Construction method of tumor organoid microenvironment chip model, model and application thereof

Abstract

The invention discloses a construction method of a tumor organoid microenvironment chip model, the model and application of the model. The construction method comprises the following steps: acquiring a tumor organ and preparing cell precipitate; carrying out enzymolysis and digestion treatment on the cell precipitate, and then adding an extracellular matrix to prepare a tumor organ suspension; the method comprises the following steps: adding a tumor organoid suspension into a micro-fluidic culture carrier, culturing to prepare suspensions of fibroblasts and vascular endothelial cells, respectively adding the two cell suspensions into corresponding hole sites of the micro-fluidic culture carrier, and finally culturing to obtain the tumor organoid microenvironment chip model. The model prepared by the method can more truly simulate the complex tumor microenvironment in vivo, is suitable for drug screening and drug sensitivity detection, is beneficial to improving the accuracy and reliability of research, and accelerates the transformation from laboratory research to clinical application.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to the construction of a tumor organoid model, and in particular to a method for constructing a tumor organoid microenvironment chip model, the model itself, and its application. Background Technology

[0002] Cancer is a highly structured and complex ecosystem comprising tumor cells and a large number of non-tumor cells embedded in the extracellular matrix. The tumor microenvironment (TME) includes a variety of immune cells, cancer-associated fibroblasts, endothelial cells, pericytes, and various other tissue-resident cells. This complex environment directly or indirectly influences tumor cell phenotypes through intricate signaling mechanisms mediated by soluble factors, cell-cell contacts, and extracellular matrix (ECM) remodeling, leading to high tumor heterogeneity. TME cells are involved in every stage of cancer progression, influencing tumor initiation, progression, invasion, infiltration, metastatic spread, and growth. TME cells and the molecules they secrete are now considered to play a key role in cancer pathogenesis and may represent attractive therapeutic targets. The complex interactions between tumor cells and their mediators are crucial for the rational development of effective anticancer drugs. Furthermore, the heterogeneity of cancer leads to highly diverse patient treatment responses, including treatment resistance, and this complexity has become a significant obstacle to discovering the specific mechanisms of treatment failure. Organoid models of patient-specific tumors are revolutionizing our understanding of cancer heterogeneity and its implications for personalized medicine. These advances are partly due to the ability of organoid models to stably preserve the genetic, proteomic, morphological, and pharmacological characteristics of the parent tumor in vitro, while also providing unprecedented opportunities for genomic and environmental manipulation.

[0003] However, tumor organoid models are difficult to fully represent the original tissue because they typically lack a complete microenvironment. Tumor organoids and the tumor microenvironment can be likened to "seeds and soil." Constructing tumor organoid models with a complete microenvironment for drug testing will more closely approximate the human body's actual response to drugs. Angiogenesis inhibitors are an important class of anti-tumor drugs. In 1971, Professor Folkman proposed the theory of tumor angiogenesis, pointing out that both local tumor growth and distant metastasis are inseparable from the formation of new tumor blood vessels. The growth and metastasis of solid tumors involve a transformation process from the pre-angiogenic phase to the vascularization phase. Tumor angiogenesis inhibitors can inhibit the formation of the tumor angiogenesis factor VEGF, thereby inducing spontaneous apoptosis of vascular endothelial cells and disrupting the tumor's neovascular network.

[0004] However, traditional in vitro models are difficult to detect the anti-tumor effects of such drugs. Tumor models, such as tumor cell models, tumor organoid models, and tumor aggregate models, only contain tumor cells and do not respond to angiogenesis inhibitors. In contrast, angiogenesis models can only reflect the effects on angiogenesis and cannot further reflect the effects on tumors.

[0005] Therefore, there is an urgent need for a comprehensive model that can better simulate the complex tumor microenvironment in vivo. This model should include not only tumor cells, but also vascular endothelial cells and other TME components, such as fibroblasts, in order to more accurately reflect the interaction between the tumor and the vascular network. Summary of the Invention

[0006] In view of this, this application provides a method for constructing a tumor organoid microenvironment (TME) chip model, the model itself, and its applications. The model constructed using the method described in this application possesses a highly realistic tumor microenvironment, allowing researchers to more realistically evaluate the efficacy of drugs targeting tumor angiogenesis, such as angiogenesis inhibitors, and observe how these drugs affect angiogenesis and how these changes, in turn, affect tumor growth and metastasis. Such a comprehensive model is expected to become an important tool for evaluating the effectiveness of novel anticancer therapies and may help accelerate the preclinical drug screening process, improving the success rate of drug development. Simultaneously, this also provides a foundation for the development of personalized medicine strategies, as patient-specific TME models can help predict the response of specific patients to specific treatment regimens, thereby guiding more precise treatment selection.

