Establishment method of a 3D microsphere model based on HepaRG differentiated liver and application thereof

Abstract

This disclosure relates to a method for establishing a HepaRG-based differentiated liver 3D microsphere model and its applications. Specifically, this disclosure provides a chemically defined differentiation medium formulation that does not require DMSO and contains hepatocyte growth factor, dexamethasone, and hepatoprotectin M. Using this medium, HepaRG cells can be efficiently induced to form compact, long-lived 3D microspheres in ultra-low adsorption 96-well plates or in conjunction with high-throughput 3D printing technology. After differentiation and maturation, the model can stably express hepatocyte markers, key drug-metabolizing enzymes, and nuclear receptors at high levels. The liver 3D microsphere model established by this disclosure can effectively simulate hepatocyte function in vivo and is suitable for high-throughput drug hepatotoxicity screening. The results show a high degree of consistency with clinical hepatotoxicity data, and it has important application value in the field of drug safety evaluation.

Description

Technical Field

[0001] This disclosure relates to the field of biomedicine. Specifically, this disclosure relates to a method for constructing a 3D liver microsphere model derived from HepaRG and its application in drug hepatotoxicity detection. Background Technology

[0002] In drug development and safety evaluation, hepatotoxicity testing is a crucial step. HepaRG cells, due to their ability to express abundant phase I drug-metabolizing enzymes (such as CYP3A4), phase II enzymes, and drug transport proteins, are superior to commonly used hepatocyte models such as HepG2. They can more realistically simulate the metabolic process of drugs in the human liver, thereby more accurately detecting the toxicity of hepatotoxic substances that require metabolic activation (such as acetaminophen).

[0003] However, the traditional HepaRG two-dimensional (2D) differentiation model has significant drawbacks. First, its differentiation cycle is as long as approximately two weeks, and the differentiation process heavily relies on dimethyl sulfoxide (DMSO) induction. DMSO itself, as a chemical solvent, may be toxic to cells and interfere with the interpretation of experimental results. Second, the 2D culture model cannot simulate the three-dimensional microenvironment of cells in vivo, resulting in lower expression levels of liver-specific functions (such as drug-metabolizing enzymes and nuclear receptors) compared to the 3D culture model. Furthermore, the traditional culture model has poor compatibility with automated, high-throughput screening equipment, limiting its application in large-scale early drug screening.

[0004] Therefore, there is an urgent need in this field for a HepaRG hepatocyte model that can overcome DMSO dependence, has a short differentiation cycle, is more functionally mature, and is suitable for high-throughput screening. Summary of the Invention

[0005] One objective of this disclosure is to overcome the aforementioned shortcomings of existing HepaRG 2D differentiation models and provide a novel differentiation culture medium and method for forming mature 3D microspheres without DMSO induction. This disclosure also provides a HepaRG liver 3D microsphere model constructed based on the above method, which should have a longer survival time and higher hepatocyte maturity (manifested as high expression of key drug-metabolizing enzymes, transporters, and nuclear receptors). Another objective of this disclosure is to combine the 3D microsphere model with high-throughput preparation technologies (such as 3D bioprinting) to establish a highly efficient drug hepatotoxicity detection platform.

[0006] Three-dimensional culture medium In view of the above-mentioned needs in the art, this disclosure provides a three-dimensional culture medium comprising or consisting of the following: Basic culture medium 5 ng / mL to 50 ng / mL HGF 0.1μM to 1μM dexamethasone 5 ng / mL to 50 ng / mL of tumor suppressor M 10 U / ml to 1000 U / ml penicillin, and Streptomycin from 0.01 mg / ml to 1 mg / ml; The cell culture medium does not contain EGF.

[0007] In some embodiments of the three-dimensional culture medium, the basal culture medium is a hepatocyte maintenance medium selected from: Lonza HCM medium, Gibco hepatoZYME-SFM medium, and Sciencell HM medium.

