Method for constructing three-dimensional liver microsphere model and application thereof

Abstract

The invention relates to a method for constructing a three-dimensional liver microsphere model and application thereof. The cynomolgus monkey primary hepatocytes are inoculated into a culture container with an ultralow adsorption surface, the volume of a cell culture medium in the container is 10-30% of the total volume of the cell culture medium used for culture according to 100%, after first culture is carried out, the remaining volume of the cell culture medium is supplemented, second culture is carried out, and the three-dimensional liver microsphere model is obtained. The brand-new method for constructing the three-dimensional liver microsphere model is designed, the three-dimensional liver microsphere model can be rapidly prepared only by using cynomolgus monkey primary hepatocytes, in addition, by further controlling the culture process, the high-quality three-dimensional liver microsphere model can be further rapidly prepared, and the method is suitable for large-scale popularization and application. The built model can better simulate the in-vivo environment and the drug reaction, and can be used as an ideal model for researching the long-term effects of drug metabolism, toxic reaction and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell microspheres and drug evaluation, and relates to a method for constructing a three-dimensional liver microsphere model and its application. Background Art

[0002] The liver is an important target organ for drug toxicity and plays a key role in the metabolism of most drugs. Drug-induced liver injury (DILI) is an important cause of acute and chronic liver diseases and a key safety issue in new drug research and development. Therefore, drug hepatotoxicity evaluation is a key step in toxicity evaluation during the drug research and development process.

[0003] Currently, the models used for drug safety evaluation include in vivo experimental animal models and in vitro models. In vivo evaluation models usually use various experimental animals, which not only have a long experimental cycle, consume a lot of financial resources, have ethical problems, but also have species differences from humans. In vitro evaluation models often use monolayer hepatocyte culture models, which are rapid in detection. Two-dimensional (2D) in vitro cell models have advantages such as high throughput and low cost compared to traditional toxicity tests using animals as carriers, but there are still problems such as lack of metabolic enzyme activity and limited maintenance of cell activity, being limited to the detection of acute drug toxicity and not suitable for long-term repeated dosing studies.

[0004] Therefore, establishing a hepatotoxicity evaluation system that can not only retain the material structure basis of the in vivo cell microenvironment, simulate various functions of the in vivo liver tissue, achieve long-term repeated dosing, but also be efficient, accurate, intuitive, and controllable when conducting drug safety evaluation is the key research direction in current drug hepatotoxicity research.

[0005] With the development of technology, in recent years, three-dimensional (3D) models have, to a certain extent, solved the above problems. Hepatic spheroids are the most commonly used 3D culture models at present. The formation of spheroids is caused by the self-aggregation of hepatocytes and does not require technical interventions such as non-adhesive surfaces and gravitational adhesion. For example, CN116396920A discloses a primary hepatocyte co-culture three-dimensional liver microsphere model, its preparation method and application. The three-dimensional liver microsphere model is spherical, and the three-dimensional liver microsphere model contains hepatocytes and non-parenchymal liver cells; the non-parenchymal liver cells include hepatic sinusoidal endothelial cells, Kupffer cells, and hepatic stellate cells; the inoculation concentration percentages of hepatocytes and non-parenchymal liver cells in the three-dimensional liver microsphere model are (67±5)%:(33±5)%. However, the application verification and mechanism exploration of in vitro liver 3D models in drug repeated dosing toxicity evaluation are still unclear and require more verification and exploration. Moreover, the current methods for constructing three-dimensional liver microsphere models mostly require multiple initial cells, are complex in operation, and have a long culture time.

[0006] In summary, how to efficiently and quickly construct a three-dimensional liver microsphere model that can be effectively used for drug toxicity evaluation remains one of the problems urgently to be solved in this field. Summary of the Invention

[0007] In view of the deficiencies of the prior art and the actual needs, the present invention provides a method for constructing a three-dimensional liver microsphere model and its application, and develops a method for simply and quickly constructing a three-dimensional liver microsphere model that can be effectively used for drug toxicity evaluation based on primary cynomolgus monkey hepatocytes, providing an ideal model for studying long-term effects such as drug metabolism and toxic reactions.

