cultivation success rate The success rate is low and highly dependent on tumor type, sample quality, and transportation conditions. The success rate is relatively low. Flux and Scalability Low throughput: The number of experimental data points that can be generated per sample is limited, making standardization difficult. High throughput: can amplify a large number of organoids from a single sample. Genetic stability It maintains high genetic stability and is highly consistent with the original tumor within a short period of culture. After long-term passage, genetic drift (overgrowth of the dominant cell population) may occur, leading to increased differences from the original tumor. Main applications • Studying the tumor microenvironment (TME) (e.g., immunotherapy, drug resistance mechanisms) • Short-term drug susceptibility testing • Angiogenesis studies • Host-tumor interactions • Disease modeling and mechanism research • Gene editing (e.g., CRISPR) • Developmental biology research • Biobanks Advantages and disadvantages Advantages: Preserves the complete TME, more closely resembling the actual state in vivo, making it the gold standard for immunotherapy research; Disadvantages: Difficult to culture, short cycle time, high heterogeneity leading to significant challenges in reproducibility, and low throughput. Advantages: Partial long-term amplification, good stability, high throughput, and relatively good reproducibility; Disadvantages: Low culture success rate, lack of natural TME, loss of some heterogeneity of the original tumor, and significant bias in drug screening and personalized drug sensitivity testing results.

[0013] It is evident that tumor explant models have demonstrated superior adaptability and applicability compared to organoid models in personalized drug sensitivity testing, making them a research hotspot in this field. However, due to the complex composition of their culture systems and the lack of an immune environment, these models still have certain limitations in reproducing patients' drug sensitivity and resistance. Therefore, there is an urgent need in this field for a drug sensitivity screening model that can balance efficiency and accuracy, providing a more refined and reliable experimental platform for drug sensitivity screening and offering valuable support for anti-tumor drug development and clinical translation. Summary of the Invention

[0014] Therefore, the technical problem to be solved by the present invention is to provide a culture medium and method for constructing explants for liver cancer or cholangiocarcinoma, and its application in drug sensitivity screening and detection of liver cancer and cholangiocarcinoma.

[0015] To solve the above-mentioned technical problems, the present invention provides a culture medium for constructing explants for liver cancer or cholangiocarcinoma. The culture medium includes a basal culture medium and the following components at the following concentrations: B27 medium additive 1X-10X, N-acetylcysteine ​​1μM-1M, nicotinamide 1nM-1M, ALK5 inhibitor 1nM-1M, FGF10 recombinant protein 1ng / ml-1g / ml, ROCK inhibitor 1nM-1M, EGF recombinant protein 1ng / ml-1g / ml, FGF7 recombinant protein 1ng / ml-1g / ml, gastrin 1nM-1M, serum 2% v / v-20% v / v, and penicillin-streptomycin 1X-20X.

[0016] In some preferred embodiments, the culture medium further includes a component of the following concentration: Spirulina exosomes 1 nM-1 M.

[0017] This invention also discloses a method for constructing explants for liver cancer or bile duct cancer, comprising the following steps:

[0018] (1) Obtain liver cancer or bile duct cancer tissue samples from patients and preprocess them to obtain tissue blocks;

[0019] (2) Place the cell support system into the cell culture plate and add the culture medium for constructing liver cancer and cholangiocarcinoma explants and peripheral blood mononuclear cells (PBMCs) to form a PBMC-explant co-culture system;

[0020] (3) Immerse the tissue block in the PBMC-explant co-culture system and incubate it to obtain the desired result.

[0021] Specifically, in the method for constructing explants for liver cancer or bile duct cancer, in step (1), the pretreatment solution of the pretreatment step includes DMEM / F-12 basal culture medium, and the following concentration of components are added: ROCK inhibitor (i.e., Y-27632 small molecule inhibitor) 10nM-10mM.

[0022] Specifically, in the method for constructing explants for liver cancer or bile duct cancer, step (1) further includes a step of preserving and cleaning the liver cancer tissue sample from the patient; wherein,

[0023] The tissue preservation solution used in the tissue preservation step includes DMEM / F-12 basal culture medium, with the following concentrations of added components: fetal bovine serum 2% v / v-50% v / v, penicillin-streptomycin 1X-20X;

[0024] The cleaning solution used in the cleaning step includes physiological saline and contains the following concentrations of penicillin-streptomycin 1X-20X.

[0025] Specifically, in the method for constructing explants for liver cancer or bile duct cancer, in step (2), the cell support system includes a metal scaffold and filter paper;

[0026] The metal support is a cylindrical stainless steel structure with a hollow interior and a hollow surface.

[0027] The filter paper is highly absorbent.

[0028] Specifically, in the method for constructing explants for liver cancer or bile duct cancer, the spirulina exosomes are exosomes obtained from spirulina using conventional methods in the art.

[0029] Specifically, in the method for constructing explants for liver cancer or bile duct cancer, the conditions for the incubation and culture step (3) include: 35-38℃, 5% CO2 culture.

[0030] The present invention also discloses an explant for liver cancer or bile duct cancer, which is cultured by the method for constructing explants for liver cancer or bile duct cancer.

[0031] This invention also discloses the application of the liver cancer or bile duct cancer explants in the field of drug sensitivity testing, wherein the drug is a drug for treating cell proliferative diseases;

[0032] Specifically, the proliferative diseases mentioned include liver cancer or bile duct cancer.

[0033] Specifically, in the application of the liver cancer or bile duct cancer explants in the field of drug sensitivity testing, the drug sensitivity testing includes the steps of mixing the antitumor drug to be tested with the liver cancer explants and performing drug administration and culture, as well as detecting the cell viability of the liver cancer explants and calculating the tumor inhibition rate of the antitumor drug.

[0034] Specifically, the drug sensitivity test also includes the step of setting up a blank control group;

[0035] Preferably, the type and concentration of the antitumor drug are adjusted according to clinical needs.

[0036] One aspect of the present invention is to provide a detailed method for constructing explants for liver cancer or bile duct cancer, comprising the following steps:

[0037] (1) Obtain liver cancer or bile duct cancer tissue samples from patients. The above tissues should be collected within half an hour after the patient's surgical resection or biopsy, and the tissue samples should be processed within 6-8 hours. During transportation, the tissues should be soaked in tissue preservation solution and stored at low temperature.