[0007] In a first aspect, this application provides a method for constructing a tumor organoid microenvironment chip model, the method comprising the following steps: S1. Obtain primary or passaged tumor organoids and prepare tumor organoid cell precipitates. S2. After enzymatic hydrolysis and digestion of the tumor organoid cell precipitate described in step S1.1, add extracellular matrix and mix well to prepare tumor organoid suspension; S3. Using a microfluidic culture carrier that includes at least a central pore and two side pores, the tumor organoid suspension described in step S2 is added to the central pore. After being cultured into a gel, tumor organoid culture medium is added to the central pore of the microfluidic culture carrier and to the sample wells on both sides adjacent to the central pore, and then cultured. S4. Obtain tumor-associated fibroblasts and tumor-associated vascular endothelial cells, and prepare fibroblast suspensions and endothelial cell suspensions using a mixed culture medium; the mixed culture medium includes components for culturing tumor organoids and components for culturing endothelial cells; S5. Take the culture medium from the fibroblast suspension described in step S4 and add it to the two side wells of the microfluidic culture carrier described in step S3; take the culture medium from the endothelial cell suspension described in step S4 and add it to the middle well of the microfluidic culture carrier described in step S3, and then culture to obtain the tumor organoid microenvironment chip model.

[0008] The tumor-associated fibroblasts in step S4 include one of the following: primary fibroblasts extracted from tumor tissue, fibroblasts differentiated from corresponding tumor tissue by stem cells, primary fibroblasts extracted from other tumor or normal tissues (with or without induction), and fibroblast cell lines (with or without induction); and / or, the tumor-associated vascular endothelial cells in step S4 include one of the following: primary vascular endothelial cells extracted from corresponding tumor tissue, vascular endothelial cells differentiated from corresponding tumor tissue by stem cells, primary vascular endothelial cells extracted from other tumor or normal tissues, and vascular endothelial cell lines.

[0009] By adopting the above technical solution, this application provides a tumor organoid microenvironment chip model that can highly simulate the tumor microenvironment. This model not only includes tumor cells but also incorporates multiple cell types such as vascular endothelial cells and tumor-associated fibroblasts. Combined with microfluidic chip design, it simulates the environmental conditions of blood flow in vivo, thereby enabling a more realistic evaluation of the efficacy of drugs targeting tumor angiogenesis, such as angiogenesis inhibitors, and observation of how drugs affect angiogenesis and how these changes, in turn, affect tumor growth and metastasis. This effectively solves the problem that traditional in vitro models cannot comprehensively reflect the anti-tumor effects of drugs. This model simplifies experimental procedures, helps accelerate the preclinical drug development process, improves the success rate of drug development, and also provides strong technical support for the development of personalized medicine strategies. This significantly improves the accuracy and reliability of drug screening, reduces uncertainties and costs in the drug development process, and accelerates the translation from laboratory discovery to clinical application.

[0010] The tumor organoid microenvironment chip model provided in this application can use a variety of tumor-associated fibroblasts and vascular endothelial cells in step S4, including but not limited to primary cells directly extracted from tumor tissue, specific tumor tissue cells differentiated from stem cells, and primary cells extracted from other tumors or normal tissues, or fibroblast and vascular endothelial cell lines that have not undergone specific induction or have undergone specific induction. This allows the model to more accurately simulate the complex tumor microenvironment in vivo, which not only enhances the understanding of tumor growth, angiogenesis and their interaction mechanisms, but also greatly improves the accuracy and reliability of drug sensitivity test results. This provides a solid foundation for the evaluation of novel anticancer therapies and the development of personalized treatment plans, and helps to reduce the cost and uncertainty of drug development and accelerate its translation from laboratory to clinical application.

[0011] Optionally, the tumor-associated fibroblasts described in step S4 are treated with mitomycin C.

[0012] Optionally, the tumor organoids mentioned in step S1 include one of the following: lung cancer organoids, colorectal cancer organoids, pancreatic cancer organoids, or liver cancer organoids.

[0013] By adopting the above technical solution, the tumor organoid microenvironment chip model provided in this application can select different types of tumor organoids, including lung cancer organoids, colorectal cancer organoids, pancreatic cancer organoids, or liver cancer organoids, in step S1. This allows the model to conduct highly realistic studies targeting specific types of cancer. Using this highly customized chip model, researchers can more realistically evaluate the efficacy of drugs targeting specific tumor angiogenesis, such as angiogenesis inhibitors, and observe how drugs affect angiogenesis and how these changes, in turn, affect the growth and metastasis of specific cancers. This approach not only enhances the understanding of the growth mechanisms of specific cancers but also greatly improves the accuracy and reliability of drug screening, providing a solid foundation for the evaluation of novel anticancer therapies and the development of personalized treatment plans. It also helps reduce the cost and uncertainty of drug development and accelerates its translation from laboratory discovery to clinical application.

[0014] Optionally, the extracellular matrix in step S2 includes a hydrogel material; The hydrogel material includes one of the following: matrix gel, natural biomaterial, and synthetic biomaterial.

[0015] Optionally, the natural biomaterial is selected from protein hydrogels; the synthetic biomaterial is selected from GelMA hydrogels.