[0008] To achieve this purpose, the present invention adopts the following technical solutions:

[0009] In the first aspect, the present invention provides a method for constructing a three-dimensional liver microsphere model, the method comprising:

[0010] Inoculating primary cynomolgus monkey hepatocytes into a culture container with an ultra-low adsorption surface, the volume of the cell culture medium in the container is 10-30% (for example, it can be 11%, 12%, 13%, 14%, 15%, 20%, 25%, 26%, 27%, 28% or 29%, etc.) of the total volume of the cell culture medium used for culture according to 100%, after the first culture, supplement the remaining volume of the cell culture medium, and perform the second culture to obtain the three-dimensional liver microsphere model.

[0011] In the present invention, a new method for constructing a three-dimensional liver microsphere model is designed. For primary cynomolgus monkey hepatocytes, a specific culture method is designed, and a specific two-step culture process is adopted, which can realize the rapid preparation of a three-dimensional liver microsphere model only by using primary cynomolgus monkey hepatocytes.

[0012] In the present invention, primary hepatocytes refer to hepatocytes directly extracted and isolated from the liver of cynomolgus monkeys.

[0013] Preferably, the density of the inoculated primary cynomolgus monkey hepatocytes is 1500-3000 cells / well, for example, it can be 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800 or 2900 cells / well, etc.

[0014] In the present invention, for primary cynomolgus monkey hepatocytes, a specific inoculation density is designed, which can effectively prepare a three-dimensional liver microsphere model.

[0015] Preferably, the time of the first culture is 24-48 h, for example, it can be 25, 26, 27, 28, 29, 30, 35, 40, 41, 42, 43, 44, 45, 46 or 47 h, etc.

[0016] In the present invention, by designing a specific first culture time, the formation of hepatocyte spheroids can be further rapidly promoted.

[0017] Preferably, the total volume of the cell culture medium used for the culture is 0.1 - 0.3 mL, for example, it can be 0.15, 0.2 or 0.25 mL, etc.

[0018] Preferably, the culture vessel with an ultra - low adsorption surface includes an ultra - low adsorption well plate.

[0019] Preferably, the ultra - low adsorption well plate includes a multi - well cell culture plate.

[0020] Preferably, the multi - well cell culture plate includes a 96 - well cell culture plate.

[0021] Preferably, the culture conditions for the first culture and the second culture are each independently 36 - 38 °C, 4 - 6% CO 2 .

[0022] The present invention realizes the rapid construction of a three - dimensional liver microsphere model through designing a specific two - step culture process. It can be understood that the general hepatocyte culture media in the art are applicable to the present invention.

[0023] Preferably, the cell culture medium contains glucose, glutamine, vitamin C, inorganic salts, epidermal growth factor and insulin.

[0024] As a preferred technical solution, the method for constructing the three - dimensional liver microsphere model includes the following steps:

[0025] (1) Inoculate cynomolgus monkey primary hepatocytes into a culture vessel with an ultra - low adsorption surface. The volume of the cell culture medium in the vessel is 10 - 30% of the total volume of the cell culture medium used for the culture according to 100%. The density of cynomolgus monkey primary hepatocytes after inoculation is 1500 - 3000 cells / well. After the first culture for 24 - 48 h;

[0026] (2) Supplement the remaining volume of the cell culture medium and conduct the second culture to obtain the three - dimensional liver microsphere model.

[0027] In a second aspect, the present invention provides a three - dimensional liver microsphere model, which is constructed by the method for constructing a three - dimensional liver microsphere model described in the first aspect.

[0028] The present invention constructs a co - culture 3D liver microsphere model of cynomolgus monkey primary hepatocytes, which can better simulate the in - vivo environment and drug response, can conduct long - term co - culture of different types of hepatocytes, and can maintain cell viability, metabolic activity and related hepatocyte functions for several weeks. This makes the model an ideal model for studying long - term effects such as drug metabolism and toxicity reactions.

[0029] In a third aspect, the present invention provides an application of the three-dimensional liver microsphere model described in the second aspect in drug research and development.

[0030] It can be understood that the application includes any one of the evaluation of clinical drug-induced hepatotoxicity, the prediction of the liver retention / excretion of nanodrugs, the assessment of the risk of drug-induced cholestasis, allowing long-term repeated dosing toxicity tests, the prediction of chronic drug toxic effects, or the evaluation of drug clearance rate, etc.