[0038] Tissue samples were processed by transferring them to a laminar flow hood and placing them on ice. They were rinsed with a cleaning solution and then immersed in a pretreatment solution. Fractions with high malignancy and good activity were selected for physical shearing, ultimately reducing the sample size to 0.5-2 mm. 3 Tissue blocks of similar size;

[0039] (2) Place the required support system in the cell culture plate and add a certain amount of culture medium for constructing explants for liver cancer or cholangiocarcinoma to each well; then use human peripheral blood lymphocyte separation medium to separate peripheral blood mononuclear cells (PBMCs) from diluted human peripheral blood samples by density gradient centrifugation; collect the boundary layer rich in PBMCs at a density of 1×10⁻⁶ cells per well. 3 -1×10 4 A PBMC cell density was seeded into the wells of a culture plate to establish a PBMC-explant co-culture system;

[0040] (3) Place the trimmed tissue blocks in the center of the filter paper of the cell culture plate according to the group, ensuring that the culture medium can completely submerge the tissue blocks. After the tissue blocks are laid out, seal the outer ring of the cell culture plate with physiological saline and place it in an incubator at 37°C and 5% CO2 for culture.

[0041] The support system described in step (2) consists of a metal support and filter paper. The metal support is a cylindrical stainless steel structure with a hollow interior and a hollow surface, and the filter paper is highly absorbent.

[0042] Another aspect of the present invention provides an application of explants from liver cancer or bile duct cancer in drug sensitivity testing, comprising the following steps:

[0043] (1) The liver cancer or bile duct cancer explants were placed in an incubator at 37°C and 5% CO2 and the different wells in the cell culture plate were divided into a blank control group and a drug administration group;

[0044] (2) Add the target concentration of antitumor drug to the culture wells of the drug administration group as needed, and place the cell culture plate in an incubator at 37°C and 5% CO2 for culture;

[0045] (3) After culturing for 2-20 days after drug administration, the cell viability of the explants was detected and photographed and recorded, and the tumor inhibition rate of different drugs was calculated.

[0046] The drugs and their concentrations used in the testing should be adjusted according to clinical needs.

[0047] The preferred application of this invention is liver cancer or bile duct cancer.

[0048] The culture medium for constructing explants for liver cancer or cholangiocarcinoma described in this invention, through screening for a system suitable for tissue development in liver cancer or cholangiocarcinoma patients, not only successfully constructed highly intact liver cancer and cholangiocarcinoma explants, but also exhibited high cell growth activity. Furthermore, due to the similarities in many micro-features between liver cancer and cholangiocarcinoma, the culture medium described in this invention has excellent clinical efficacy for the construction of explants for both liver cancer and cholangiocarcinoma.

[0049] The method for constructing explants for liver cancer or cholangiocarcinoma described in this invention utilizes surgically removed tumor tissue from liver cancer or cholangiocarcinoma patients. Through explant culture methods, especially optimized explant culture media, a highly active and structurally intact explant model for liver cancer or cholangiocarcinoma can be constructed. Compared with traditional cell line culture methods, the method for constructing explants for liver cancer or cholangiocarcinoma described in this invention can effectively improve the success rate and stability of explant construction, effectively avoid gene mutations and phenotypic changes that may occur in tumor cells during in vitro culture, and more realistically reflect the biological characteristics of tumor cells in vivo. This allows for the establishment of a drug sensitivity screening model that more closely resembles the physiological state of the human body and accurately reflects drug efficacy.

[0050] The method for constructing explants for liver cancer or cholangiocarcinoma described in this invention provides an explant model that accurately simulates tumor cell growth and drug response in vivo, offering a more precise experimental platform for drug screening. By studying the effects of different drugs on explants for liver cancer or cholangiocarcinoma, the efficacy of drugs can be effectively predicted, and drugs effective against individual patient tumor cells can be screened, thereby improving treatment outcomes.

[0051] The method for constructing explants for liver cancer or bile duct cancer described in this invention can utilize the patient's own tumor tissue to construct an explant model, conduct individualized drug sensitivity screening, and through drug sensitivity analysis of the patient's individual tumor cells, more precise treatment plans can be formulated, thereby improving the patient's survival rate and quality of life.

[0052] The method for constructing explants for liver cancer or cholangiocarcinoma described in this invention, using the explant models constructed according to this invention for drug sensitivity testing, can provide precise clinical references for personalized treatment. By simulating the response of tumor cells to different drugs in vitro, the explants constructed according to this invention can predict the patient's response to specific chemotherapy drugs and screen for the optimal treatment regimen. This accurate predictive ability is of great significance in guiding clinical medication, helping to improve treatment efficacy and reduce treatment risks.

[0053] The explants for liver cancer or bile duct cancer constructed by the method of this invention perfectly preserve the tissue structure, spatial heterogeneity, and real intercellular interactions of the primary tumor. Not only is the modeling cycle short and long-term culture possible, but the success rate can also reach almost 100%. In particular, the explant model constructed by this invention can be cryopreserved and thawed, making it suitable for establishing biobanks and solving the shortcomings of traditional explant models that cannot be cryopreserved and thawed for biological samples.

[0054] The advantages of the tumor explant model constructed based on the culture medium and method of the present invention compared with the traditional tumor explant model are listed in Table 2 below.

[0055] Table 2

[0056] Feature Dimension Traditional tumor explant models This invention constructs a tumor explant model Sources and preparation Physical cutting: Directly cutting small pieces of tissue. Physical cutting: Directly cutting small pieces of tissue. Cellular composition Highly complex and heterogeneous: • Multiple tumor cell subsets • Immune cells (T cells, B cells, macrophages, etc.) • Fibroblasts • Vascular endothelial cells • Extracellular matrix (ECM) Highly complex and heterogeneous: • Multiple tumor cell subsets • Immune cells (T cells, B cells, macrophages, etc.) • Fibroblasts • Vascular endothelial cells • Extracellular matrix (ECM) Organizational structure It perfectly preserves the tissue structure, spatial heterogeneity, and authentic cell-to-cell interactions of the primary tumor. It perfectly preserves the tissue structure, spatial heterogeneity, and authentic cell-to-cell interactions of the primary tumor. cultivation cycle Short-term (usually 1-2 weeks) • Difficult to passage for long periods • Prone to overgrowth of normal fibroblasts Short modeling cycle, long culture cycle; enables cryopreservation and thawing; allows for the establishment of biobanks. Cultivation success rate The success rate is low and highly dependent on tumor type, sample quality, and transportation conditions. The success rate is high, almost 100%. Flux and Scalability Low throughput: The number of experimental data points that can be generated per sample is limited, making standardization difficult. Medium throughput: Each sample can generate relatively abundant experimental data points, which can be standardized. Genetic stability It maintains high genetic stability and is highly consistent with the original tumor within a short period of culture. It maintains high genetic stability and is highly consistent with the original tumor within a short period of culture. Main applications • Studying the tumor microenvironment (TME) (e.g., immunotherapy, drug resistance mechanisms) • Short-term drug susceptibility testing • Angiogenesis studies • Host-tumor interactions • Research on the tumor microenvironment (TME) (e.g., immunotherapy, drug resistance mechanisms) • High-throughput drug screening and personalized drug sensitivity testing • Angiogenesis research • Host-tumor interactions • Biobanks Advantages and disadvantages Advantages: Preserves the complete TME, more closely resembling the actual state in vivo, making it the gold standard for immunotherapy research; Disadvantages: Difficult to culture, short cycle time, high heterogeneity leading to significant challenges in reproducibility, and low throughput. Advantages: Preserves the complete TME, more closely resembling the actual in vivo state, making it the gold standard for immunotherapy research, and also highly suitable for evaluating the efficacy of various types of anti-tumor drugs; easy to culture, moderate culture cycle. Disadvantages: moderate throughput. Attached Figure Description

