Three-dimensional intestinal cancer cell model sensitive to anti-cancer drugs and construction method and application thereof

Abstract

The invention relates to a three-dimensional intestinal cancer cell model sensitive to anti-cancer drugs as well as a construction method and application thereof, and relates to the technical field of biological medicines. The method comprises the following steps: inoculating a culture medium containing an extracellular matrix with a colorectal cancer cell line and fiber cells, and carrying out three-dimensional cell co-culture to obtain the three-dimensional colorectal cancer cell model sensitive to anti-cancer drugs. According to the present invention, the three-dimensional intestinal cancer cell model sensitive to the anti-cancer drug is established by using the intestinal cancer cell line through the in vitro three-dimensional co-culture, and the anti-cancer drug is screened and evaluated with high throughput, such that the efficacy of the anti-cancer drug can be accurately and rapidly evaluated, the development of the new drug can be accelerated, the drug cost can be reduced, and the treatment scheme can be optimized.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a three-dimensional intestinal cancer cell model sensitive to anti-cancer drugs, a construction method thereof, and an application thereof. Background Art

[0002] Since the sensitivity of intestinal cancer cell lines from different individuals to anti-cancer drugs varies, some cell lines may highly express drug targets, making it easier for chemotherapy drugs to bind to them and exert their effects, showing higher sensitivity; while some other cell lines may lowly express or mutate drug targets, resulting in ineffective drug action and drug resistance. By using a human intestinal cancer cell line sensitive to anti-cancer drugs, the activity screening and efficacy evaluation of newly developed chemotherapy drugs can be carried out quickly and efficiently, accelerating the R & D process of new chemotherapy drugs; at the same time, the sensitive cell line can be used to evaluate the combined effects of chemotherapy drugs and other therapies, such as targeted therapy and immunotherapy, to explore synergistic effects and provide more effective therapeutic drugs for clinical use. In addition, in the pre-clinical stage of drug R & D, through experiments on sensitive cell lines, the efficacy and potential toxic side effects of drugs can be evaluated more accurately, which helps to predict the response of patients to drugs, provides important reference basis for drugs to enter clinical trials, improves the success rate of drug R & D, and reduces R & D costs. In view of this, the present invention provides a three-dimensional intestinal cancer cell model sensitive to anti-cancer drugs, a construction method thereof, and an application thereof. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a three-dimensional intestinal cancer cell model sensitive to anti-cancer drugs, a construction method thereof, and an application thereof. The purpose is to construct a three-dimensional intestinal cancer cell model sensitive to anti-cancer drugs for high-throughput screening and evaluation of anti-cancer drugs.

[0004] The technical solution of the present invention for solving the above technical problems is as follows:

[0005] In the first aspect, a construction method of a three-dimensional intestinal cancer cell model sensitive to anti-cancer drugs includes the following steps:

[0006] Inoculate an intestinal cancer cell line and fibroblasts into a culture medium containing an extracellular matrix, and perform three-dimensional cell co-culture to obtain a three-dimensional intestinal cancer cell model sensitive to anti-cancer drugs.

[0007] Among them, the intestinal cancer cell line includes human colorectal cancer cell line DLD-1, human colon adenocarcinoma cell line SW480, human colon cancer cell line LOVO, and so on.

[0008] Among them, the extracellular matrix can be a fibrous or granular solid extracellular matrix composed of cellulose-poly-L-lysine copolymer. This solid extracellular matrix is composed of nanofibers with a nanofiber gap of less than 5 micrometers, insoluble in water, having a low density, allowing cell adhesion and free movement on its surface, allowing growth on the fiber surface and between fiber voids, and allowing cells to use it to build an extracellular framework structure required for three-dimensional cell growth; at the same time, it establishes and maintains a stable internal environment highly similar to that in the body for cells; different from existing microcarriers, cells do not completely rely on adhesion to the fiber surface for growth, and will not grow inside the fibers, so it is easier to carry out nutrient and metabolic waste exchange and maintain the consistency of the entire culture system; this extracellular matrix does not adsorb dyes and antibodies and does not produce spontaneous fluorescence, thus not affecting the detection, analysis and identification of the cultured cells.