[0031] Compared with the prior art, the present invention has at least the following beneficial effects:

[0032] The present invention designs a new method for constructing a three-dimensional liver microsphere model. For cynomolgus monkey primary hepatocytes, a specific culture method is designed, and a specific two-step culture process is adopted, which can realize the rapid preparation of a three-dimensional liver microsphere model only using cynomolgus monkey primary hepatocytes. In addition, by further controlling the culture process, a high-quality three-dimensional liver microsphere model can be further rapidly prepared. The constructed model can better simulate the in vivo environment and the response to drugs, can perform long-term co-culture of different types of hepatocytes, and can maintain cell viability, metabolic activity, and related hepatocyte functions for several weeks, and can be used as an ideal model for studying long-term effects such as drug metabolism and toxic reactions. Description of the Drawings

[0033] Figure 1 It is a cell morphology diagram of the process of forming liver microspheres by the two-step culture method of cynomolgus monkey primary hepatocytes. The scale bar is 200 μm, 10×;

[0034] Figure 2 It is a result diagram of albumin levels in 2D and 3D culture models;

[0035] Figure 3 It is a result diagram of urea secretion levels in 2D and 3D culture models;

[0036] Figure 4 It is a comparison result diagram of the inducibility response of liver drug enzymes (CYP1A and CYP2B) in 2D and 3D hepatocyte models;

[0037] Figure 5 It is a diagram of the morphological changes of 3D microspheres after continuous action of different compounds, 200 μm, 10×;

[0038] Figure 6 It is a result diagram of evaluating the toxicity of amiodarone hydrochloride by 2D hepatocytes and 3D liver microsphere models;

[0039] Figure 7 It is a result diagram of evaluating the hepatotoxicity of isoniazid by 2D hepatocytes and 3D liver microsphere models. Detailed Embodiments

[0040] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings and through specific embodiments. However, the following examples are merely simple examples of the present invention and do not represent or limit the scope of the protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0041] For those not specifying specific techniques or conditions 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 reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0042] In the specific embodiment of the present invention, the cell culture medium used is (Jianshun Biology, 66020-23029).

[0043] The primary hepatocytes of cynomolgus monkeys were prepared by isolating the liver of cynomolgus monkeys.

[0044] Example 1

[0045] In this example, a three-dimensional liver microsphere model was constructed.

[0046] (1) The primary hepatocytes of cynomolgus monkeys were inoculated into a 96-well cell culture plate. The volume of the cell culture medium in each well was 10% (10 μL) of the total volume of the cell culture medium used for cultivation, calculated as 100%. The density of the primary hepatocytes of cynomolgus monkeys after inoculation was 2000 cells / well. After the first cultivation for 24 h in an incubator at 37 °C and 5% CO 2 ;

[0047] (2) The remaining volume of the cell culture medium (90 μL) was supplemented, and the second cultivation was carried out in an incubator at 37 °C and 5% CO 2 to obtain the three-dimensional liver microsphere model.

[0048] Example 2

[0049] In this example, a three-dimensional liver microsphere model was constructed.

[0050] (1) The primary hepatocytes of cynomolgus monkeys were inoculated into a 96-well cell culture plate. The volume of the cell culture medium in each well was 20% (20 μL) of the total volume of the cell culture medium used for cultivation, calculated as 100%. The density of the primary hepatocytes of cynomolgus monkeys after inoculation was 3000 cells / well. After the first cultivation for 24 h in an incubator at 37 °C and 5% CO 2 ;

[0051] (2) The remaining volume of the cell culture medium (80 μL) was supplemented, and the second cultivation was carried out in an incubator at 37 °C and 5% CO 2 to obtain the three-dimensional liver microsphere model.

[0052] Example 3

[0053] In this example, a three-dimensional liver microsphere model is constructed.

[0054] (1) Inoculate primary cynomolgus monkey hepatocytes into a 96-well cell culture plate. The volume of the cell culture medium in each well is 30% (30 μL) of the total volume of the cell culture medium used for cultivation, calculated as 100%. The density of the primary cynomolgus monkey hepatocytes after inoculation is 2,000 cells / well. Incubate in an incubator at 37 °C and 5% CO 2 for 48 h for the first cultivation;

[0055] (2) Supplement the remaining volume of the cell culture medium (70 μL), and conduct the second cultivation in an incubator at 37 °C and 5% CO 2 to obtain the three-dimensional liver microsphere model.

[0056] Example 4

[0057] In this example, a three-dimensional liver microsphere model is constructed.

[0058] Compared with Example 1, the only difference is that the density of the primary cynomolgus monkey hepatocytes after inoculation in step (1) is 1,000 cells / well.