[0016] By adopting the above technical solution, the extracellular matrix used in step S2 of the tumor organoid microenvironment chip model provided in this application includes hydrogel materials, which include at least one of matrix gel, natural biomaterials, and synthetic biomaterials. This design allows the model to better simulate the physicochemical properties of the extracellular matrix in vivo, providing a more realistic three-dimensional growth environment for tumor organoids. By selecting appropriate hydrogel materials, not only can the long-term stable culture of tumor organoids be promoted, but the interaction between tumor cells and their surrounding microenvironment can also be simulated more accurately, thereby enhancing the understanding of tumor growth, angiogenesis, and their interaction mechanisms. This method not only improves the accuracy and reliability of drug sensitivity test results but also provides a solid foundation for the evaluation of novel anticancer therapies and the development of personalized treatment plans, and helps reduce the cost and uncertainty of drug development, accelerating its translation from laboratory discovery to clinical application.

[0017] Optionally, the tumor organoid culture medium in step S3 includes a tumor organoid culture medium corresponding to the tumor organoid; the corresponding tumor organoid culture medium includes one of lung cancer organoid culture medium, colorectal cancer organoid culture medium, pancreatic cancer organoid culture medium or liver cancer organoid culture medium.

[0018] Optionally, the components for culturing tumor organoids in step S4 include tumor organoid culture medium; the components for culturing endothelial cells include endothelial cell culture medium.

[0019] Optionally, the microfluidic culture medium in step S3 includes a high-throughput drug sensitivity chip; the manufacturer and model of the high-throughput drug sensitivity chip are as follows: (AVATARGET, cat no: OCG0201).

[0020] Secondly, this application provides a microenvironment organoid model, which is constructed using the construction method provided in this application.

[0021] By adopting the above-described technical solution, the microenvironment organoid model provided in this application is obtained based on the aforementioned construction method. This model integrates multiple cell types, including but not limited to tumor organoids (such as lung cancer organoids, colorectal cancer organoids, pancreatic cancer organoids, or liver cancer organoids), tumor-associated fibroblasts, and vascular endothelial cells. It uses hydrogel materials, including matrix gel, natural biomaterials, or synthetic biomaterials, as the extracellular matrix to simulate the real tumor microenvironment. This highly realistic model not only more accurately reflects the complexity of tumor growth, angiogenesis, and their interactions but also provides drug sensitivity test results that are closer to clinical reality. Compared to traditional in vitro models, this microenvironment organoid model significantly improves the accuracy and reliability of drug screening, providing strong technical support for the development of novel anticancer therapies and the implementation of personalized medicine strategies. Furthermore, this model simplifies experimental procedures, helps accelerate the preclinical drug development process, increases the success rate of drug development, reduces uncertainties and costs in the drug development process, and accelerates the translation from laboratory discovery to clinical application.

[0022] Optional application of microenvironment organoid models in antitumor drug development.

[0023] By adopting the above technical solutions, the microenvironment organoid model provided in this application can significantly improve the efficiency and accuracy of drug screening in the development of anti-tumor drugs.

[0024] Optionally, the application includes applications in tumor drug screening or tumor drug sensitivity testing.

[0025] By employing the above-described technical solution, the microenvironment organoid model provided in this application can play a crucial role in tumor drug screening or tumor drug sensitivity testing. This model not only includes various types of tumor organoids (such as lung cancer organoids, colorectal cancer organoids, pancreatic cancer organoids, or liver cancer organoids), but also integrates multiple cell types, including tumor-associated fibroblasts and vascular endothelial cells. It utilizes hydrogel materials, including matrix gel, natural biomaterials, or synthetic biomaterials, as the extracellular matrix, thereby creating a highly realistic tumor microenvironment. This highly realistic microenvironment organoid model can more accurately simulate the interaction between the tumor and its surrounding microenvironment in vivo, enabling results that more closely resemble clinical realities during drug screening and drug sensitivity testing. Specifically, in tumor drug screening, this model can be used to evaluate newly developed anti-tumor drugs, particularly those targeting angiogenesis, such as angiogenesis inhibitors, to observe how these drugs affect tumor growth, angiogenesis, and tumor cell behavior. In tumor drug sensitivity testing, this model can facilitate personalized medicine. By establishing a microenvironment organoid model on tumor samples from specific patients, the effectiveness of different drugs on the patient's tumor can be tested, thereby guiding physicians to select the most appropriate treatment plan for the patient. In summary, this microenvironment organoid model provides a more precise and reliable tool for the development of anti-tumor drugs, helps improve the efficiency and success rate of drug screening, and provides strong technical support for achieving personalized tumor treatment.

[0026] Thirdly, this application provides a composition for constructing a tumor organoid microenvironment chip model, comprising a tumor organoid culture medium and a mixed culture medium; The tumor organoid culture medium is used to culture tumor organoid suspensions; The mixed culture medium includes components for culturing tumor organoids and components for culturing endothelial cells; the mixed culture medium is used to resuspend tumor-associated fibroblasts and tumor-associated vascular endothelial cells to obtain fibroblast suspensions and endothelial cell suspensions, respectively.