[0009] The beneficial effects of the present invention are as follows: Compared with other drug screening models, the present invention uses an intestinal cancer cell line to establish a three-dimensional intestinal cancer cell model sensitive to anticancer drugs through in vitro three-dimensional co-culture, and conducts high-throughput screening and evaluation of anticancer drugs, which can accurately and quickly evaluate the efficacy of anticancer drugs, accelerate the development of new drugs, reduce the cost of drug use, and optimize the treatment plan.

[0010] On the basis of the above technical solutions, the present invention can also be improved as follows.

[0011] Further, it includes the following specific steps: inoculate an intestinal cancer cell line and fibroblast cells in a well plate, add a culture medium containing an extracellular matrix to the well plate, and perform three-dimensional cell co-culture to obtain a three-dimensional intestinal cancer cell model sensitive to anticancer drugs;

[0012] The total cell concentration inoculated in the well plate is 1.0 - 3.0×10 6 cells / mL, and the inoculation number ratio of the intestinal cancer cell line to the fibroblast cells is 2 - 4:1.

[0013] Further, the volume ratio of the extracellular matrix to the culture medium in the culture medium containing the extracellular matrix is 1:2 - 4;

[0014] The conditions for the three-dimensional cell co-culture are: 37°C, 5% CO2, and culture for 5 - 7 days.

[0015] Further, the intestinal cancer cell line includes at least one of the human colorectal cancer cell line DLD-1, the human colon adenocarcinoma cell line SW480, and the human colon cancer cell line LOVO.

[0016] In the second aspect, a three-dimensional intestinal cancer cell model sensitive to anticancer drugs, and the three-dimensional intestinal cancer cell model is constructed by the construction method described above.

[0017] Thirdly, an application of a three-dimensional intestinal cancer cell model sensitive to anti-cancer drugs, which is to use the three-dimensional intestinal cancer cell model sensitive to anti-cancer drugs in the evaluation and screening of anti-cancer drugs.

[0018] Fourthly, a screening method of a three-dimensional intestinal cancer cell model for anti-cancer drugs comprises the following steps:

[0019] (1) Cell plating: Resuspend and count the three-dimensional intestinal cancer cell model sensitive to anti-cancer drugs with a cell culture medium to obtain a cell suspension. After counting, transfer the cell suspension to a well plate to obtain a well plate for cell plating.

[0020] (2) Drug sensitivity detection: Perform drug sensitivity detection of chemotherapeutic drugs in the well plate for cell plating, and obtain a detection result of cell viability by detecting cell viability.

[0021] Further, step (1) is specifically: Resuspend and count the three-dimensional intestinal cancer cell model sensitive to anti-cancer drugs with a cell culture medium to obtain a cell suspension; after counting, add an extracellular matrix to the cell suspension, mix well and transfer to a well plate to obtain a well plate for cell plating; the volume ratio of the cell suspension to the extracellular matrix is 0.5 - 1.5:0.5 - 1.5.

[0022] Further, the anti-cancer drugs in step (2) include chemotherapeutic drugs.

[0023] Further, the cell viability detection is performed using any one of the CCK-8 method, the MTT CCK-8 method, and the CyQUANT method. Description of the Drawings

[0024] Figure 1 It is a microscopic result diagram of the effect of the chemotherapeutic drug of the present invention on normal intestinal cells CCD481;

[0025] Figure 2 It is a result diagram of the effect of the chemotherapeutic drug of the present invention on normal intestinal cells CCD481;

[0026] Figure 3 It is a microscopic result diagram of the effect of the chemotherapeutic drug of the present invention on human colorectal cancer cell line DLD-1;

[0027] Figure 4 It is a diagram of the effect of the chemotherapeutic drug of the present invention on normal intestinal cells CCD481;

[0028] Figure 5 It is a microscopic result diagram of the effect of the chemotherapeutic drug of the present invention on human colon cancer cell line LOVO;

[0029] Figure 6 It is a result of the effect of the chemotherapeutic drug of the present invention on human colon cancer cell line LOVO;

[0030] Figure 7 This is the microscopic result diagram of the effect of the chemotherapy drug of the present invention on human colon adenocarcinoma cell line SW480;

[0031] Figure 8 This is the result of the effect of the chemotherapy drug of the present invention on human colon adenocarcinoma cell line SW480. Detailed implementation mode

[0032] The principles and features of the present invention are described below. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention. For those technical or conditions not specified in the examples, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be purchased through regular channels.

[0033] Examples

[0034] 1. Key experimental reagents and materials.

[0035] 1.1. Experimental reagents.