[0059] Example 5

[0060] In this example, a three-dimensional liver microsphere model is constructed.

[0061] Compared with Example 1, the only difference is that the density of the primary cynomolgus monkey hepatocytes after inoculation in step (1) is 4,000 cells / well.

[0062] Example 6

[0063] In this example, a three-dimensional liver microsphere model is constructed.

[0064] Compared with Example 1, the only difference is that the time of the first cultivation in step (1) is 12 h.

[0065] Example 7

[0066] In this example, a three-dimensional liver microsphere model is constructed.

[0067] Compared with Example 1, the only difference is that the time of the first cultivation in step (1) is 72 h.

[0068] Comparative Example 1

[0069] In this comparative example, a three-dimensional liver microsphere model is constructed.

[0070] Compared with Example 1, the only difference is that the primary cynomolgus monkey hepatocytes are inoculated into a 96-well cell culture plate, and all the culture medium (100 μL) is directly used without stepwise cultivation.

[0071] Comparative Example 2

[0072] This comparative example constructs a three-dimensional liver microsphere model.

[0073] Primary hepatocytes of cynomolgus monkeys were used for 2D culture, specifically including:

[0074] Take out the cell cryopreservation tube and immediately place it in a 37 °C water bath (note that the water surface should preferably cover the cryopreservation liquid level as much as possible, but do not touch the cryopreservation tube cap); gently shake the cryopreservation tube for about 1 min 45 s (the time is for reference, and the specific time depends on the remaining ice crystal volume), thaw until a small part (about the size of a soybean) of ice crystals remains in the tube, disinfect the surface of the cryopreservation tube with 75% alcohol, and quickly transfer it to a biosafety cabinet; quickly pour the liquid in the cryopreservation tube into 40 mL of pre-warmed resuscitation medium at one time; centrifuge at room temperature, select a centrifugal force and time of 120 g for 5 min, set the acceleration speed to 5, the deceleration speed to 1, and turn off the brake mode; add 2 mL of the corresponding plating medium, gently flick the bottom of the centrifuge tube to resuspend the cells and make the volume up to 3 mL, and use trypan blue staining to measure the number of viable cells and cell viability, and adjust the viable cell density to 0.6×10 6 / mL, and plate.

[0075] Test example

[0076] This test example analyzes the three-dimensional liver microsphere models prepared in each example and comparative example.

[0077] (1) Compare the formation time of the liver microsphere model

[0078] Use an optical microscope to observe the morphology during the formation process of liver microspheres.

[0079] The formation process of the liver microspheres of primary hepatocytes of cynomolgus monkeys in Example 1 is as Figure 1 shown. In Examples 1-3, liver microspheres of primary hepatocytes of cynomolgus monkeys can be formed in 3 days, while in Comparative Example 1, liver microspheres of primary hepatocytes of cynomolgus monkeys take 8 days to form. It can be seen that the two-step culture method designed in the present invention can significantly improve the formation speed of the three-dimensional liver microsphere model.

[0080] In Examples 4, 5, 6, and 7, the liver microspheres of primary hepatocytes of cynomolgus monkeys form spheres in 5 days, indicating that the present invention specifically designs a specific initial seeding density and the first-stage culture time, which can further improve the formation speed of the three-dimensional liver microsphere model.

[0081] (2) Preliminary verification of the model

[0082] 1) Albumin and urea were detected. At different time points of cell culture, the cell culture supernatant was collected, and the albumin and urea contents were evaluated respectively.

[0083] Experimental method: Cells were cultured in 2D (Comparative Example 2) and 3D microspheres (Example 1) respectively. For the 2D culture model, cell culture supernatants were collected on the 1st, 2nd, 3rd, and 5th days after inoculation. For the 3D model, cell culture supernatants were collected on the 1st, 2nd, 3rd, 5th, 7th, and 10th days after inoculation. An albumin assay kit and a urea assay kit were used to detect the albumin expression level and urea secretion level. The results are shown respectively as Figure 2 and Figure 3 shown.

[0084] Experimental conclusion: After culturing in 3D for different days, the secretion of albumin and urea reached a relatively high level; and both were significantly higher than the 2D culture level, and this level was maintained until 10 days after culturing, indicating that the 3D model could maintain a high level of albumin and urea secretion for a long time and maintain its liver function.