[0027] By employing the above technical solution, this application provides a composition for constructing a tumor organoid microenvironment chip model. The composition includes a tumor organoid culture medium and a mixed culture medium. The tumor organoid culture medium is specifically used to culture tumor organoid suspensions, ensuring the healthy growth of tumor organoids within the chip model. The mixed culture medium contains components for culturing tumor organoids and components for culturing endothelial cells. This mixed culture medium is used to resuspend tumor-associated fibroblasts and tumor-associated vascular endothelial cells, thereby obtaining fibroblast suspensions and endothelial cell suspensions, respectively.

[0028] This composition is designed to better simulate the complex tumor microenvironment in vivo when constructing tumor organoid microarray models. By using specialized culture media, the growth states of different cell types can be optimized, ensuring the survival and functional performance of tumor organoids, fibroblasts, and vascular endothelial cells within the microenvironment. This not only improves the model's realism but also enhances its application value in drug screening and drug sensitivity testing, enabling researchers to more accurately assess the efficacy of anti-tumor drugs and providing a scientific basis for developing personalized medicine strategies. Furthermore, this composition simplifies experimental procedures, improves experimental consistency and reproducibility, thereby accelerating the translation from laboratory research to clinical application.

[0029] Optionally, the tumor organoid culture medium includes: the tumor organoid culture medium includes a tumor organoid culture medium corresponding to the tumor organoid; the corresponding tumor organoid culture medium includes pancreatic cancer organoid culture medium or colorectal cancer organoid culture medium.

[0030] Optionally, in the mixed culture medium, the component for culturing tumor organoids includes tumor organoid culture medium; and the component for culturing endothelial cells includes endothelial cell culture medium.

[0031] Optionally, the composition further includes an extracellular matrix; the extracellular matrix includes a hydrogel material; the hydrogel material includes at least one of matrix gel, natural biomaterial, and synthetic biomaterial.

[0032] Optionally, the natural biomaterial is selected from protein hydrogels; the synthetic biomaterial is selected from GelMA hydrogels.

[0033] In summary, the present invention has the following beneficial technical effects: This application provides a tumor organoid microenvironment chip model that highly simulates the tumor microenvironment. This model not only includes tumor cells but also incorporates multiple cell types such as vascular endothelial cells and tumor-associated fibroblasts. Combined with microfluidic chip design, it simulates the environmental conditions of blood flow in vivo, thereby enabling a more realistic evaluation of the efficacy of drugs targeting tumor angiogenesis, such as angiogenesis inhibitors. It also allows observation of how drugs affect angiogenesis and how these changes, in turn, affect tumor growth and metastasis, effectively solving the problem that traditional in vitro models cannot comprehensively reflect the anti-tumor effects of drugs. This model simplifies experimental procedures, helps accelerate the preclinical drug development process, improves the success rate of drug development, and provides strong technical support for the development of personalized medicine strategies. This significantly improves the accuracy and reliability of drug screening, reduces uncertainties and costs in the drug development process, and accelerates the translation from laboratory discovery to clinical application. The microenvironment organoid model provided in this application is obtained based on the above-described construction method. This model integrates multiple cell types, including but not limited to tumor organoids (such as lung cancer organoids, colorectal cancer organoids, pancreatic cancer organoids, or liver cancer organoids), tumor-associated fibroblasts, and vascular endothelial cells. It utilizes hydrogel materials, including matrix gel, natural biomaterials, or synthetic biomaterials, as the extracellular matrix to simulate the real tumor microenvironment. This highly realistic model not only more accurately reflects the complexity of tumor growth, angiogenesis, and their interactions but also provides drug sensitivity test results that are closer to clinical reality. Compared to traditional in vitro models, this microenvironment organoid model significantly improves the accuracy and reliability of drug screening, providing strong technical support for the development of novel anticancer therapies and the implementation of personalized medicine strategies. Furthermore, this model simplifies experimental procedures, helps accelerate the preclinical drug development process, increases the success rate of drug development, reduces uncertainties and costs in the drug development process, and accelerates the translation from laboratory discovery to clinical application. This application provides a composition for constructing a tumor organoid microenvironment chip model, comprising a tumor organoid culture medium and a mixed culture medium. The tumor organoid culture medium is used to culture tumor organoid suspensions, ensuring their healthy growth within the microarray model. The mixed culture medium contains components required for culturing tumor organoids and endothelial cells, used to prepare fibroblast and endothelial cell suspensions. This composition is designed to optimize cell growth, improve model fidelity, and enhance its application value in drug screening and drug sensitivity testing, providing a scientific basis for evaluating the efficacy of anti-tumor drugs and personalized medicine strategies. Furthermore, this composition simplifies experimental procedures, improves experimental consistency and reproducibility, and accelerates the translation from laboratory research to clinical application. Attached Figure Description To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0034] Figure 1 This is an overall structural diagram of the high-throughput drug sensitivity chip in step S3 of Embodiment 1 of this application; Figure 2 This is a structural diagram of each unit of the high-throughput drug sensitivity chip in step S3 of embodiment 1 of this application; Figure 3 This is a unit diagram showing the addition of pancreatic cancer-associated fibroblast suspension and HUVEC cell suspension to the left and right sample wells of the high-throughput drug sensitivity chip in step S6 of embodiment 1 of this application. Figure 4 The left side shows the state of the pancreatic cancer organoid microenvironment model on day 1, day 3, and day 5 after adding drug-containing culture medium during the drug sensitivity test. Figure 4 The right side is the control group, which is a blank experiment without the addition of drug-containing culture medium; Figure 5 and Figure 6 These are two statistical graphs, one showing the results of detecting the lysate from Experiment 1 using a chemiluminescent microplate reader; Figure 7 The left side shows the state of the colorectal cancer organoid microenvironment model on day 1, day 3, and day 5 after adding drug-containing culture medium during the drug sensitivity test. Figure 7 The right side is the control group, which is a blank experiment without the addition of drug-containing culture medium; Figure 8 and Figure 9 These are two statistical graphs, one showing the results of detecting the lysate from Experiment 2 using a chemiluminescent microplate reader. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0036] The solution of this application will be described below with reference to the following specific embodiments. Unless otherwise specified, the raw materials used in the following embodiments are all from commercially available products, and the devices or equipment used are all purchased from conventional market sales channels.

