Construction method of in-vitro three-dimensional co-culture liver cancer cell model and drug effect detection application

Abstract

The invention relates to a construction method and efficacy detection application of an in-vitro three-dimensional co-culture hepatoma carcinoma cell model, and relates to the technical field of three-dimensional culture of hepatoma carcinoma cells, the construction method comprises the following steps: co-culturing human hepatoma carcinoma cells and co-culture cells in a culture medium containing a novel solid extracellular matrix, the co-culture cells comprise at least one of human fibrocytes, mesenchymal stem cells and hepatic stellate cells. Human liver cancer cells and co-culture cells are subjected to in-vitro three-dimensional co-culture through a brand-new culture system, so that an in-vitro anti-cancer drug efficacy detection model is established, the model supports high-throughput screening and evaluation of anti-cancer drugs, the efficacy of the anti-cancer drugs can be accurately and rapidly evaluated, development of new drugs is accelerated, and the development cost of the anti-cancer drugs is reduced. The medication cost is reduced, and the treatment scheme is optimized.

Description

Technical Field

[0001] The present invention relates to the technical field of three-dimensional culture of liver cancer cells, and specifically relates to a method for constructing an in vitro three-dimensional co-culture liver cancer cell model and its application in drug efficacy detection. Background Art

[0002] In the process of drug research and development, compared with tumor cells growing three-dimensionally in vivo, the traditional planar-growing tumor cell model lacks a stable internal environment, and even more lacks cell function differentiation and cell-cell interaction. Therefore, the 2D anti-cancer drug efficacy detection model is overly sensitive to anti-cancer drugs, and there are huge differences in drug responses compared with in vivo tumors. Compared with the 2D model, due to the cell structure, differentiation, and function of 3D-cultured cells being closer to those in vivo tissues, the drug response in the 3D model can better reflect its real effect in vivo. However, for the 3D tumor sphere model established by the traditional Matrigel method, due to the interference of the hydrogel on drug penetration, cell communication, cell migration, and the consistency of the microenvironment within the system, it is impossible to more comprehensively evaluate the drug efficacy. In view of this, the present invention provides a method for constructing an in vitro three-dimensional co-culture liver cancer cell model and its application in drug efficacy detection. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a method for constructing an in vitro three-dimensional co-culture liver cancer cell model and its application in drug efficacy detection. The purpose is to prove that the anti-cancer drug efficacy detection model established by the method of the present invention can provide a scientific basis for clinical anti-cancer drug screening and efficacy evaluation.

[0004] The technical solution of the present invention to solve the above technical problem is as follows:

[0005] In the first aspect, a method for constructing an in vitro three-dimensional co-culture liver cancer cell model, comprising the following steps:

[0006] Co-culture human liver cancer cells and co-culture cells in a culture medium containing a novel solid extracellular matrix; the co-culture cells include at least one of human fibroblasts, mesenchymal stem cells, and hepatic stellate cells.

[0007] Among them, the novel solid extracellular matrix can be a fibrous or granular solid extracellular matrix composed of cellulose-L-lysine polymers. This solid extracellular matrix is composed of nanofibers with nanofiber gaps less than 5 micrometers; it is insoluble in water, has a low density, allows cell adhesion and free movement on its surface, allows growth on the fiber surface and in the fiber voids, and allows 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.

[0008] The beneficial effects of the present invention are as follows:

[0009] (1) The present invention establishes an in vitro anti-cancer drug efficacy detection model by performing three-dimensional co-culture of human liver cancer cells and co-cultured cells in vitro through a new culture system; the in vitro anti-cancer drug efficacy detection model is highly similar to in vivo solid tumors in terms of the types of its constituent cells, cell differentiation and function, cell interaction and migration, and the microenvironment in which the cells are located is highly stable and consistent; therefore, compared with the existing liver cancer models established by two-dimensional planar culture or three-dimensional culture techniques represented by Matrigel, the method for establishing this model is simpler, has higher repeatability, and the evaluation of the effects of anti-cancer drugs is also more accurate.