[0057] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0058] Figure 1 This is a flowchart of the method for constructing explants for liver cancer or bile duct cancer according to the present invention;

[0059] Figure 2 The results of morphological observation of parental tissues and explants in Experiment Example 2 (400×);

[0060] Figure 3 The lesion morphology before and after medication in the clinical consistency trial of Experiment Example 3;

[0061] Figure 4 The results of drug sensitivity testing in the clinical consistency experiment of Experiment Example 3 are as follows;

[0062] Figure 5 The images show the measured values ​​of liver cancer explants under different drug tests in the drug sensitivity test of Experiment Example 4.

[0063] Figure 6 The results of drug sensitivity testing are shown for the liver cancer explants constructed in the drug sensitivity test of Experiment Example 4.

[0064] Figure 7 This is a measured image of the bile duct carcinoma explant under different drug tests in the drug sensitivity test of Experiment Example 7;

[0065] Figure 8 The morphological changes of liver cancer cells and bile duct cancer cells before and after cryopreservation in Experiment Example 9. Detailed Implementation

[0066] Figure 1 This is a flowchart of the method for constructing explants for liver cancer or bile duct cancer according to the present invention. In the following embodiments, the present invention provides a method for constructing liver cancer explants, comprising the following steps:

[0067] (1) Obtain liver cancer or bile duct cancer tissue samples from patients and preprocess them to obtain tissue blocks;

[0068] (2) Place the cell support system into the cell culture plate and add complete culture medium for liver cancer or bile duct cancer explants and peripheral blood mononuclear cells (PBMCs) to form a PBMC-explant co-culture system;

[0069] The complete culture medium for explants of liver cancer or cholangiocarcinoma includes a basal culture medium and the following components at the following concentrations: B27 medium additive 1X-10X, N-acetylcysteine ​​1μM-1M, nicotinamide 1nM-1M, ALK5 inhibitor (A83-01 inhibitor) 1nM-1M, FGF10 recombinant protein 1ng / ml-1g / ml, ROCK inhibitor (Y-27632 small molecule inhibitor) 1nM-1M, EGF recombinant protein 1ng / ml-1g / ml, FGF7 recombinant protein 1ng / ml-1g / ml, gastrin 1nM-1M, serum 2% v / v-20% v / v, and penicillin-streptomycin 1X-20X;

[0070] In some preferred embodiments, the culture medium for constructing hepatocellular carcinoma or cholangiocarcinoma explants further includes a component at a concentration of 1 nM-1 M spirulina exosomes. This invention, by adding suitable active ingredients to the complete culture medium for hepatocellular carcinoma or cholangiocarcinoma explants, can better achieve the proliferation of hepatocellular carcinoma or cholangiocarcinoma explants.

[0071] (3) Immerse the tissue block in the PBMC-explant co-culture system and incubate it.

[0072] In the method for constructing explants for liver cancer or cholangiocarcinoma described in this invention, liver cancer or cholangiocarcinoma tissue is used as the matrix material. By screening and optimizing the culture medium, a liver cancer or cholangiocarcinoma explant model with high activity and complete structure can be constructed, which can more realistically reflect the biological characteristics of tumor cells in vivo.

[0073] As a feasible specific implementation method, the method for constructing explants for liver cancer or bile duct cancer specifically includes the following steps:

[0074] (1) Obtain liver cancer or bile duct cancer tumor tissue samples from patients. The above tissues should be collected within half an hour after surgical resection or biopsy. The tissue samples should be processed within 6-8 hours. During transportation, the tissues should be soaked in tissue preservation solution and stored at low temperature. To process the tissue samples, transfer the obtained tissue samples to a laminar flow hood, place them on ice, rinse them with cleaning solution, and then soak them in pretreatment solution. Select the parts with higher malignancy and better activity for physical shearing. The final processing is 1 mm. 3 Tissue blocks of similar size;

[0075] (2) Place the required support system in the cell culture plate and add 500 μL of complete culture medium for hepatocellular carcinoma or cholangiocarcinoma explants to each well; then use human peripheral blood lymphocyte separation medium to separate peripheral blood mononuclear cells (PBMCs) from diluted human peripheral blood samples by density gradient centrifugation; collect the boundary layer rich in PBMCs at 1 × 10⁻⁶ cells per well. 3 -1×10 4 A PBMC cell density was seeded into the wells of a culture plate to establish a PBMC-explant co-culture system;

[0076] The explant culture medium for liver cancer or cholangiocarcinoma includes a basal culture medium and the following components at the following concentrations: B27 medium additive 1X-10X, N-acetylcysteine ​​1μM-1M, nicotinamide 1nM-1M, ALK5 inhibitor (A83-01 inhibitor) 1nM-1M, FGF10 recombinant protein 1ng / ml-1g / ml, ROCK inhibitor (Y-27632 small molecule inhibitor) 1nM-1M, EGF recombinant protein 1ng / ml-1g / ml, FGF7 recombinant protein 1ng / ml-1g / ml, gastrin 1nM-1M, serum 2% v / v-20% v / v, and penicillin-streptomycin 1X-20X;

[0077] In some preferred embodiments, the culture medium for constructing explants for liver cancer or cholangiocarcinoma further includes the following components at concentrations: Spirulina exosomes 1 nM-1 M;

[0078] (3) Place the trimmed tissue blocks in the center of the filter paper of the cell culture plate according to the group, ensuring that the culture medium can completely submerge the tissue blocks. After the tissue blocks are laid out, seal the outer ring of the cell culture plate with physiological saline and place it in an incubator at 37°C and 5% CO2 for culture.

[0079] In some specific embodiments, in step (1), the pretreatment solution of the pretreatment step includes DMEM / F-12 basal culture medium, and the following concentration of component is added: ROCK inhibitor (Y-27632 small molecule inhibitor) 10nM-10mM.

[0080] In some specific embodiments, step (1) further includes the steps of preserving and cleaning the patient or cholangiocarcinoma / hepatocellular carcinoma tissue sample;

[0081] The tissue preservation solution used in the tissue preservation step includes DMEM / F-12 basal medium, with the following components added at concentrations: fetal bovine serum 2% v / v-50% v / v, penicillin-streptomycin 1X-20X;

[0082] The cleaning solution used in the cleaning step includes physiological saline and contains the following concentrations of penicillin-streptomycin 1X-20X.