[0037] Material description: TrypleE enzyme: Manufacturer: Gibco; Product number: 12605-028; Pancreatic cancer organoid culture medium: Manufacturer: Jiangsu Aiweide Biotechnology Co., Ltd.; Product No.: MPA0101; High-throughput drug sensitivity chip: Manufacturer: Jiangsu Aiweide Biotechnology Co., Ltd.; Product No.: OCG0201; Pancreatic cancer organoid culture medium: Manufacturer: Jiangsu Aiweide Biotechnology Co., Ltd.; Product No.: MPA0101; Endothelial cell culture medium: Manufacturer: ScienCell; Product No.: 1001; Gemcitabine: Manufacturer: Selleck; Product No.: S1714; Tegafur: Manufacturer: Selleck; Product No.: S1300; CTG Detection Kit: Manufacturer: Promega; Product Number: G7571; Colorectal cancer organoid culture medium: Manufacturer: Jiangsu Aiweide Biotechnology Co., Ltd.; Product No.: MCO0101; SN-38 Manufacturer: Selleck; Part Number: S4908; Raltitrexed: Manufacturer: Selleck; Product No.: S1192; Matrix adhesive: Manufacturer: Corning; Item No.: 356231.

[0038] Example 1 This embodiment provides a pancreatic cancer organoid microenvironment model, the construction method of which includes the following steps: S1. Select pancreatic cancer organoids to be passaged, add pre-cooled PBS to the culture plate, add 1 mL of PBS to each well, collect it into a centrifuge tube, and refrigerate at 4°C for 15 min; centrifuge (centrifugation speed is 1000 rpm, centrifugation time is 5 min), and remove the supernatant to obtain pancreatic cancer organoid precipitate. S2. Add TrypleE enzyme to the pancreatic cancer organoid precipitate obtained in step S1 and digest for 10 min. After digestion into single cells, centrifuge (centrifugation speed is 1000 rpm and centrifugation time is 5 min). Remove the supernatant and resuspend in pancreatic cancer organoid culture medium. Perform cell counting to obtain pancreatic cancer organoids. S3. Centrifuge the pancreatic cancer organoids obtained in step S2 (centrifugation speed: 1000 rpm, centrifugation time: 5 min) to obtain pancreatic cancer organoid precipitate. Add an appropriate amount of extracellular matrix, mix well, and then add an appropriate amount of organoid suspension (1.5 × 10⁻⁶) to the central well of the high-throughput drug sensitivity chip. 5Cells / ml of matrix gel were incubated at 37°C and 5% CO2. After gel formation, 50 μL of pancreatic cancer organoid culture medium was added to the left, middle, and right sample wells of the drug sensitivity chip (the drug sensitivity chip here is one of the drug sensitivity chips provided by application numbers 2023105307566, 2023211335836, 2023105302350, and 2023302759312, and the drug sensitivity chip used in this application is the one with application number 202310530235.0). The culture was then incubated at 37°C and 5% CO2 for 4 days. S4. After 4 days of culture, pancreatic cancer-associated fibroblasts were harvested. Specifically, these are immortalized human pancreatic cancer-associated fibroblasts (treated with mitomycin C at 1 μg / mL for 6 hours beforehand). The cells were digested into single cells (using TryPLE digestion solution), resuspended in a mixed culture medium (a 1:1 mixture of pancreatic cancer organoid culture medium and endothelial cell culture medium), counted, and adjusted to a suitable density (4 × 10⁻⁶ cells / mL). 5 (cells / ml) S5. Collect HUVEC vascular endothelial cells, digest them into single cells, resuspend them in a mixed culture medium (1:1 mixture of pancreatic cancer organoid culture medium and endothelial cell culture medium), count them, and adjust to an appropriate density (density of 4 × 10⁻⁶ cells / year). 5 (cells / ml) S6. Remove all culture medium from the culture chamber, add 50 μL of pancreatic cancer-associated fibroblast suspension (20,000 cells per well) to the left and right sample wells of the high-throughput drug sensitivity chip, and add 50 μL of HUVEC cell suspension (20,000 cells per well) to the middle sample well; then incubate at 37°C and 5% CO2 for 1 day. S7. After culturing for 1 day, a pancreatic cancer organoid microenvironment model was prepared and used for drug sensitivity testing.