[0010] (2) The present invention establishes an in vitro anti-cancer drug efficacy detection model by performing three-dimensional co-culture of human liver cancer cells and co-cultured cells in vitro through a new culture system. This model supports high-throughput screening and evaluation of anti-cancer drugs, can accurately and quickly evaluate the efficacy of anti-cancer drugs, accelerate the development of new drugs, reduce the cost of medication, and optimize the treatment plan.

[0011] On the basis of the above technical solutions, the present invention can also be improved as follows.

[0012] Further, the co-cultured cells are composed of human fibroblasts, mesenchymal stem cells, and hepatic stellate cells;

[0013] Or, the co-cultured cells are composed of human fibroblasts and mesenchymal stem cells;

[0014] Or, the co-cultured cells are composed of mesenchymal stem cells and hepatic stellate cells;

[0015] Or, the co-cultured cells are composed of hepatic stellate cells;

[0016] Alternatively, the co-cultured cells consist of human fibroblasts;

[0017] Alternatively, the co-cultured cells consist of mesenchymal stem cells.

[0018] Furthermore, when the co-cultured cells consist of human fibroblasts, mesenchymal stem cells and hepatic stellate cells; the inoculation number ratio of the human hepatoma cells to the co-cultured cells is 3:0.5 - 3.5; the inoculation number ratio of the human fibroblasts, the mesenchymal stem cells to the hepatic stellate cells is 0.5 - 1.5:0.5 - 1.5:0.5 - 1.5;

[0019] Alternatively, when the co-cultured cells consist of mesenchymal stem cells and hepatic stellate cells; the inoculation number ratio of the human hepatoma cells to the co-cultured cells is 3:0.5 - 3.5; the inoculation number ratio of the mesenchymal stem cells to the hepatic stellate cells is 0.5 - 1.5:0.5 - 1.5;

[0020] Alternatively, when the co-cultured cells consist of human fibroblasts and mesenchymal stem cells; the inoculation number ratio of the human hepatoma cells to the co-cultured cells is 3:0.5 - 2.5; the inoculation number ratio of the human fibroblasts to the mesenchymal stem cells is 0.5 - 1.5:0.5 - 1.5;

[0021] Alternatively, when the co-cultured cells consist of hepatic stellate cells; the inoculation number ratio of the human hepatoma cells to the hepatic stellate cells is 3:0.5 - 1.5;

[0022] Alternatively, when the co-cultured cells consist of human fibroblasts; the inoculation number ratio of the human hepatoma cells to the human fibroblasts is 3:0.5 - 1.5;

[0023] Alternatively, when the co-cultured cells consist of mesenchymal stem cells; the inoculation number ratio of the human hepatoma cells to the mesenchymal stem cells is 3:0.5 - 1.5.

[0024] Furthermore, in the medium containing the novel solid extracellular matrix, the volume ratio of the novel solid extracellular matrix to the medium is 0.5 - 1.5:3.

[0025] Furthermore, the density of the total inoculated cells in the medium containing the novel solid extracellular matrix is (1 - 3)×10 6 cells / mL.

[0026] Furthermore, the conditions for co-culture are: 37°C, 5% CO2, culture for 5 - 7 d.

[0027] In a second aspect, an in vitro three-dimensional co-culture hepatoma cell model, the in vitro three-dimensional co-culture hepatoma cell model is constructed by the construction method described above.

[0028] The third aspect is the application of the in vitro three-dimensional co-culture liver cancer cell model, which is used in the efficacy testing of anti-liver cancer drugs.