[0085] 2) Effects of inducers on CYP1A and CYP2B in 2D / 3D models

[0086] The CYP1A inducer omeprazole (OME) and the CYP2B inducer phenobarbital (PHE) were dissolved in DMSO to prepare stock solutions. On the 3rd day after inoculation in the 2D model and on the 3rd, 5th, 7th, and 10th days of culture in the 3D model, OME (100 μmol·L -1 ) and PHE (1 mmol·L -1 ) were administered respectively, and a solvent (0.1% DMSO) control group was set up for each. After incubating in a medium containing 10% FBS for 24 h, the cells were washed 3 times with PBS, and the metabolite content was detected by mass spectrometry.

[0087] The results are shown as Figure 4 shown. In both the 2D and 3D models, after treatment with the inducer (OME), the CYP1A activity was up-regulated; after treatment with the inducer (PHE), the CYP2B activity was up-regulated; after culturing for 3 days, the CYP1A activity and CYP2B induction level of the 3D-cultured cells were significantly higher than those of the 2D culture. The 2D culture was limited by time and could only be detected for 3 days, while the 3D model could be cultured for a long time and maintain a relatively high hepatic drug-metabolizing enzyme activity.

[0088] (3) Model application test

[0089] To further verify the application effect of the model constructed by the present invention, taking the three-dimensional liver microsphere model prepared in Example 1 as an example for verification, and taking the 2D model prepared in Comparative Example 2 as a control, the specific experiments included:

[0090] 1) Morphological changes of 3D microspheres after continuous action of amiodarone hydrochloride and isoniazid

[0091] Experimental method: Isoniazid and amiodarone hydrochloride will be administered to the 3D model on the 4th day after inoculation. A control group of 0.1% DMSO will be set up. The administration concentrations of the isoniazid group are 100, 200, 400, 600, 800, and 1000 μmol·L -1 , and the administration concentrations of amiodarone hydrochloride are 10, 20, 40, 100, 150, 200, and 250 μmol·L -1 . After administration, incubate for 24 h under the conditions of 37 °C and 5% CO 2 . Take pictures with an optical microscope to observe the morphology of the microspheres.

[0092] The results are as Figure 5 shown. Amiodarone hydrochloride (AMI) is a drug that causes acute liver damage or hepatotoxicity. The microspheres show a loose state even at low concentrations. Isoniazid (ISO) is a drug that causes chronic liver damage or hepatotoxicity. Even at a concentration as high as 1000 μmol·L -1 , the microspheres are still very compact, indicating that the 3D microspheres constructed in the present invention can be effectively applied to the analysis of hepatotoxic drugs.

[0093] 2) Evaluation of the hepatotoxicity of amiodarone hydrochloride and isoniazid using 2D hepatocytes and 3D liver microsphere models

[0094] Experimental method:

[0095] Evaluation of drug toxicity using 2D liver models:

[0096] In a 96-well culture plate, inoculate 2000 primary crab-eating macaque hepatocytes in each well. Set up a control group of 0.1% DMSO and amiodarone hydrochloride and isoniazid administration groups. The administration concentrations of the isoniazid group are 100, 200, 400, 600, 800, and 1000 μmol·L -1 , and the administration concentrations of amiodarone hydrochloride are 10, 20, 40, 100, 150, 200, and 250 μmol·L -1 . After administration, incubate for 24 h under the conditions of 37 °C and 5% CO 2 . Then add an equal volume of ATP detection reagent to each well, gently shake, and equilibrate at room temperature for 10 min. Take the reaction solution in a 96-well fluorescence detection plate and measure the absorbance value of each well with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the cytotoxicity (%) = (LDH experimental release amount) × 100%.

[0097] Evaluation of repeated-dose drug toxicity using 3D liver models:

[0098] The 3D model will be administered isoniazid and amiodarone hydrochloride on the 4th day after inoculation, and a control group will be set up. The drug will be administered continuously 3 times at intervals of 24 hours. The administration concentration is the same as that listed in "Evaluating Drug Toxicity with 2D Liver Model". The supernatant will be taken 24 hours after each drug administration to measure the absorbance value. After repeating the drug administration 3 times, the cytotoxicity will be calculated according to the method in "Evaluating Drug Toxicity with 2D Liver Model" to obtain the effect of the drug on cell proliferation inhibition.