[0083] In some specific embodiments, in step (2), the cell support system includes a metal scaffold and filter paper;

[0084] The metal support is a cylindrical stainless steel structure with a hollow interior and a hollow surface.

[0085] The filter paper is highly absorbent.

[0086] Specifically, in the method for constructing explants for liver cancer or cholangiocarcinoma, the spirulina exosomes are exosomes obtained from spirulina using conventional methods in the art. The extraction method for the spirulina exosomes can be a conventional gradient centrifugation method.

[0087] As an example, in the following embodiments of the present invention, the method for preparing the added Spirulina exosomes includes the following steps:

[0088] (1) Take fresh spirulina, wash it, add 6 times its weight of PBS buffer and mix it, and perform pulping under normal parameters. Collect the filtrate after filtration.

[0089] (2) At 2℃, the filtrate obtained above was centrifuged at 2500 rpm for 20 min, filtered again and the supernatant was collected;

[0090] (3) At the same time, at 2℃, the collected supernatant was centrifuged at 8000rpm for 30min, filtered again and the supernatant was collected;

[0091] (4) At the same time, the collected supernatant was centrifuged at 12000 rpm for 40 min at 2℃, filtered again and the supernatant was collected.

[0092] (5) Continue to filter the collected supernatant through a 0.45μm microporous membrane to obtain Spirulina exosomes.

[0093] In some specific implementations, the conditions for the incubation and cultivation step (3) include: 35-38℃, 5% CO2 cultivation.

[0094] In the following embodiments of the present invention, a liver cancer or bile duct cancer explant obtained by the construction method is also provided.

[0095] In the following embodiments of the present invention, the application of the liver cancer or bile duct cancer explant in the field of drug sensitivity testing is also provided, wherein the drug is a drug for treating liver cancer or bile duct cancer.

[0096] In some specific embodiments, the drug sensitivity test includes the steps of mixing the antitumor drug to be tested with the liver cancer or bile duct cancer explant and performing drug administration culture, as well as detecting the cell viability of the liver cancer or bile duct cancer explant and calculating the tumor inhibition rate of the antitumor drug.

[0097] As an exemplary specific implementation, the application of the liver cancer or bile duct cancer explants of the present invention in drug sensitivity testing includes the following steps:

[0098] (1) The liver cancer or bile duct cancer explants were placed in an incubator at 37°C and 5% CO2 and the different wells in the cell culture plate were divided into a blank control group and a drug administration group;

[0099] (2) Add the target concentration of antitumor drug to the culture wells of the drug administration group as needed, and place the cell culture plate in an incubator at 37°C and 5% CO2 for culture;

[0100] (3) After culturing for 2-20 days after drug administration, the cell viability of the explants was detected and photographed and recorded, and the tumor inhibition rate of different drugs was calculated.

[0101] The drugs and their concentrations used in the testing should be adjusted according to clinical needs.

[0102] The following specific implementation examples illustrate the embodiments of the present invention. Those skilled in the art can easily complete the operations according to the guidance of this specification and understand the advantages of the present invention. Based on the embodiments of the present invention, those skilled in the art, without inventive effort, are all within the scope of protection of the present invention.

[0103] In the following embodiments of the present invention, the B27 culture medium additive involved can be any commercially available product in the art, regardless of brand; the ALK5 inhibitor is a commercially available A83-01 inhibitor, such as MCE; the ROCK inhibitor is a commercially available Y-27632 small molecule inhibitor, such as MCE.

[0104] It should be noted that in the following embodiments of the present invention, the amount of each component added is expressed in concentration units known in the art, such as M, mM, g / ml, % v / v, or X. Wherein, the concentration multiple X represents a multiple relative to the working concentration. For example, penicillin-streptomycin 10X means that the concentration of penicillin-streptomycin is concentrated to 10 times its conventional working concentration. It can be diluted accordingly during preparation. Other units, such as those for B27 culture medium additives, express the same meaning.

[0105] Example 1

[0106] like Figure 1 The process shown in this embodiment, the method for constructing liver cancer explants, specifically includes the following steps:

[0107] (1) Obtain liver cancer tumor tissue samples from patients. The above tissues should be collected within half an hour after surgical resection or biopsy. The tissue samples should be processed within 4-8 hours. During transportation, the tissues should be soaked in tissue preservation solution and stored at low temperature. To process the tissue samples, the obtained tissue samples should be transferred to a laminar flow hood, placed on ice, rinsed with cleaning solution, and then soaked in pretreatment solution. The parts with higher malignancy and better activity should be physically sheared. The final processing should be 1 mm. 3 Tissue blocks of similar size; among them,

[0108] The tissue preservation solution includes DMEM / F-12 basal medium, supplemented with 20% fetal bovine serum v / v and penicillin-streptomycin 10X;

[0109] The cleaning solution includes physiological saline and is supplemented with penicillin-streptomycin 10X;

[0110] The pretreatment solution includes DMEM / F-12 basal culture medium with 5 mM of Y-27632 small molecule inhibitor added;

[0111] (2) Place the required cell support system in the cell culture plate and add 500 μL of complete culture medium for liver cancer explants to each well;

[0112] The complete culture medium for liver cancer explants includes DMEM / F-12 basal medium and the following components at the following concentrations: B27 medium additive 5X, N-acetylcysteine ​​0.5M, nicotinamide 0.5M, A83-01 0.5M, FGF10 recombinant protein 0.5g / ml, Y-27632 small molecule inhibitor 0.5M, EGF recombinant protein 0.5g / ml, FGF7 recombinant protein 0.5g / ml, gastrin 0.5M, serum 10% v / v, and penicillin-streptomycin 10X;

[0113] The cell support system includes a metal scaffold and filter paper;

[0114] The metal support is a cylindrical stainless steel structure with a hollow interior and a hollow surface.

[0115] The filter paper is highly absorbent filter paper;

[0116] Subsequently, peripheral blood mononuclear cells (PBMCs) in diluted human peripheral blood samples were separated using density gradient centrifugation with human peripheral blood lymphocyte separation medium. PBMC-rich boundary layers were collected at 5 × 10⁶ cells per well. 3 A PBMC cell density was seeded into the wells of a culture plate to establish a PBMC-explant co-culture system;

[0117] (3) The trimmed tissue blocks are laid flat in the center of the filter paper of the cell culture plate according to the group, ensuring that the culture medium can completely submerge the tissue blocks. After laying, the outer ring of the cell culture plate is sealed with physiological saline and placed in an incubator at 37°C and 5% CO2 for incubation.