[0039] See the overall structural diagram of the high-throughput drug sensitivity chip in step S3 of this embodiment. Figure 1 .

[0040] In this embodiment, the structural diagram of each unit of the high-throughput drug sensitivity chip in step S3 is shown below. Figure 2 .

[0041] In this embodiment, in step S6, the unit diagram after adding pancreatic cancer-associated fibroblast suspension and HUVEC cell suspension to the left and right sample wells of the high-throughput drug sensitivity chip is shown below. Figure 3 .

[0042] Experiment 1: Drug sensitivity test; This experiment used the pancreatic cancer organoid microenvironment model prepared in Example 1 to test drug sensitivity. The specific method is as follows: (1) Prepare a suitable concentration of drug-containing culture medium (the culture medium is a 1:1 mixture of pancreatic cancer organoid culture medium and endothelial culture medium): gemcitabine (gemcitabine 1 μM), tegafur (tegafur 46 μM). (2) Observe and record the growth status of the pancreatic cancer organoid microenvironment model. Remove the culture medium from all chambers of the high-throughput drug sensitivity chip. Add 40 μL of the drug-containing culture medium prepared in step (1) to the left, right, and middle sample wells of the high-throughput drug sensitivity chip, respectively. Place the chip in a swing perfusion apparatus at 37°C and 5% CO2 and perform flow culture at 7°C / 30 min. The day of drug addition is designated as Day 0.

[0043] (3) On the third day, observe and record the growth status of the pancreatic cancer organoid microenvironment model, and add 20 μL of drug-containing culture medium to the left, right and middle sample wells of the high-throughput drug sensitivity chip respectively; (4) On day 5, observe and record the growth status of the pancreatic cancer organoid microenvironment model and perform activity detection using a CTG detection kit. Add 50 μL CTG detection reagent to the left, right and middle sample wells of the high-throughput drug sensitivity chip, respectively, and shake for 60 min using a shaker; (5) After incubation, use a chemiluminescent microplate reader to detect and read the values ​​of the middle wells of the drug sensitivity chip.

[0044] In this embodiment, Figure 4 The left side shows the state of the pancreatic cancer organoid microenvironment model on day 1, day 3, and day 5 after adding drug-containing culture medium during the drug sensitivity test. Figure 4 The right side shows the control group, which is a blank experiment without the addition of drug-containing culture medium.

[0045] Figure 5 and Figure 6 These are two statistical graphs, one showing the results of detecting the lysate from Experiment 1 using a chemiluminescent microplate reader.

[0046] The results of this experiment are as follows: Activity results of pancreatic cancer microenvironment model: gemcitabine: 27.96, 32.16, 32.76; tegafur: 69.55, 70.76, 82.08.

[0047] Activity results of pancreatic cancer organoid models: gemcitabine: 4.77, 2.57, 6.63; tegafur: 46.74, 52.17, 36.83.

[0048] Results analysis: From Figures 5-6The statistical charts show that gemcitabine and tegafur have certain killing effects in the drug sensitivity test of the pancreatic cancer organoid microenvironment model; gemcitabine and tegafur have stronger killing effects in the drug sensitivity test of pancreatic cancer organoids; the pancreatic cancer organoid microenvironment model constructed in this application has a certain degree of drug resistance compared with pancreatic cancer organoids.

[0049] Example 2 This embodiment provides a method for constructing a colorectal cancer microenvironment model in a high-throughput drug sensitivity chip. The method for constructing the model is as follows: S1. Select colorectal cancer organoids to be passaged, add pre-cooled PBS to the culture plate, add 1 mL of PBS to each well, collect in centrifuge tubes, and refrigerate at 4°C for 15 min; centrifuge (centrifugation speed is 1000 rpm, centrifugation time is 5 min), remove the supernatant, and obtain colorectal cancer organoid precipitate. S2. Add TrypleE enzyme and digest for 10 min to form single cells. Centrifuge (centrifugation speed 1000 rpm, centrifugation time 5 min), remove supernatant, add intestinal cancer organoid culture medium for resuspending, and count cells. S3. Centrifuge the colorectal cancer organoids (centrifugation speed 1000 rpm, centrifugation time 5 min) to obtain colorectal cancer organoid precipitate, add extracellular matrix, mix well, and then add organoid suspension (1.5 × 10⁻⁶) to the central well of the high-throughput drug sensitivity chip. 5 (cells / ml matrix gel), and incubated in a 37℃, 5% CO2 incubator. After gel formation, add 50μL of colorectal cancer organoid culture medium to the left, middle and right sample wells of the drug sensitivity chip, and incubate in a 37℃, 5% CO2 incubator for 4 days. S4. After 4 days of culture, collect colorectal cancer-associated fibroblasts. Specifically, these are human colorectal cancer-associated immortalized fibroblasts (fibroblasts pre-treated with mitomycin C, 1 μg / mL for 6 hours). Digest them into single cells (using TryPLE digestion solution), resuspend them in a mixed culture medium (a 1:1 mixture of colorectal cancer organoid culture medium and endothelial cell culture medium), count them, and adjust the density to an appropriate level (4 × 10⁻⁶ cells / mL). 5 (cells / ml) S5. Collect HUVEC vascular endothelial cells, digest them into single cells (using TryPLE digestion solution), resuspend them in a mixed culture medium (a 1:1 mixture of colon cancer organoid culture medium and endothelial cell culture medium), count them, and adjust to an appropriate density (density of 4 × 10⁻⁶ cells / year). 5 (cells / ml) S6. Remove all culture medium from the culture chamber, add 50 μL of colorectal cancer-associated fibroblast suspension (20,000 cells per well) to the left and right sample wells of the high-throughput drug sensitivity chip, and add 50 μL of HUVEC cell suspension (20,000 HUVEC cells per well) to the middle sample well; then incubate at 37°C and 5% CO2 for 1 day. S7. After culturing for 1 day, a colorectal cancer microenvironment model was constructed in a high-throughput drug sensitivity chip and used for drug sensitivity testing.