[0036] The sources of the experimental reagents are shown in Table 1:

[0037] Table 1

[0038]

[0039] The preparation method of the extracellular matrix in the form of fibers or granular solids composed of cellulose-poly-L-lysine copolymer includes the following steps:

[0040] Step 1, selective oxidation:

[0041] Dissolve 1.0 mol of cellulose (size: 250 microns) in 16200 mL of deionized water, stir well to form a suspension, and then add 1.0 mol of sodium periodate. Adjust the pH to 2.0 - 2.5 with 1M HCl. Then, keep the mixture in the dark at 40 °C and stir for 48 hours. The product is washed with deionized water to remove salts (conductivity lower than 10 μs).

[0042] Step 2, nucleophilic carbonyl addition reaction to form Schiff base:

[0043] The insoluble 2,3-dialdehyde cellulose after the oxidation reaction is uniformly suspended in 2 liters of deionized water, and the pH is adjusted to 2.0 - 2.5 with 1M HCl. The oxidized cellulose suspension is directly dropped into 1 liter of aqueous solution containing 250 grams of poly-L-lysine under continuous stirring (300 RPM), with a pH of 4.0. React at 25 °C for 4 hours, adjust the pH of the reaction system to 8.0 with 1M NaOH, and then react at 25 °C for 4 hours.

[0044] Step 3: Reduce the Schiff base (C=N) to a secondary amine (C-NH):

[0045] Adjust the pH to 6 with 1M HCl, add 3 mol of sodium triacetoxyborohydride, and continue the reaction at 25 °C for 36 hours. After adjusting the pH of the reaction system to 8.0 with 1M NaOH, add 1 mol of sodium borohydride and continue the reaction at 25 °C for 24 hours. The product can be washed with deionized water to remove salts (conductivity below 10 μs). Allow natural precipitation for 24 hours and remove the supernatant. Sterilize under high temperature and high pressure.

[0046] 1.2. Instrumentation and equipment.

[0047] The descriptions of the instrumentation and equipment are shown in Table 2:

[0048] Table 2

[0049] Instrument Name Supplier Model Microplate Reader Beijing Pulang New Technology Co., Ltd. DNM-9602G Carbon Dioxide Incubator Thermo Fisher Scientific 3111

[0050] 2. Method for constructing a three-dimensional intestinal cancer cell model sensitive to anticancer drugs.

[0051] The method for constructing a three-dimensional intestinal cancer cell model sensitive to anticancer drugs includes the following steps:

[0052] The method for constructing a three-dimensional intestinal cancer cell model sensitive to anticancer drugs includes the following steps:

[0053] Inoculate an intestinal cancer cell line and fibroblasts in a culture medium containing an extracellular matrix, perform three-dimensional cell co-culture, and obtain a three-dimensional intestinal cancer cell model sensitive to anticancer drugs.

[0054] Among them, the intestinal cancer cell line includes human colorectal cancer cell line DLD-1, human colon adenocarcinoma cell line SW480, human colon cancer cell line LOVO, etc.

[0055] Preferably, this embodiment includes the following specific steps: Inoculate an intestinal cancer cell line and fibroblasts in a well plate, add a culture medium containing an extracellular matrix to the well plate, perform three-dimensional cell co-culture, and obtain a three-dimensional intestinal cancer cell model sensitive to anticancer drugs;

[0056] The total cell concentration inoculated in the well plate is 1.0 - 3.0×10 6 cells / mL, such as 1.0×10 6 cells / mL, 2.0×10 6 cells / mL, 3.0×10 6 cells / mL, etc., and the ratio of the number of the intestinal cancer cell line to the number of fibroblasts inoculated is 2 - 4:1, such as 2:1, 3:1, 4:1, etc.

[0057] Further, the volume ratio of the extracellular matrix to the culture medium in the culture medium containing the extracellular matrix is 1:2-4, such as 1:2, 1:3, 1:4, etc.;

[0058] The conditions for the three-dimensional cell co-culture are: 37°C, 5% CO2, culture for 5-7 days, such as 5 days, 6 days, 7 days, etc.

[0059] This example also relates to the application of a three-dimensional intestinal cancer cell model sensitive to anticancer drugs, and uses the three-dimensional intestinal cancer cell model sensitive to anticancer drugs in the evaluation and screening of anticancer drugs.