[0118] Example 2

[0119] The method for constructing liver cancer explants described in this embodiment includes the following steps:

[0120] (1) Obtain liver cancer tumor tissue samples from patients. The above tissues should be collected within half an hour after surgical resection or biopsy. The tissue samples should be processed within 6-8 hours. During transportation, the tissues should be soaked in tissue preservation solution and stored at low temperature. To process the tissue samples, the obtained tissue samples should be transferred to a laminar flow hood, placed on ice, rinsed with cleaning solution, and then soaked in pretreatment solution. The parts with higher malignancy and better activity should be physically sheared. The final processing should be 1 mm. 3 Tissue blocks of similar size; among them,

[0121] The tissue preservation solution includes DMEM / F-12 basal medium, supplemented with 2% fetal bovine serum v / v and penicillin-streptomycin 20X;

[0122] The cleaning solution includes physiological saline and is supplemented with penicillin-streptomycin 1X;

[0123] The pretreatment solution includes DMEM / F-12 basal medium with 10 nM of Y-27632 small molecule inhibitor added;

[0124] (2) Place the required cell support system in the cell culture plate and add 500 μL of complete culture medium for liver cancer explants to each well;

[0125] The complete culture medium for liver cancer explants includes DMEM / F-12 basal medium and the following components at the following concentrations: B27 medium additive 1X, N-acetylcysteine ​​1M, nicotinamide 1nM, A83-01 inhibitor 1M, FGF10 recombinant protein 1ng / ml, Y-27632 small molecule inhibitor 1M, EGF recombinant protein 1ng / ml, FGF7 recombinant protein 1g / ml, gastrin 1nM, serum 20% v / v, and penicillin-streptomycin 1X;

[0126] The cell support system includes a metal scaffold and filter paper;

[0127] The metal support is a cylindrical stainless steel structure with a hollow interior and a hollow surface.

[0128] The filter paper is highly absorbent filter paper;

[0129] Subsequently, peripheral blood mononuclear cells (PBMCs) in diluted human peripheral blood samples were separated using density gradient centrifugation with human peripheral blood lymphocyte separation medium. PBMC-rich boundary layers were collected at 5 × 10⁶ cells per well. 3 A PBMC cell density was seeded into the wells of a culture plate to establish a PBMC-explant co-culture system;

[0130] (3) The trimmed tissue blocks are laid flat in the center of the filter paper of the cell culture plate according to the group, ensuring that the culture medium can completely submerge the tissue blocks. After laying, the outer ring of the cell culture plate is sealed with physiological saline and placed in an incubator at 37°C and 5% CO2 for incubation.

[0131] Example 3

[0132] The method for constructing liver cancer explants described in this embodiment includes the following steps:

[0133] (1) Obtain liver cancer tumor tissue samples from patients. The above tissues should be collected within half an hour after surgical resection or biopsy. The tissue samples should be processed within 6-8 hours. During transportation, the tissues should be soaked in tissue preservation solution and stored at low temperature. To process the tissue samples, the obtained tissue samples should be transferred to a laminar flow hood, placed on ice, rinsed with cleaning solution, and then soaked in pretreatment solution. The parts with higher malignancy and better activity should be physically sheared. The final processing should be 1 mm. 3 Tissue blocks of similar size; among them,

[0134] The tissue preservation solution includes DMEM / F-12 basal medium, supplemented with 50% fetal bovine serum v / v and penicillin-streptomycin 1X;

[0135] The cleaning solution includes physiological saline and is supplemented with penicillin-streptomycin 20X;

[0136] The pretreatment solution includes DMEM / F-12 basal medium with 10 nM of Y-27632 small molecule inhibitor added;

[0137] (2) Place the required cell support system in the cell culture plate and add 500 μL of complete culture medium for liver cancer explants to each well;

[0138] The complete culture medium for liver cancer explants includes DMEM / F-12 basal medium and the following components at the following concentrations: B27 medium additive 10X, N-acetylcysteine ​​1μM, nicotinamide 1M, A83-01 inhibitor 1nM, FGF10 recombinant protein 1g / ml, Y-27632 small molecule inhibitor 1nM, EGF recombinant protein 1g / ml, FGF7 recombinant protein 1ng / ml, gastrin 1M, serum 2% v / v, and penicillin-streptomycin 20X;

[0139] The cell support system includes a metal scaffold and filter paper;

[0140] The metal support is a cylindrical stainless steel structure with a hollow interior and a hollow surface.

[0141] The filter paper is highly absorbent filter paper;

[0142] Subsequently, peripheral blood mononuclear cells (PBMCs) in diluted human peripheral blood samples were separated using density gradient centrifugation with human peripheral blood lymphocyte separation medium. PBMC-rich boundary layers were collected at 5 × 10⁶ cells per well. 3 A PBMC cell density was seeded into the wells of a culture plate to establish a PBMC-explant co-culture system;

[0143] (3) The trimmed tissue blocks are laid flat in the center of the filter paper of the cell culture plate according to the group, ensuring that the culture medium can completely submerge the tissue blocks. After laying, the outer ring of the cell culture plate is sealed with physiological saline and placed in an incubator at 37°C and 5% CO2 for incubation.

[0144] Example 4

[0145] The method for constructing liver cancer explants in this embodiment is the same as in Embodiment 1, except that 0.5M of Spirulina exosomes are added to the complete culture medium for liver cancer explants.

[0146] Example 5

[0147] like Figure 1 The process shown in this embodiment is to construct and culture cholangiocarcinoma explants. The method is the same as in Embodiment 1, except that a cholangiocarcinoma tumor tissue sample from a patient is taken for the same culture.

[0148] Example 6

[0149] like Figure 1 The process shown in this embodiment is for the construction and culture of cholangiocarcinoma explants. The method is the same as in embodiment 2, except that a cholangiocarcinoma tumor tissue sample from a patient is taken for the same culture.

[0150] Example 7

[0151] like Figure 1 The process shown in this embodiment is to construct and culture bile duct cancer explants. The method is the same as in embodiment 3, except that bile duct cancer tumor tissue samples from patients are used for the same culture.

[0152] Example 8

[0153] like Figure 1 The process shown in this embodiment is to construct and culture bile duct cancer explants. The method is the same as in embodiment 4, except that bile duct cancer tumor tissue samples from patients are used for the same culture.

[0154] Comparative Example 1

[0155] The method for constructing liver cancer explants in this comparative example is the same as in Example 4, except that an equal amount of cyanobacterial exosomes is added to the complete culture medium for the liver cancer explants instead of the spirulina exosomes. The extraction method for the cyanobacterial exosomes is the same as that for the spirulina exosomes.

[0156] Comparative Example 2

[0157] The method for constructing liver cancer explants in this comparative example is the same as in Example 4, except that an equal amount of carrot exosomes is added to the complete culture medium for the liver cancer explants instead of the spirulina exosomes. The extraction method for the carrot exosomes is the same as the extraction method for the spirulina exosomes.