[0029] Among them, liver cancer cells in the body do not exist in isolation, but are in a complex tumor microenvironment and interact with surrounding cells; in the co-culture system, co-cultured cells can affect the drug resistance of liver cancer cells by secreting various cytokines and growth factors; for example, fibroblasts can secrete some factors such as transforming growth factor-β (TGF-β) to induce liver cancer cells to produce drug resistance, activate drug resistance-related signaling pathways in liver cancer cells, and make liver cancer cells resistant to chemotherapy drugs; mesenchymal cells can secrete a variety of growth factors, cytokines, such as hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), etc. These factors may activate related signaling pathways in liver cancer cells and promote The proliferation of liver cancer cells and co-culture may change the drug sensitivity of liver cancer cells; the specific roles of mesenchymal cells are as follows: on the one hand, some factors secreted by mesenchymal cells may induce epithelial-mesenchymal transition of liver cancer cells, making liver cancer cells acquire stronger drug resistance; on the other hand, mesenchymal cells and liver cancer cells combine with each other to provide a physical barrier for liver cancer cells, hindering the contact between drugs and liver cancer cells, thereby causing liver cancer cells to develop resistance to chemotherapy drugs; hepatic stellate cells originate from liver tissue and will proliferate in large numbers when the liver is damaged, but there are currently limited clinical drugs for the treatment of liver fibrosis, and their efficacy has certain limitations; therefore, drug screening for hepatic stellate cells is of great significance for the development of new anti-liver fibrosis drugs.

[0030] In a fourth aspect, a drug efficacy detection method based on an in vitro three-dimensional co-cultured liver cancer cell model comprises the following steps:

[0031] (1) Cell plating: resuspending and counting the in vitro three-dimensional co-culture liver cancer cell model in a human liver cancer cell culture medium to obtain a cell suspension; adding a novel solid extracellular matrix to the cell suspension, mixing and transferring to a well plate to obtain a well plate for cell plating;

[0032] (2) Drug sensitivity test: The efficacy of anti-liver cancer drugs is tested in the well plate where the cells are plated by detecting cell viability.

[0033] Furthermore, the density of cells in the cell suspension is 5000-20000 cells / mL; and the volume ratio of the cell suspension to the novel solid extracellular matrix is ​​2:0.5-1.5. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1This is a comparison result diagram of the 2D and 3D drug screening of the liver cancer cell line of the present invention under a microscope; among them, A is the co-culture of the liver cancer cell line and hepatic stellate cells, E is the 3D culture of the liver cancer cell line, F is the 2D culture of the liver cancer cell line, and G is the 2D culture of the liver cancer cell line + hepatic stellate cells;

[0035] Figure 2 This is the comparison result of the 2D and 3D drug screening of the liver cancer cell line of the present invention; among them, a is the co-culture of the liver cancer cell line and hepatic stellate cells, e is the 3D culture of the liver cancer cell line, f is the 2D culture of the liver cancer cell line, and g is the 2D culture of the liver cancer cell line + hepatic stellate cells;

[0036] Figure 3 This is a comparison result diagram of the 3D co-culture drug screening of the liver cancer cell line of the present invention under a microscope; among them, B is the co-culture of the liver cancer cell line and human fibroblasts, C is the co-culture of the liver cancer cell line and mesenchymal stem cells, and D is the co-culture of the liver cancer cell line, human fibroblasts, and human mesenchymal stem cells;

[0037] Figure 4 This is the comparison result of the 3D co-culture drug screening of the liver cancer cell line of the present invention; among them, b is the co-culture of the liver cancer cell line and human fibroblasts, c is the co-culture of the liver cancer cell line and mesenchymal stem cells, and d is the co-culture of the liver cancer cell line, human fibroblasts, and human mesenchymal stem cells;

[0038] Figure 5 This is a scanning electron micrograph of the fibrous or granular solid extracellular matrix structure formed by the cellulose-L-lysine polymer of the present invention. Among them, A-C are scanning electron micrographs of the dried and dehydrated fibrous or granular solid extracellular matrix at different electron microscope scales. Detailed implementation mode

[0039] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not intended to limit the scope of the present invention. For those not specified in the examples in terms of specific techniques or conditions, the techniques or conditions described in the literature in this field or according to the product specifications are followed. For reagents or instruments not specified in terms of the manufacturer, they are all conventional products that can be purchased through regular channels.

[0040] Example

[0041] 1. Experimental reagents and materials.

[0042] 1.1. Experimental reagents.

[0043] The sources of the experimental reagents are shown in Table 1:

[0044] Table 1

[0045]

[0046]

[0047] In the following examples, a method for preparing a fibrous or particulate solid extracellular matrix composed of cellulose-L-lysine polymer (i.e., an organoid-based extracellular matrix) is provided, which includes the following steps:

[0048] Step 1, selective oxidation:

[0049] Dissolve 1.0 mol of cellulose (size: 250 μm) 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, allow the mixture to react under stirring in the dark at 40 °C for 48 hours. The product is washed with deionized water to remove salts (conductivity below 10 μs).