[0050] Experiment 2: Drug sensitivity test; This experiment used the colorectal cancer organoid microenvironment model prepared in Example 2 for drug sensitivity testing. The specific method is as follows: (1) Prepare a suitable concentration of drug-containing culture medium (a 1:1 mixture of colorectal cancer organoid culture medium and endothelial culture medium): SN-38 (SN-38 90 nM), raltitrexed (raltitrexed 1.5 μM). (2) Observe the growth status of the model, remove the culture medium from all chambers of the high-throughput drug sensitivity chip, and add 40 μL of drug-containing culture medium to the left, right and middle sample wells of the high-throughput drug sensitivity chip, respectively. Place the chip in a swing perfusion apparatus at 37°C and 5% CO2 incubator for flow culture at 7°C / 30 min. The day of drug addition is designated as Day 0.

[0051] (3) On the third day, observe and record the growth status of the colorectal cancer organoid microenvironment model, and add 20 μL of drug-containing culture medium to the left, right and middle sample wells of the high-throughput drug sensitivity chip respectively; (4) On day 5, observe and record the growth status of the colorectal cancer organoid microenvironment model and perform activity detection using a CTG detection kit. Add 50 μL of CTG detection reagent to the left, right and middle sample wells of the high-throughput drug sensitivity chip, and shake for 60 min using a shaker; (5) After incubation, use a chemiluminescent microplate reader to detect and read the values ​​of the middle wells of the drug sensitivity chip.

[0052] In this experiment, Figure 7 The left side shows the state of the colorectal cancer organoid microenvironment model on day 1, day 3, and day 5 after adding drug-containing culture medium during the drug sensitivity test. Figure 7 The right side shows the control group, which is a blank experiment without the addition of drug-containing culture medium.

[0053] Figure 8 and Figure 9 These are two statistical graphs, one showing the results of detecting the lysate from Experiment 2 using a chemiluminescent microplate reader.

[0054] The results of this experiment are as follows: Activity results of colorectal cancer microenvironment model: SN-38: 15.23, 22.45, 23.14; Raltitrexed: 52.18, 58.47, 61.39; SN-38+Raltitrexed: 14.75, 15.48, 14.97.

[0055] Activity results of colorectal cancer organoid models: SN-38: 6.05, 11.57, 13.67; raltitrexed: 75.37, 82.89, 83.45; SN-38+raltitrexed: 17.86, 15.13, 15.66.

[0056] Results analysis: From Figures 8-9 The statistical charts show that, in the drug sensitivity testing of the colorectal cancer organoid microenvironment model, SN-38 + raltitrexed (a combination drug) has a greater killing effect on SN-38 and raltitrexed alone; in the drug sensitivity testing of colorectal cancer organoids, SN-38 + raltitrexed (a combination drug) has no combined effect on SN-38 and raltitrexed alone; the colorectal cancer organoid microenvironment model constructed in this application has a certain degree of accuracy in drug sensitivity testing compared with colorectal cancer organoids.

[0057] In summary, the drug sensitivity test results show that the constructed pancreatic cancer organoid microenvironment models and colorectal cancer organoid microenvironment models can more accurately simulate the in vivo tumor microenvironment, thus significantly improving the accuracy and reliability of drug sensitivity testing. In the pancreatic cancer organoid microenvironment model, gemcitabine and tegafur both showed some killing effects, while in the pancreatic cancer organoid model, these two drugs showed stronger killing effects, indicating that this microenvironment model has a certain degree of drug resistance compared to the organoid model alone. In the colorectal cancer organoid microenvironment model, SN-38 and raltitrexed showed different degrees of inhibitory effects when used alone, while their combined use showed a stronger inhibitory effect. The strong killing effect, especially in the microenvironment model, shows that the combined drug therapy is more effective than the monotherapy. This indicates that the constructed colorectal cancer organoid microenvironment model can more realistically reflect the actual efficacy of drugs in complex microenvironments and has higher accuracy in drug sensitivity testing. These experimental results collectively demonstrate that by constructing a tumor organoid microenvironment chip model containing multiple cell types and extracellular matrix, not only can the effects of monotherapy and combination therapy be evaluated more accurately, but potential treatment combinations can also be efficiently screened, thereby greatly improving the efficiency of drug screening and providing a scientific basis for developing personalized treatment plans. Ultimately, this will help accelerate the development of new drugs and their translation into clinical applications. The above description is only a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the principles of this application should be included within the scope of protection of this application.