[0060] This example also relates to a method for screening anticancer drugs using a three-dimensional intestinal cancer cell model, which includes the following steps:

[0061] (1) Cell seeding: Resuspend and count the three-dimensional intestinal cancer cell model sensitive to anticancer drugs with a cell culture medium to obtain a cell suspension. After counting, transfer the cell suspension to a well plate to obtain a well plate with cell seeding.

[0062] (2) Drug sensitivity detection: Perform drug sensitivity detection of chemotherapeutic drugs in the well plate with cell seeding, and obtain the detection result of cell viability by detecting cell viability.

[0063] Preferably in this example, step (1) is specifically: Resuspend and count the three-dimensional intestinal cancer cell model sensitive to anticancer drugs with a cell culture medium to obtain a cell suspension; after counting, add an extracellular matrix to the cell suspension, mix well and transfer it to a well plate to obtain a well plate with cell seeding; the volume ratio of the cell suspension to the extracellular matrix is 0.5-1.5:0.5-1.5, such as 0.5:1.5, 1:1, 1.5:0.5, etc.

[0064] Preferably in this example, the density of cells in the cell suspension is 0.5-2×10 4 cells / mL, such as 0.5×10 4 cells / mL, 1×10 4 cells / mL, 2×10 4 cells / mL, etc.;

[0065] Preferably in this example, the anticancer drugs include chemotherapeutic drugs; such as fluorouracil, irinotecan, oxaliplatin, etc.; the cell viability detection uses any one of the CCK-8 method, MTT CCK-8 method, and CyQUANT method for detection.

[0066] 3. Verification experiment.

[0067] 3.1. Cell culture:

[0068] 1) 2D cell culture: Take out the cryopreserved cell lines (intestinal cancer cells, intestinal epithelial cells, human fibroblasts) from liquid nitrogen, quickly put them into a 37°C water bath and gently shake to rapidly thaw the cells. After thawing, transfer them into a 15 mL centrifuge tube, add 10 mL of pre-warmed basal medium (1640 +

[0069] 10% FBS), centrifuge at room temperature at 400 g for 5 minutes, aspirate and discard the supernatant, and perform cell counting. The initial seeding concentration of the cells is 1.0×10 6 cells per flask. Add 6 mL of basal medium to resuspend the cells, and then inoculate the cells into a T75 flask and place it in an incubator for standby culture. Regularly observe the cell morphology and growth status. When the cells are in the logarithmic growth phase, perform subculture to maintain cell viability and proliferation ability.

[0070] 2) 3D cell culture: Digest the 2D intestinal cancer cell line in the logarithmic growth phase and count the cells. Adjust the cell concentration to 2.0×10 6 cells / mL, inoculate the cells into a low-attachment 96-well plate, add 50 μL of extracellular matrix (basic extracellular matrix for intestinal cancer organoids) to each well, and supplement with intestinal cancer medium (intestinal cancer organoid medium) to 150 μL.

[0071] 3) 3D cell co-culture: Inoculate the digested cells according to a ratio of 3:1 of intestinal cancer cell line and human fibroblasts, with a total cell number of 2.0×10 6 cells / mL. Inoculate the cells into a low-attachment 96-well plate, add 50 μL of extracellular matrix to each well, and supplement with intestinal cancer medium to 150 μL. During the culture process, perform medium change and subculture according to the cell growth and cell fusion status. Select the successfully cultured organoids for drug sensitivity experiments. The samples should show the organoid morphology and have a diameter between 100 - 200 μm.

[0072] 3.2. Drug sensitivity screening:

[0073] 1) Cell seeding: Resuspend and count the cells with intestinal cancer cell medium, with approximately 10,000 live cells per well. After thoroughly mixing the extracellular matrix and cell suspension at a ratio of 1:2, use a single-channel pipette to dispense the suspension into a 96-well plate at 90 μL / well. Set 6 wells for negative control, and the rest are drug wells. After thoroughly mixing the extracellular matrix and cell suspension at a ratio of 1:1, use a single-channel pipette to dispense the suspension into a 96-well plate at 90 μL / well as blank control wells, usually set 6 wells. Gently tap the periphery of the well plate to evenly distribute the cell suspension at the bottom.