[0050] Step 2, nucleophilic carbonyl addition reaction to form Schiff base:

[0051] The insoluble 2,3-dialdehyde cellulose after the oxidation reaction is uniformly suspended in 2000 mL of deionized water, and the pH is adjusted to 2.0 - 2.5 with 1M HCl. The oxidized cellulose suspension is directly added dropwise to 1 liter of 2.5M lysine aqueous solution under continuous stirring (300 RPM), with a pH of 4.0. React at 25 °C for 4 hours, and then adjust the pH of the reaction system to 8.0 with 1M NaOH and react at 25 °C for 4 hours.

[0052] Step 3, reduce the Schiff base (C=N) to secondary amine (C-NH):

[0053] Adjust the pH to 6 with 1M HCl, add 3 mol of sodium triacetoxyborohydride, and continue to react 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 to react 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 by autoclaving.

[0054] As can be seen from Figure 5 A, the major axis of the fibrous or particulate solid extracellular matrix after drying and dehydration is in the range of 100 - 1000 μm. As shown in Figure 5 B and C, the fibrous or particulate solid extracellular matrix is formed by cross-linking of nanofibers to form a porous network structure, and the gap between nanofibers is less than 5 μm; it presents a fibrous or short film-like structure in appearance.

[0055] The selectively oxidized polysaccharide is insoluble and in the form of solid fibers or particles with a fiber length or particle major axis not less than 1 μm in size. The aldehyde moiety is generated by selectively oxidizing the hydroxyl groups on C2 and C3 of the glucose units, and the oxidation rate of the glucose units contained in the polysaccharide is not more than 80% and the oxidation does not produce more carboxyl groups than aldehyde groups or cause the breakage of the polysaccharide chain. The polypeptide includes one or more of collagen, gelatin, and polylysine; the extracellular matrix composed of cellulose-L-lysine polymer is an insoluble solid fiber or particle with a porous multi-layer network structure.

[0056] 1.2. Instrumentation.

[0057] The description of the instrumentation is shown in Table 2:

[0058] Table 2

[0059] Instrument Name Supplier Model Microplate Reader Beijing Pulang New Technology Co., Ltd. DNM-9602G Carbon Dioxide Incubator Thermo Fisher Scientific 3111

[0060] 2. In vitro three-dimensional co-culture human hepatocellular carcinoma cell model.

[0061] The in vitro three-dimensional co-culture human hepatocellular carcinoma cell model is obtained by the following construction method:

[0062] Co-culture human hepatocellular carcinoma cells and co-culture cells in a medium containing a novel solid extracellular matrix; the co-culture cells include at least one of human fibroblasts, mesenchymal stem cells, and hepatic stellate cells.

[0063] Preferably in this embodiment, the co-culture cells are composed of human fibroblasts, mesenchymal stem cells, and hepatic stellate cells;

[0064] Or, the co-culture cells are composed of human fibroblasts and mesenchymal stem cells;

[0065] Or, the co-culture cells are composed of mesenchymal stem cells and hepatic stellate cells;

[0066] Or, the co-culture cells are composed of hepatic stellate cells;

[0067] Or, the co-culture cells are composed of human fibroblasts;

[0068] Or, the co-culture cells are composed of mesenchymal stem cells.

[0069] Preferably, in this embodiment, when the co-cultured cells are composed of human fibroblasts, mesenchymal stem cells, and hepatic stellate cells, the inoculation number ratio of the human hepatoma cells to the co-cultured cells is 3: 0.5 - 3.5, such as 3: 0.5, 3: 1, 3: 2, 3: 3, etc.; the inoculation number ratio of the human fibroblasts, the mesenchymal stem cells, and the hepatic stellate cells is 0.5 - 1.5: 0.5 - 1.5: 0.5 - 1.5, such as 1.5: 0.5: 1.5, 1: 1: 1, 0.5: 1.5: 0.5, etc.