[0158] Comparative Example 3

[0159] The method for constructing liver cancer explants in this comparative example is the same as in Example 4, except that an equal amount of soybean exosomes is added to the complete culture medium for the liver cancer explants instead of the spirulina exosomes. The extraction method for the soybean exosomes is the same as the extraction method for the spirulina exosomes.

[0160] Comparative Example 4

[0161] The method for constructing liver cancer explants in this comparative example is the same as in Example 4, except that an equal amount of aloe vera exosomes is added to the complete culture medium for the liver cancer explants instead of the spirulina exosomes. The extraction method for the aloe vera exosomes is the same as the extraction method for the spirulina exosomes.

[0162] Comparative Example 5

[0163] The method for constructing liver cancer explants in this comparative example is the same as in Example 4, except that an equal amount of spirulina enzymatic hydrolysate is added to the complete culture medium for liver cancer explants instead of the spirulina exosomes. The spirulina enzymatic hydrolysate is obtained by enzymatic hydrolysis of spirulina after pulping with a complex enzyme consisting of cellulase, lipase, pectinase, and protease (mass ratio 5:2:0.5:1).

[0164] Comparative Example 6

[0165] The method for constructing liver cancer explants in this comparative example is the same as in Example 4, except that an equal amount of spirulina (crushed) is added to the complete culture medium for liver cancer explants instead of the spirulina exosomes.

[0166] Comparative Example 7

[0167] The method for constructing cholangiocarcinoma explants in this comparative example is the same as in Example 8, except that an equal amount of cyanobacterial exosomes is added to the complete culture medium for the cholangiocarcinoma explants instead of the spirulina exosomes. The extraction method for the cyanobacterial exosomes is the same as that for the spirulina exosomes.

[0168] Comparative Example 8

[0169] The method for constructing cholangiocarcinoma explants in this comparative example is the same as in Example 8, except that an equal amount of carrot exosomes is added to the complete culture medium for the cholangiocarcinoma explants instead of the spirulina exosomes. The extraction method for the carrot exosomes is the same as the extraction method for the spirulina exosomes.

[0170] Comparative Example 9

[0171] The method for constructing cholangiocarcinoma explants in this comparative example is the same as in Example 8, except that an equal amount of soybean exosomes is added to the complete culture medium for the cholangiocarcinoma explants instead of the spirulina exosomes. The extraction method for the soybean exosomes is the same as the extraction method for the spirulina exosomes.

[0172] Comparative Example 10

[0173] The method for constructing cholangiocarcinoma explants in this comparative example is the same as in Example 8, except that an equal amount of aloe vera exosomes is added to the complete culture medium for the cholangiocarcinoma explants instead of the spirulina exosomes. The extraction method for the aloe vera exosomes is the same as the extraction method for the spirulina exosomes.

[0174] Comparative Example 11

[0175] The method for constructing the cholangiocarcinoma explants in this comparative example is the same as in Example 8, except that an equal amount of spirulina enzymatic hydrolysate is added to the complete culture medium for the cholangiocarcinoma explants instead of the spirulina exosomes. The spirulina enzymatic hydrolysate is obtained by enzymatic hydrolysis of spirulina after pulping with a complex enzyme consisting of cellulase, lipase, pectinase, and protease (mass ratio 5:2:0.5:1).

[0176] Comparative Example 12

[0177] The method for constructing cholangiocarcinoma explants in this comparative example is the same as in Example 8, except that an equal amount of spirulina (crushed) is added to the complete culture medium for the cholangiocarcinoma explants instead of the spirulina exosomes.

[0178] Experimental Example

[0179] 1. Detection of explant viability in liver cancer culture

[0180] Liver cancer explant cells cultured under the scheme of Example 1 (in vitro culture day=5) were used for activity detection, and fresh liver cancer tumor tissue obtained before culture was used as the control group (day=0). The activity data of cells in the experimental group and the control group were observed, and the results are shown in Table 3 below.

[0181] Table 3

[0182]

[0183] It is evident that the in vitro induction culture method described in this invention can induce the proliferation of tumor tissue, proving that the in vitro culture system described in this invention has been successfully constructed.

[0184] In this experimental example, the original fresh liver cancer tumor tissue from the same batch was used as a control, and cells were cultured for different times under the protocols of Example 4 and Comparative Examples 1-5, respectively. The survival rate of the explant cells after culture relative to the original control group cells was also measured, and the results are shown in Table 4 below.

[0185] Table 4

[0186]

[0187] As can be seen, the scheme in Example 4 not only further improved cell proliferation activity but also accelerated the proliferation rate. In contrast, other exosomes selected in the comparative scheme did not have the effect of improving cell proliferation activity. Furthermore, directly using spirulina hydrolysate or spirulina powder for explant culture did not significantly promote cell proliferation. The reason for this is speculated to be that the cells constructed in this scheme require the formation of complete, organ-like structures, and the requirements for promoting proliferation rate differ from those of general cell proliferation, thus exhibiting a significant difference.

[0188] 2. Morphology of explants for liver cancer

[0189] In this experimental example, morphological analysis was performed on the liver cancer explants cultured in Example 1. The results are detailed in the appendix. Figure 2 The results are shown.

[0190] As can be seen, compared with parental tissue, the explants constructed by the method described in this invention can form complete, organ-like structures, and can be used as models for drug sensitivity testing of liver cancer. They possess complete structures and exhibit good application results. In this experimental example, the explants cultured based on the scheme in Example 4 also have complete structures.

[0191] 3. Clinical Consistency Experiment of Hepatocellular Carcinoma Explants

[0192] This experimental case study is based on drug sensitivity testing of explants for liver cancer in clinical patients and guides clinical drug use.

[0193] This experimental case involved a 65-year-old male patient diagnosed with prostate cancer via biopsy in April 2021. The tumor measured 50×40×30mm, had a Gleason score of 5+4=9, and was assigned to ISUP group 4. From April 2021 to May 2022, after multiple lines of endocrine therapy, the patient developed elevated PSA levels (more than 20 ng / ml), liver metastasis, and was diagnosed with metastatic castration-resistant prostate cancer (mCRPC), indicating disease progression.

[0194] The patient was enrolled in a clinical trial of a new drug in July 2022 and received oral TQB3720 for 45 days, but the liver metastases continued to progress. Details of the patient's lesions as of August 22, 2022, are attached. Figure 3 Type A has dimensions of 49×65mm. According to the formula V=L×W 2 The volume of the lesion before medication was calculated as V1 = 81153.8 × 0.52.

[0195] The patient underwent a liver metastasis biopsy on August 23, 2021. The liver cancer tissue sample was cultured as an explant according to the method in Example 1, and the constructed explant was used for drug sensitivity testing.