[0074] 2) Drug exposure: After observing the good growth of the organoids / cells under the microscope, a drug addition experiment is carried out. Before adding the drug, observe the status of each well, eliminate abnormal wells, and take photos for recording. The experiment sets up a negative control group, a blank group, and a drug test group. The blank group has 6 replicates per plate, without adding drugs, and 10 μL of culture medium is supplemented; the negative control group inoculates the organoids / cells, without adding drugs, and 10 μL of culture medium is supplemented; the drug test group inoculates the organoids / cells and supplements 10 μL of the drug solution.

[0075] 3) Preparation of the drug stock solution plate: Dilute the drug stock solution on the drug plate according to the requirements, and then perform gradient dilution. There are a total of 6 concentration gradients. Pipette 10 μL of the drug solution into the cell culture plate. After adding the drug, place it in the incubator and let it stand for 72 h according to the experimental requirements.

[0076] 4) Drug sensitivity detection: After drug addition and incubation, a drug sensitivity detection experiment is carried out. Take out the culture plate, observe it under the microscope, eliminate abnormal wells, add 10 μL of CCK8, incubate for 4 h. The detection conditions are room temperature (about 25 °C), wavelength 450 nm, shake for 2 min to promote cell lysis, and incubate for 8 min to make the luminescence signal stable. After the detection is completed, export the data, eliminate abnormal wells, and calculate the cell survival rate: Survival rate (%) = ((OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group)) × 100%. Use GraphPad Prism 8 software to plot the dose-response curve and calculate the IC50 value to compare the sensitivity of different cell lines to chemotherapeutic drugs.

[0077] 3.3. Results of the drug sensitivity experiment.

[0078] 3.1. Results of the action of chemotherapeutic drugs on normal intestinal cells CCD481:

[0079] Table 3 Results of the action of chemotherapeutic drugs on normal intestinal cells CCD481

[0080]

[0081] Figure 1 and Figure 2 The results in Table 3 and the drug sensitivity results show that chemotherapeutic drugs such as fluorouracil basically do not kill normal intestinal cells, confirming that CCD481 can be used as a control for drug-resistant cell lines.

[0082] 4.2. Results of the action of chemotherapeutic drugs on human colorectal cancer cell line DLD-1:

[0083] Table 4 Results of the action of chemotherapeutic drugs on human colorectal cancer cell line DLD-1

[0084]

[0085] Figure 3 and Figure 4The drug sensitivity results in Table 4 showed that the chemotherapeutic drug oxaliplatin could significantly inhibit the proliferation of DLD-1, and with the increase of drug concentration, the activity continued to decline. After adding human fibroblasts, the drug resistance could be improved, and the trend was basically the same as that of 3D culture.

[0086] 4.3 Results of the effects of chemotherapeutic drugs on the human colon cancer cell line LOVO:

[0087] Table 5 Results of the effects of chemotherapeutic drugs on the human colon cancer cell line LOVO

[0088]

[0089] Figure 5 and Figure 6 The drug sensitivity results in Table 5 showed that LOVO was highly sensitive to chemotherapeutic drugs, and the trend of the drug on 2D cells was basically the same as that of 3D culture.

[0090] 4.4 Results of the effects of chemotherapeutic drugs on the human colon adenocarcinoma cell line SW480:

[0091] Table 6 Results of the effects of chemotherapeutic drugs on the human colon adenocarcinoma cell line SW480

[0092]

[0093] Figure 7 and Figure 8 The drug sensitivity results in Table 6 showed that SW480 was less sensitive to chemotherapeutic drugs and had strong drug resistance.

[0094] When screening drugs for the treatment of colorectal cancer, the CCD-841 cell line is often used as a normal cell control. By observing the morphological changes of cells after drug treatment and comparing the effects of drugs on CCD-841 cells and colorectal cancer cell lines, it is possible to understand whether the drugs have adverse effects on normal cells, providing an important basis for the safety evaluation of drugs.

[0095] The DLD-1 cells are derived from clinical colorectal cancer patients, and their biological behaviors and responses to drugs can reflect the clinical actual situation to a certain extent. When screening platinum drugs, we found that oxaliplatin could significantly inhibit the proliferation of DLD-1 cells and induce apoptosis, which provided an experimental basis for the application of platinum drugs in the treatment of colorectal cancer.

[0096] Meanwhile, the LOVO cell line showed high sensitivity to all three chemotherapeutic drugs. In particular, oxaliplatin had a strong killing effect on LOVO cells, could dose-dependently inhibit the proliferation of LOVO cells, and induce apoptosis, providing an experimental basis for the application of platinum drugs in the chemotherapy of colon cancer. Therefore, the LOVO cell line can be preferentially used in chemotherapy drug research trials. The SW480 cell line showed low sensitivity to chemotherapeutic drugs and can be used for the study of drug resistance mechanisms.