[0070] When the co-cultured cells are composed of mesenchymal stem cells and hepatic stellate cells, the inoculation number ratio of the human hepatoma cells to the co-cultured cells is 3: 0.5 - 3.5, such as 3: 0.5, 3: 1, 3: 2, etc.; the inoculation number ratio of the mesenchymal stem cells to the hepatic stellate cells is 0.5 - 1.5: 0.5 - 1.5, such as 0.5: 1.5, 1: 1, etc.

[0071] Or, when the co-cultured cells are composed of human fibroblasts and mesenchymal stem cells, the inoculation number ratio of the human hepatoma cells to the co-cultured cells is 3: 0.5 - 2.5, such as 3: 0.5, 3: 1, 3: 2, etc.; the inoculation number ratio of the human fibroblasts to the mesenchymal stem cells is 0.5 - 1.5: 0.5 - 1.5, such as 0.5: 1.5, 1: 1, etc.

[0072] Or, when the co-cultured cells are composed of hepatic stellate cells, the inoculation number ratio of the human hepatoma cells to the hepatic stellate cells is 3: 0.5 - 1.5, such as 3: 0.5, 3: 1, 3: 1.5, etc.

[0073] Or, when the co-cultured cells are composed of human fibroblasts, the inoculation number ratio of the human hepatoma cells to the human fibroblasts is 3: 0.5 - 1.5, such as 3: 0.5, 3: 1, 3: 1.5, etc.

[0074] Or, when the co-cultured cells are composed of mesenchymal stem cells, the inoculation number ratio of the human hepatoma cells to the mesenchymal stem cells is 3: 0.5 - 1.5, such as 3: 0.5, 3: 1, 3: 1.5, etc.

[0075] Preferably, in this embodiment, in the medium containing the novel solid extracellular matrix, the volume ratio of the novel solid extracellular matrix to the medium is 0.5 - 1.5: 3, such as 0.5: 3, 1: 3, 1.5: 3, etc. The density of the total inoculated cells in the medium containing the novel solid extracellular matrix is (1 - 3)×10 6 cells / mL, such as 1×10 6 cells / mL, 2×10 6cells / mL, 3×10 6 cells / mL, etc. The co - culture conditions in step (1) are: 37°C, 5% CO2, culture for 5 - 7 days, such as 5 days, 6 days, 7 days, etc.

[0076] This example also relates to a method for detecting the efficacy based on an in vitro three - dimensional co - culture hepatocellular carcinoma cell model, including the following steps:

[0077] (1) Cell seeding: Resuspend and count the in vitro three - dimensional co - culture hepatocellular carcinoma cell model with a human hepatocellular carcinoma cell medium to obtain a cell suspension; add a novel solid extracellular matrix to the cell suspension, mix well and transfer it to a well plate to obtain the well plate for cell seeding.

[0078] (2) Drug sensitivity detection: Perform the efficacy detection of anti - hepatocellular carcinoma drugs in the well plate of the cell - seeded well plate by detecting cell viability.

[0079] Preferably in this example, the density of cells in the cell suspension is 5000 - 20000 cells / well, such as 5000 cells / well, 10000 cells / well, 20000 cells / well, etc. The volume ratio of the cell suspension to the novel solid extracellular matrix is 2:0.5 - 1.5, such as 2:0.5, 2:1, 2:1.5, etc.

[0080] 3. Experimental method.

[0081] 3.1. 2D cell line culture:

[0082] Cell resuscitation; Take out the cryopreserved cell lines (Hepg2, hepatic stellate cells) from liquid nitrogen, quickly put them into a 37°C water bath and gently shake to rapidly melt the cells. After melting, transfer them into a 15 - mL centrifuge tube, add 10 mL of pre - warmed basal medium (1640 + 10% FBS), centrifuge at room temperature, centrifuge at 400 g for 5 minutes, aspirate the supernatant, and perform cell counting;

[0083] Cell culture: The initial seeding concentration of cells is 1.0×10 6 cells / bottle. Add 6 mL of basal medium (1640 + 10% FBS) to resuspend the cells, and then inoculate the cells into a T75 bottle and place it in an incubator for standby culture.