[0099] Amiodarone hydrochloride (AMI) is a drug that causes acute liver damage or hepatotoxicity. The hepatotoxicity shown under both 2D and 3D models is dose-dependent ( Figure 6 ); under the 3D culture model, the toxicity shown by amiodarone hydrochloride is time / drug administration frequency-dependent and positively dose-related. This may be related to the higher metabolic enzyme activity of the drug in the 3D culture model, resulting in faster drug metabolism. It may also be because the 3D cultured cells form cell spheres, simulating the in vivo physiological results and slowing down the direct penetration of the drug. This result shows that the 3D model has the potential for more repeated drug administrations.

[0100] Isoniazid (ISO) is a drug that causes chronic liver damage or hepatotoxicity. The obvious dose-dependent characteristics of its toxicity can only be shown under the 3D model (72 hours) ( Figure 7 ); in 2D culture, no cytotoxicity was detected when the isoniazid administration concentration was as high as 1 mmol / L, while its toxicity could be detected in the established 3D model, showing time-dependence and positive dose-relatedness.

[0101] In summary, the present invention designs a new method for constructing a three-dimensional liver microsphere model. For primary cynomolgus monkey hepatocytes, a specific culture method is designed, and a specific two-step culture process is adopted, which can realize the rapid preparation of a three-dimensional liver microsphere model only using primary cynomolgus monkey hepatocytes. In addition, by further controlling the culture process, a high-quality three-dimensional liver microsphere model can be prepared more quickly. The constructed model can better simulate the in vivo environment and the drug response, can perform long-term co-culture of different types of hepatocytes, and can maintain cell viability, metabolic activity, and related hepatocyte functions for several weeks. It can be used as an ideal model for studying the long-term effects such as drug metabolism and toxic reactions.

[0102] The applicant declares that the above is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A method for constructing a three-dimensional liver microsphere model, characterized in that: The method comprises: The primary hepatocytes of cynomolgus monkeys are inoculated into a culture container with an ultra-low adsorption surface, wherein the volume of the cell culture medium in the container is 10% to 30% of the total volume of the cell culture medium used for culture, and after the first culture, the remaining volume of the cell culture medium is supplemented and the second culture is performed to obtain the three-dimensional liver microsphere model.

2. The method for constructing a three-dimensional liver microsphere model according to claim 1, characterized in that: The density of the cynomolgus monkey primary hepatocytes after inoculation is 1500 to 3000 cells / well; Preferably, the first culture time is 24 to 48 hours.

3. The method for constructing a three-dimensional liver microsphere model according to claim 1 or 2, characterized in that: The total volume of the cell culture medium used in the culture is 0.1-0.3 mL.

4. The method for constructing a three-dimensional liver microsphere model according to any one of claims 1 to 3, characterized in that: The culture container with an ultra-low adsorption surface comprises an ultra-low adsorption well plate; Preferably, the ultra-low adsorption well plate comprises a multi-well cell culture plate.

5. The method for constructing a three-dimensional liver microsphere model according to any one of claims 1 to 4, characterized in that: The culture conditions of the first culture and the second culture are independently 36-38° C. and 4-6% CO 2 .

6. The method for constructing a three-dimensional liver microsphere model according to any one of claims 1 to 5, characterized in that: The cell culture medium contains glucose, glutamine, vitamin C, inorganic salts, epidermal growth factor and insulin.

7. The method for constructing a three-dimensional liver microsphere model according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: (1) Inoculating cynomolgus monkey primary hepatocytes in a culture container with an ultra-low adsorption surface, wherein the volume of the cell culture medium in the container is 10% to 30% of the total volume of the cell culture medium used for culture, and the density of the cynomolgus monkey primary hepatocytes after inoculation is 1500 to 3000 cells / well, and performing the first culture for 24 to 48 hours; (2) Supplementing the remaining volume of cell culture medium and performing a second culture to obtain the three-dimensional liver microsphere model.

8. A three-dimensional liver microsphere model, characterized in that: The three-dimensional liver microsphere model is constructed by the method for constructing a three-dimensional liver microsphere model according to any one of claims 1 to 7.

9. Use of the three-dimensional liver microsphere model according to claim 8 in drug development.

10. The use according to claim 9, characterized in that: The applications include any one of the evaluation of clinical drug-induced hepatotoxicity, prediction of liver retention / elimination of nanomedicines, risk assessment of drug-induced cholestasis, toxicity testing that allows long-term repeated administration, prediction of chronic drug toxic effects, or evaluation of drug clearance.

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