[0196] In this experimental example, the drug sensitivity test based on the explant specifically includes the following steps:

[0197] (1) The constructed liver cancer explants were placed in an incubator at 37°C and 5% CO2 and cultured. Different wells in the cell culture plate were divided into a blank control group and a drug administration group.

[0198] (2) Add the target concentration (5 mg / mL) of antitumor drug to the culture wells of the drug administration group as needed, and place the cell culture plate in an incubator at 37°C and 5% CO2 for culture;

[0199] (3) After culturing for 3 days after drug administration, the cell viability of the explants was detected by MTT staining and photographed. The tumor inhibition rate of different drugs was then calculated.

[0200] In this experimental example, the protocol and results for antitumor drug detection are shown in Table 5 below. The measured results of the drug sensitivity test are detailed in the appendix. Figure 4 .

[0201] Table 5

[0202]

[0203] Based on the drug sensitivity results shown in Table 3, it can be seen that the patient has sensitive and feasible drugs, indicating that drugs such as docetaxel and capecitabine are sensitive. Clinically, medication selection can be made based on the above results and the patient's physical condition.

[0204] Based on the results of the drug sensitivity test, the clinical administration regimen was formulated as follows: docetaxel 120 mg + capecitabine 1.5 g, orally twice daily, D1-14.

[0205] The patient underwent a follow-up examination after four cycles of medication, and a CT scan was performed in November 2022 (see attached results). Figure 3 As shown in Figure B, the liver lesion measures 42 × 54 mm. Similarly, according to the formula V = L × W... 2 Calculate the lesion volume V2 after 90 days of medication using ×0.52, which is V2 = 49533.12.

[0206] It is evident that after medication adjustments based on drug sensitivity testing, the patient's liver lesions decreased, the in vivo tumor inhibition rate was calculated as 1 - V2 / V1 = 39.0%, the ZPS score was 2, the efficacy assessment was SD, and the condition was stable, demonstrating the effectiveness of the dosing regimen.

[0207] It is evident that the explants constructed using the method of this invention achieve very high accuracy in clinical organ drug sensitivity testing, indicating that the explants constructed using the method of this invention can be fully applied to clinical oncology drug sensitivity testing and can be used for clinical drug use guidance.

[0208] 4. In vitro drug sensitivity test of liver cancer explants

[0209] In this experimental example, liver cancer tissue cells were taken from the patient, and liver cancer explants were constructed according to the method in Example 1 above, and in vitro drug sensitivity tests were performed on them.

[0210] In this experimental example, the drugs to be tested for drug sensitivity include raltitrexed, donafenib, epirubicin, irinotecan, trifluuridine tipiridine, anlotinib, apatinib, and fruquintinib, and the concentrations used are those used clinically.

[0211] The drug sensitivity test described in this experimental example includes the following steps:

[0212] (1) The constructed liver cancer explants were placed in an incubator at 37°C and 5% CO2 and cultured. Different wells in the cell culture plate were divided into a blank control group and a drug administration group.

[0213] (2) Add the target concentration (5 mg / mL) of antitumor drug to the culture wells of the drug administration group as needed, and place the cell culture plate in an incubator at 37°C and 5% CO2 for culture;

[0214] (3) Three days after drug administration and culture, the cell viability of the explants was detected by MTT staining and photographed. The drug inhibition rate (IR) was calculated based on the gray values ​​before and after staining. For details, please refer to the appendix. Figure 5-6 .

[0215] In this experimental example, the final data obtained were as follows: raltitrexed had an inhibition rate of 35.5%, donafenib had an inhibition rate of 11.1%, epirubicin had an inhibition rate of 40.8%, irinotecan had an inhibition rate of 49.6%, trifluuridine teppirimidine had an inhibition rate of 0.9%, anlotinib had an inhibition rate of 11.4%, apatinib had an inhibition rate of 16.8%, and fruquintinib had an inhibition rate of 1.6%.

[0216] Clinically, the drug sensitivity test results can be used to formulate a clinical medication plan for the patient. Clinical efficacy has shown that the medication plan guided by the drug sensitivity test can effectively improve the patient's lesions, and the clinical guidance is completely accurate.

[0217] 5. Clinical medication

[0218] Under the present invention, drug sensitivity tests have been conducted on the lesions of 346 patients in clinical practice, and targeted clinical medication guidance has been provided. Clinical observation and medication feedback show that the method for drug sensitivity testing based on the constructed liver cancer explants (under Examples 1-4) of the present invention has a clinical efficacy rate of up to 100%. This also demonstrates that the explants constructed in this invention have a 100% accuracy rate for clinical sensitivity testing and can be fully used for guiding clinical medication.

[0219] 6. Detection of the viability of bile duct carcinoma explants in culture

[0220] The activity of cholangiocarcinoma explant cells cultured under the scheme of Example 5 (in vitro culture day=5) was tested, and fresh liver cancer tumor tissue obtained before culture was used as the control group (day=0). The activity data of cells in the experimental group and the control group were observed, and the results are shown in Table 6 below.

[0221] Table 6

[0222]

[0223] It is evident that the in vitro induction culture method described in this invention can induce the proliferation of tumor tissue, which also proves that the in vitro culture system described in this invention has been successfully constructed.

[0224] In this experimental example, the original fresh cholangiocarcinoma tumor tissue from the same batch was used as a control, and cells were cultured for different times under the protocols of Example 8 and Comparative Examples 5-8, respectively. The survival rate of the explant cells after culture relative to the original control group cells was also measured, and the results are shown in Table 7 below.

[0225] Table 7

[0226]

[0227] It is evident that the scheme in Example 8 not only further enhanced cell proliferation activity but also resulted in a faster proliferation rate. In contrast, other exosomes selected in the comparative scheme did not exhibit the effect of enhancing cell proliferation activity. Furthermore, directly using spirulina hydrolysate or spirulina powder for explant culture did not significantly promote cell proliferation. The reason for this is speculated to be that the cell construction in this scheme requires the formation of a complete, organ-like structure, which differs from the requirements for promoting cell proliferation rate compared to general cell proliferation, thus exhibiting significant differences; in particular, different explants also showed different effects in promoting proliferation.

[0228] 7. Clinical consistency study of explants for cholangiocarcinoma

[0229] This experimental case study is based on the drug sensitivity test of explants for cholangiocarcinoma in clinical patients, and guides the clinical medication.

[0230] In this experimental case, a clinical patient diagnosed with cholangiocarcinoma was selected. A cholangiocarcinoma tissue sample from the patient was taken and cultured as an explant according to the method in Example 5. The constructed explants were then subjected to drug sensitivity tests.