[0084] 3.2. 3D cell line culture:

[0085] Hepatocellular carcinoma cells: Digest and count the hepatocellular carcinoma cell line in the logarithmic growth phase. 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 organoid basal extracellular matrix to each well, supplement the hepatocellular carcinoma organoid medium to 150 μL, and continue 2D culture for the remaining cells.

[0086] 3.3. 3D Co - culture:

[0087] The 3D co - culture includes the following steps:

[0088] (1) Cell grouping:

[0089] During the co - culture stage, the cells are divided into the following groups: a is the co - culture of hepatocellular carcinoma cell line and hepatic stellate cells; b is the co - culture of hepatocellular carcinoma cell line and human fibroblasts; c is the co - culture of hepatocellular carcinoma cell line and mesenchymal stem cells; d is the co - culture of hepatocellular carcinoma cell line, human fibroblasts, and human mesenchymal stem cells; e is the 3D culture of hepatocellular carcinoma cell line; f is the 2D culture of hepatocellular carcinoma cell line; g is the 2D culture of hepatocellular carcinoma cell line + hepatic stellate cells.

[0090] (2) Co - culture of hepatocellular carcinoma cell line and hepatic stellate cells: The digested cells are inoculated at a ratio of 3:1 of hepatocellular carcinoma cell line to hepatic stellate cells, and the total cell number is 2.0×10 6 cells / mL. The cells are inoculated into a low - attachment 96 - well plate, and 50 μL of organoid basal extracellular matrix is added to each well, and the hepatocellular carcinoma organoid medium is supplemented to 150 μL. During the culture process, medium replacement and passage are carried out according to the cell growth and cell fusion status. Select the successfully cultured organoids for the drug sensitivity experiment. The samples should show the organoid morphology, and the diameter is between 100 - 200 μm.

[0091] (3) The remaining grouping operations are the same as above. Among them, for group d, after digestion, the ratio of the co - culture of hepatocellular carcinoma cell line, human fibroblasts, and human mesenchymal stem cells is changed to 3:1:1.

[0092] 3.4. Drug sensitivity screening:

[0093] The drug sensitivity screening includes the following steps:

[0094] (1) Cell grouping: The same as the co - culture stage.

[0095] (2) Cell seeding: Resuspend and count the cells with the corresponding cancer medium, about 10000 viable cells / 96 - well plate. After thoroughly mixing the extracellular matrix and the cell suspension at a volume ratio of 1:2, use a single - channel pipette to aliquot the suspension into the 96 - well plate at 90 μL / well. Set 6 - 12 wells for negative control, and the rest are drug wells. After thoroughly mixing the extracellular matrix and the cell suspension at a volume ratio of 1:1, use a single - channel pipette to aliquot the suspension into the 96 - well plate at 90 μL / well as blank control wells, generally set 6 wells. Gently tap the periphery of the well plate to make the cell suspension evenly distributed at the bottom.

[0096] (3) Drug exposure: After observing the good growth of the organoids / cells under the microscope, the drug addition experiment is carried out. Before adding the drug, observe the status of each well, eliminate the abnormal wells, and take photos for record. Among them, 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.

[0097] (4) 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 to confirm whether the highest drug usage concentration is correct. The drug addition plan needs to be checked before adding the drug. One person adds the drug and another person confirms. After adding the drug, place it in the incubator and let it stand for 72 h according to the experimental requirements.

[0098] (5) Drug sensitivity detection: After drug addition and incubation, perform the drug sensitivity detection experiment. Take out the culture plate, observe it under the microscope, eliminate the abnormal wells, add 10 μL of CCK8, incubate for 4 h, the detection conditions are room temperature (about 25 °) and 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 the abnormal wells (the reasons need to be explained), and perform data analysis.

[0099] 4. Results of the drug sensitivity experiment.

[0100] 4.1. Comparison results of 2D and 3D drug screening of the hepatocellular carcinoma cell line.