[0097] In addition, the experimental results suggest that if a certain drug has a significant inhibitory effect on intestinal cancer cells but has little effect on CCD-841 cells, it indicates that the drug has good selectivity and is more likely to become an effective therapeutic drug.

[0098] In summary, the present invention screens out an intestinal cancer cell line more suitable for chemotherapy drug research by analyzing the drug sensitivity screening results of different intestinal cancer cells to chemotherapeutic drugs.

[0099] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for constructing a three-dimensional intestinal cancer cell model sensitive to anti-cancer drugs, characterized in that, It includes the following steps: Inoculate an intestinal cancer cell line and fibroblasts in a culture medium containing an extracellular matrix, and perform three-dimensional cell co-culture to obtain a three-dimensional intestinal cancer cell model sensitive to anti-cancer drugs.

2. The method for constructing a three-dimensional intestinal cancer cell model sensitive to anti-cancer drugs according to claim 1, wherein It includes the following specific steps: Inoculate an intestinal cancer cell line and fibroblasts in a well plate, add a culture medium containing an extracellular matrix to the well plate, and perform three-dimensional cell co-culture to obtain a three-dimensional intestinal cancer cell model sensitive to anti-cancer drugs; The total cell concentration inoculated in the well plate is 1.0 - 3.0×10 6 cells / mL, and the ratio of the number of the intestinal cancer cell line inoculated to the number of the fibroblast inoculated is 2 - 4:

1.

3. The method for constructing a three-dimensional intestinal cancer cell model sensitive to an anti-cancer drug according to claim 2, characterized in that In the culture medium containing an extracellular matrix, the volume ratio of the extracellular matrix to the culture medium is 1:2 - 4; The conditions for the three-dimensional cell co-culture are: 37 °C, 5% CO2, and culture for 5 - 7 days.

4. The method for constructing a three-dimensional intestinal cancer cell model sensitive to anti-cancer drugs according to claim 1, characterized in that The intestinal cancer cell line includes at least one of the human colorectal cancer cell line DLD-1, the human colon adenocarcinoma cell line SW480, and the human colon cancer cell line LOVO.

5. A three-dimensional intestinal cancer cell model sensitive to anti-cancer drugs, characterized in that, The three-dimensional intestinal cancer cell model is constructed by the construction method described in any one of claims 1 to 4.

6. Use of a three-dimensional intestinal cancer cell model sensitive to anti-cancer drugs, characterized in that, Use the three-dimensional intestinal cancer cell model sensitive to anti-cancer drugs described in claim 5 in the evaluation and screening of anti-cancer drugs.

7. A screening method for anti-cancer drugs using a three-dimensional intestinal cancer cell model, characterized in that, It includes the following steps: (1) Cell seeding: Resuspend and count the three-dimensional intestinal cancer cell model sensitive to anti-cancer drugs described in claim 5 with a cell culture medium to obtain a cell suspension. After counting, transfer the cell suspension to a well plate to obtain a well plate for cell seeding; (2) Drug sensitivity detection: Perform drug sensitivity detection of chemotherapeutic drugs in the well plate for cell seeding, and obtain the detection result of cell viability by detecting cell viability.

8. The screening method of the three-dimensional intestinal cancer cell model for anti-cancer drugs according to claim 7, characterized in that, Step (1) is specifically: Resuspend and count the three-dimensional intestinal cancer cell model sensitive to anti-cancer drugs with a cell culture medium to obtain a cell suspension; after counting, add an extracellular matrix to the cell suspension, mix well and transfer it to a well plate to obtain a well plate for cell seeding; the volume ratio of the cell suspension to the extracellular matrix is 0.5 - 1.5:0.5 - 1.

5.

9. The method for screening anti-cancer drugs using the three-dimensional intestinal cancer cell model according to claim 7, wherein The anti-cancer drugs in step (2) include chemotherapeutic drugs.

10. The method for screening anti-cancer drugs using the three-dimensional intestinal cancer cell model according to claim 7, characterized in that, The cell viability detection is performed using any one of the CCK-8 method, the MTT CCK-8 method, and the CyQUANT method.

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