[0231] In this experimental example, the drug sensitivity test based on the explant specifically includes the following steps:

[0232] (1) The constructed cholangiocarcinoma explants were placed in an incubator at 37°C and 5% CO2 and the different wells in the cell culture plate were divided into a blank control group and a drug administration group;

[0233] (2) Add the target concentration (5 mg / mL) of antitumor drug to the culture wells of the drug administration group as needed, and place the cell culture plate in an incubator at 37°C and 5% CO2 for culture;

[0234] (3) After culturing for 3 days after drug administration, the cell viability of the explants was detected by MTT staining and photographed. The tumor inhibition rate of different drugs was then calculated.

[0235] In this experimental example, the protocol and results for antitumor drug detection are shown in Table 8 below. Detailed graphs of the drug sensitivity test results are attached. Figure 7 .

[0236] Table 8

[0237]

[0238] Based on the drug sensitivity results shown in Table 6, it can be seen that the patient has the appropriate drug sensitivity, indicating that drugs such as gemcitabine + cisplatin are sensitive. Clinically, medication selection can be made based on the above results and the patient's physical condition.

[0239] In fact, after the aforementioned drug sensitivity test, the patient underwent corresponding medication adjustments, and the treatment effect was good.

[0240] 8. Clinical medication

[0241] Under the present invention, drug sensitivity tests have been conducted on the lesions of 191 patients in clinical practice, and targeted clinical medication guidance has been provided. Clinical observation and medication feedback show that the method for drug sensitivity testing based on the constructed cholangiocarcinoma explants (under Examples 5-8) of the present invention has a clinical efficacy rate of up to 100%. This also demonstrates that the explants constructed in this invention have a 100% accuracy rate in clinical sensitivity testing and can be fully used for clinical medication guidance.

[0242] 9. Cryopreservation-thawing experiment

[0243] This experiment is based on the hepatocellular carcinoma explant cells prepared under the schemes of Examples 1 and 4 above, and the cholangiocarcinoma explant cells cultured under the schemes of Examples 5 and 8, to carry out cell cryopreservation-thaw experiments.

[0244] Prepare cryopreservation solutions, digestion solutions, culture media, etc., in accordance with the standard practices for cryopreservation experiments in this field, and prepare cryopreservation tubes, cooling boxes, etc.

[0245] Select well-shaped explant micro-tissues, gently rinse with PBS, add cryopreservation solution, and aliquot into cryovials. Immediately place the cryovials in a pre-cooled (4°C) programmed cooling box for 30 minutes, then place the entire box directly into an -80°C ultra-low temperature freezer for 18-24 hours. Remove the cryovials and store them in liquid nitrogen at -120°C.

[0246] Remove the cryovial from the target cell and immediately place it in a 37°C water bath, gently shaking continuously until it thaws completely within 1-2 minutes. Aspirate the micro-tissue block and add it dropwise to a centrifuge tube containing preheated complete culture medium. After centrifugation to remove the supernatant, resuspend the precipitate in preheated complete culture medium. Inoculate the cell suspension into culture dishes containing fresh complete culture medium for thawing. Place the culture dishes in a 37°C, 5% CO2 incubator. Observe cell morphological changes 24 hours after thawing.

[0247] In this experimental example, the cell viability data of liver cancer cells and cholangiocarcinoma cells before and after cryopreservation-re-examination are shown in Table 9 below. The morphological results of the explant cells prepared in Examples 1 and 5 are shown in the appendix. Figure 8 .

[0248] Table 9

[0249]

[0250] It is evident that the liver cancer explants or bile duct cancer explants prepared under the present invention can be cryopreserved and then thawed for use, and the viability of the explant tissue is effectively guaranteed. They are suitable for establishing biobanks and solve the defects of traditional explant models that cannot be cryopreserved and thawed.

[0251] In summary, the method for constructing explants for liver cancer or bile duct cancer described in this invention utilizes surgically removed tumor tissue from liver cancer or bile duct cancer patients. Through explant culture, especially optimized explant culture media, a highly active and structurally intact explant model for liver cancer or bile duct cancer can be constructed. This not only effectively improves the success rate and stability of explant construction but also effectively avoids gene mutations and phenotypic changes that may occur in tumor cells during in vitro culture. Furthermore, it more accurately reflects the biological characteristics of tumor cells in vivo, thereby establishing a drug sensitivity screening model that is closer to the physiological state of the human body and can accurately reflect the efficacy of drugs.

[0252] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A culture medium for constructing explants for liver cancer or cholangiocarcinoma, characterized in that, The culture medium consists of a basal medium and the following components at the following concentrations: B27 medium additive 1X-10X, N-acetylcysteine ​​1μM-1M, nicotinamide 1nM-1M, ALK5 inhibitor 1nM-1M, FGF10 recombinant protein 1ng / ml-1g / ml, ROCK inhibitor 1nM-1M, EGF recombinant protein 1ng / ml-1g / ml, FGF7 recombinant protein 1ng / ml-1g / ml, gastrin 1nM-1M, serum 2% v / v-20% v / v, penicillin-streptomycin 1X-20X, and spirulina exosomes 1nM-1M.

2. A method for constructing explants for liver cancer or bile duct cancer, characterized in that, Includes the following steps: (1) Obtain liver cancer or bile duct cancer tissue samples from patients and preprocess them to obtain tissue blocks; (2) Place the cell support system into the cell culture plate and add the culture medium for constructing liver cancer and cholangiocarcinoma explants as described in claim 1; then add peripheral blood mononuclear cells (PBMCs) to form a PBMC-explant co-culture system. (3) Immerse the tissue block in the PBMC-explant co-culture system and incubate it to obtain the desired result.

3. The method for constructing explants for liver cancer or bile duct cancer according to claim 2, characterized in that, In step (1), the pretreatment solution of the pretreatment step includes DMEM / F-12 basal culture medium, and the following concentration of components are added: ROCK inhibitor 10nM-10mM.

4. The method for constructing explants for liver cancer or bile duct cancer according to claim 3, characterized in that, Step (1) further includes the steps of preserving and cleaning the liver cancer or bile duct cancer tissue sample from the patient; wherein, The tissue preservation solution used in the tissue preservation step includes DMEM / F-12 basal culture medium, with the following concentrations of added components: fetal bovine serum 2% v / v-50% v / v, penicillin-streptomycin 1X-20X; The cleaning solution used in the cleaning step includes physiological saline and contains the following concentrations of penicillin-streptomycin 1X-20X.

5. The method for constructing explants for liver cancer or bile duct cancer according to claim 2, characterized in that, In step (2), the cell support system includes a metal scaffold and filter paper; The metal support is a cylindrical stainless steel structure with a hollow interior and a hollow surface. The filter paper is highly absorbent.

6. The method for constructing explants for liver cancer or bile duct cancer according to claim 2, characterized in that, In step (3), the incubation and cultivation conditions include: 35-38℃, 5% CO2 cultivation, and cultivation time of 1-30 days.

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