[0101] Table 3 Comparison results of 2D and 3D drug screening of the hepatocellular carcinoma cell line

[0102]

[0103] In Table 3, a is the co-culture of the hepatocellular carcinoma cell line and hepatic stellate cells, e is the 3D culture of the hepatocellular carcinoma cell line, f is the 2D culture of the hepatocellular carcinoma cell line, and g is the 2D culture of the hepatocellular carcinoma cell line + hepatic stellate cells.

[0104] From Figure 1 A, E - G in Figure 2 a, e - g in

[0105] and the drug sensitivity results in Table 3, it shows that the drug resistance of hepatocellular carcinoma cells under 3D culture increases, and after adding hepatic stellate cells, the drug resistance of the cells further increases.

[0106] Table 4 Comparison results of 3D co-culture drug screening of the hepatocellular carcinoma cell line

[0107]

[0108] In Table 4, b is the co-culture of liver cancer cell line and human fibroblasts, c is the co-culture of liver cancer cell line and mesenchymal stem cells, and d is the co-culture of liver cancer cell line, human fibroblasts, and human mesenchymal stem cells.

[0109] Depend on Figure 3 Medium BD, Figure 4 The drug sensitivity results in BD and Table 4 show that human mesenchymal hepatocytes and fibroblasts can improve the drug resistance of liver cancer cells.

[0110] 5. Experimental results.

[0111] In vivo, liver cancer cells do not exist in isolation, but are in a complex tumor microenvironment, interacting with surrounding cells; in the co-culture system, other cells can affect the drug resistance of liver cancer cells by secreting various cytokines, growth factors, etc.; for example, fibroblasts can secrete some factors such as transforming growth factor-β (TGF-β) to induce liver cancer cells to produce drug resistance, activate drug resistance-related signaling pathways in liver cancer cells, and make liver cancer cells resistant to chemotherapy drugs; mesenchymal cells can secrete a variety of growth factors, cytokines, etc., such as hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), etc. These factors may activate related signaling pathways in liver cancer cells and promote the proliferation of liver cancer cells. Co-culture may change the drug sensitivity of liver cancer cells. The effects of mesenchymal cell co-culture are as follows: on the one hand, some factors secreted by mesenchymal cells may induce epithelial-mesenchymal transition in liver cancer cells, so that liver cancer cells acquire stronger drug resistance; on the other hand, mesenchymal cells and liver cancer cells combine with each other, providing a physical barrier for liver cancer cells, hindering the contact between drugs and liver cancer cells, thereby causing liver cancer cells to develop drug resistance to chemotherapy drugs. Hepatic stellate cells originate from liver tissue and proliferate in large numbers when the liver is damaged. However, there are currently limited drugs for the treatment of liver fibrosis in clinical practice, and their efficacy has certain limitations. Therefore, drug screening for hepatic stellate cells is of great significance for the development of new anti-liver fibrosis drugs. Screening drugs in this co-culture environment can discover drugs that can interfere with the interaction between fibroblasts and liver cancer cells, and these drugs may have better efficacy in vivo.

[0112] The present invention preliminarily confirms that 3D co-culture can improve the drug resistance of liver cancer cells, and after adding hepatic stellate cells, the drug resistance of cells is further improved; in addition, the addition of mesenchymal cells and fibroblasts also narrows the difference with in vivo medication to a certain extent.

[0113] In summary, the present invention screens drugs through an in vitro three-dimensional co-culture liver cancer cell model, narrows the difference with in vivo drug use, and can discover drugs that can interfere with the interaction between fibroblasts and liver cancer cells, thereby having better therapeutic effects in vivo.

[0114] Although the embodiments of the present invention have been shown and described above, it is to 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 an in vitro three-dimensional co-culture liver cancer cell model, characterized in that It includes the following steps: Co-culture human liver cancer cells and co-culture cells in a medium containing a novel solid extracellular matrix; the co-culture cells include at least one of human fibroblasts, mesenchymal stem cells, and hepatic stellate cells.