Claims

1. A method for constructing a tumor organoid microenvironment chip model, characterized in that, The construction method includes the following steps: S1. Obtain primary or passaged tumor organoids and prepare tumor organoid cell precipitates. S2. After enzymatic hydrolysis and digestion of the tumor organoid cell precipitate described in step S1.1, add extracellular matrix and mix well to prepare tumor organoid suspension; S3. Using a microfluidic culture carrier that includes at least a central pore and two side pores, the tumor organoid suspension described in step S2 is added to the central pore. After being cultured into a gel, tumor organoid culture medium is added to the central pore of the microfluidic culture carrier and to the sample wells on both sides adjacent to the central pore, and then cultured. S4. Obtain tumor-associated fibroblasts and tumor-associated vascular endothelial cells, and prepare fibroblast suspensions and endothelial cell suspensions using a mixed culture medium; the mixed culture medium includes components for culturing tumor organoids and components for culturing endothelial cells; S5. Take the culture medium from the fibroblast suspension described in step S4 and add it to the two side wells of the microfluidic culture carrier described in step S3; take the culture medium from the endothelial cell suspension described in step S4 and add it to the middle well of the microfluidic culture carrier described in step S3, and after culturing, obtain the tumor organoid microenvironment chip model. The tumor-associated fibroblasts in step S4 include one of the following: primary fibroblasts extracted from tumor tissue, fibroblasts from corresponding tumor tissue differentiated from stem cells, primary fibroblasts extracted from other tumor or normal tissues (with or without induction), and fibroblast cell lines (with or without induction). and / or; The tumor-associated vascular endothelial cells mentioned in step S4 include one of the following: primary vascular endothelial cells extracted from the corresponding tumor tissue, vascular endothelial cells from the corresponding tumor tissue differentiated from stem cells, primary vascular endothelial cells extracted from other tumors or normal tissues, and vascular endothelial cell lines.

2. The method for constructing a tumor organoid microenvironment chip model according to claim 1, characterized in that, The tumor-associated fibroblasts described in step S4 are treated with mitomycin C.

3. The construction method according to claim 1, characterized in that, The tumor organoids mentioned in step S1 include one of the following: lung cancer organoids, colorectal cancer organoids, pancreatic cancer organoids, or liver cancer organoids.

4. The construction method according to claim 1, characterized in that, The extracellular matrix described in step S2 includes a hydrogel material; The hydrogel material includes one of the following: matrix gel, natural biomaterial, and synthetic biomaterial.

5. The construction method according to claim 3, characterized in that, The tumor organoid culture medium in step S3 includes a tumor organoid culture medium corresponding to the tumor organoid; the corresponding tumor organoid culture medium includes one of lung cancer organoid culture medium, colorectal cancer organoid culture medium, pancreatic cancer organoid culture medium or liver cancer organoid culture medium.

6. The construction method according to claim 1, characterized in that, The components for culturing tumor organoids in step S4 include tumor organoid culture medium; the components for culturing endothelial cells include endothelial cell culture medium.

7. The construction method according to claim 1, characterized in that, The microfluidic culture medium described in step S3 includes a high-throughput drug sensitivity chip.

8. A microenvironment organoid model, characterized in that, It is constructed using the construction method described in any one of claims 1 to 7.

9. The application of the microenvironment organoid model of claim 9 in the development of antitumor drugs, wherein the application includes the application in tumor drug screening or tumor drug sensitivity testing.

10. A composition for constructing a tumor organoid microenvironment chip model according to any one of claims 1 to 7, characterized in that, Including tumor organoid culture media and mixed culture media; The tumor organoid culture medium includes a tumor organoid culture medium corresponding to the tumor organoids; the tumor organoid culture medium is used to culture tumor organoid suspensions; The mixed culture medium includes components for culturing tumor organoids and components for culturing endothelial cells; the mixed culture medium is used to resuspend tumor-associated fibroblasts and tumor-associated vascular endothelial cells to obtain fibroblast suspensions and endothelial cell suspensions, respectively. The mixed culture medium includes a tumor organoid culture medium as the component used for culturing tumor organoids, and an endothelial cell culture medium as the component used for culturing endothelial cells. Preferably, the corresponding tumor organoid culture medium includes pancreatic cancer organoid culture medium or colorectal cancer organoid culture medium; Preferably, the composition further includes an extracellular matrix; the extracellular matrix includes a hydrogel material; the hydrogel material includes at least one of matrix gel, natural biomaterial, and synthetic biomaterial.

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