2. The method for constructing an in vitro three-dimensional co-culture liver cancer cell model according to claim 1, characterized in that, The co-culture cells are composed of human fibroblasts, mesenchymal stem cells, and hepatic stellate cells; Alternatively, the co-culture cells are composed of human fibroblasts and mesenchymal stem cells; Alternatively, the co-culture cells are composed of mesenchymal stem cells and hepatic stellate cells; Alternatively, the co-culture cells are composed of hepatic stellate cells; Alternatively, the co-culture cells are composed of human fibroblasts; Alternatively, the co-culture cells are composed of mesenchymal stem cells.

3. The method for constructing an in vitro three-dimensional co-culture liver cancer cell model according to claim 2, characterized in that, When the co-culture cells are composed of human fibroblasts, mesenchymal stem cells, and hepatic stellate cells; the inoculation number ratio of the human liver cancer cells to the co-culture cells is 3:0.5 - 3.5; the inoculation number ratio of the human fibroblasts, the mesenchymal stem cells, and the hepatic stellate cells is 0.5 - 1.5:0.5 - 1.5:0.5 - 1.5; Alternatively, when the co-culture cells are composed of mesenchymal stem cells and hepatic stellate cells; the inoculation number ratio of the human liver cancer cells to the co-culture cells is 3:0.5 - 3.5; the inoculation number ratio of the mesenchymal stem cells to the hepatic stellate cells is 0.5 - 1.5:0.5 - 1.5; Alternatively, when the co-culture cells are composed of human fibroblasts and mesenchymal stem cells; the inoculation number ratio of the human liver cancer cells to the co-culture cells is 3:0.5 - 2.5; the inoculation number ratio of the human fibroblasts to the mesenchymal stem cells is 0.5 - 1.5:0.5 - 1.5; Alternatively, when the co-culture cells are composed of hepatic stellate cells; the inoculation number ratio of the human liver cancer cells to the hepatic stellate cells is 3:0.5 - 1.5; Alternatively, when the co-culture cells are composed of human fibroblasts; the inoculation number ratio of the human liver cancer cells to the human fibroblasts is 3:0.5 - 1.5; Alternatively, when the co-culture cells are composed of mesenchymal stem cells; the inoculation number ratio of the human liver cancer cells to the mesenchymal stem cells is 3:0.5 - 1.

5.

4. The method for constructing an in vitro three-dimensional co-culture liver cancer cell model according to claim 1, wherein, In the medium containing the novel solid extracellular matrix, the volume ratio of the novel solid extracellular matrix to the medium is 0.5 - 1.5:

3.

5. The method for constructing an in vitro three-dimensional co-culture liver cancer cell model according to claim 1, characterized in that The density of the total inoculated cells in the culture medium containing the novel solid extracellular matrix is (1-3)×10 6 cells / mL.

6. The method for constructing an in vitro three-dimensional co-culture liver cancer cell model according to claim 1, wherein, The conditions for co-culture are: 37°C, 5% CO2, and culture for 5 - 7 days.

7. An in vitro three-dimensional co-culture liver cancer cell model, characterized in that, The in vitro three-dimensional co-culture liver cancer cell model is constructed by the construction method described in any one of claims 1 to 6.

8. Application of an in vitro three-dimensional co-culture liver cancer cell model, characterized in that, Use the in vitro three-dimensional co-culture liver cancer cell model described in claim 7 for the efficacy detection of anti-liver cancer drugs.

9. A method for detecting drug efficacy based on an in vitro three-dimensional co-culture liver cancer cell model, characterized in that It includes the following steps: (1) Cell seeding: Resuspend and count the in vitro three-dimensional co-culture liver cancer cell model described in claim 7 with a human liver cancer cell medium to obtain a cell suspension; add the novel solid extracellular matrix to the cell suspension, mix well and transfer it to a well plate to obtain the well plate for cell seeding; (2) Drug sensitivity detection: Perform the efficacy detection of anti-liver cancer drugs in the well plate for cell seeding by detecting cell viability.

10. The method for detecting drug efficacy based on an in vitro three-dimensional co-culture liver cancer cell model according to claim 9, wherein, The density of cells in the cell suspension is 5000 - 20000 cells / mL; the volume ratio of the cell suspension to the novel solid extracellular matrix is 2:0.5 - 1.5.

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