[0008] In some implementations of the three-dimensional culture medium, the three-dimensional culture medium disclosed herein is specifically designed for the three-dimensional culture of HepaRG cells. The concentration ranges of each component have been optimized and screened to synergistically maintain the hepatocyte characteristics of HepaRG cells and promote the formation of functionally complete three-dimensional microspheres.

[0009] In some embodiments of the three-dimensional culture medium, the hepatocyte growth factor (HGF) is 5 ng / mL to 50 ng / mL; for example, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL; preferably 10 ng / mL.

[0010] In some implementations of the three-dimensional culture medium, dexamethasone is present at concentrations from 0.1 μM to 1 μM; for example, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, and 1.0 μM.

[0011] In some embodiments of the three-dimensional culture medium, the oncogene M (OSM) is 5 ng / mL to 50 ng / mL; for example, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL; preferably 20 ng / mL.

[0012] In some embodiments of the three-dimensional culture medium, penicillin is used at concentrations from 10 U / ml to 1000 U / ml; for example, 10 U / ml, 50 U / ml, 100 U / ml, 200 U / ml, 300 U / ml, 400 U / ml, 500 U / ml, 600 U / ml, 800 U / ml, 1000 U / ml; preferably 100 U / ml.

[0013] In some embodiments of the three-dimensional culture medium, streptomycin is used at a concentration of 0.01 mg / ml to 1 mg / ml; for example, 0.01 mg / ml, 0.05 mg / ml, 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1.0 mg / ml; preferably 0.1 mg / ml.

[0014] In some specific implementations of three-dimensional culture media, the three-dimensional culture media comprises or consists of the following: Basic culture medium 10 ng / mL HGF, 0.1 μM dexamethasone, 20 ng / mL of tumor suppressor M 100 U / ml penicillin, and 0.1 mg / ml streptomycin.

[0015] Three-dimensional culture method of hepatocytes This disclosure provides a method for three-dimensional culture of hepatocytes, including the step of contacting HepaRG cells with any of the aforementioned three-dimensional culture media.

[0016] The core of this disclosed culture method lies in utilizing the aforementioned specialized three-dimensional culture medium to provide a suitable three-dimensional growth microenvironment for HepaRG cells. This allows the cells to overcome the limitations of two-dimensional planar culture and spontaneously aggregate to form three-dimensional microspheres, thereby maintaining the mature function of hepatocytes. The "contact" can be achieved through conventional cell seeding methods (such as seeding HepaRG cells into a culture system containing the three-dimensional culture medium) or through 3D bioprinting. This method requires no complex equipment, rapidly achieves three-dimensional culture of HepaRG cells, and yields physiologically functional three-dimensional hepatocyte microspheres, suitable for both laboratory research and large-scale production.

[0017] In some specific implementation schemes of three-dimensional culture methods, the method includes the following steps: 1) The HepaRG cells are seeded on an ultra-low adsorption support (preferably an ultra-low adsorption plate) at a density of 200 to 1600 cells / well (preferably 400 cells / well); 2) Optionally, the HepaRG cells are centrifuged at 100g to 200g for 3 to 5 minutes; 3) Incubate HepaRG cells in the three-dimensional culture medium at 37°C and 5% CO2 for 7 to 20 days, preferably 9 to 14 days; 4) Obtain three-dimensional hepatocyte microspheres; Preferably, the three-dimensional culture medium is replaced every two days.

[0018] In step 1, the cell seeding density (200-1600 cells / well) is crucial. Too low a density will prevent cells from aggregating and forming complete microspheres, while too high a density will result in excessively large microspheres and internal cell hypoxia and necrosis. A seeding density of 400 cells / well is sufficient to form uniformly sized, fully functional three-dimensional microspheres. An ultra-low adsorption support (preferably an ultra-low adsorption plate) can prevent cell adhesion and promote spontaneous cell aggregation.

[0019] In step 2, centrifugation (100-200g, 3-5 minutes) is an optional step. Its purpose is to promote rapid cell aggregation and shorten the microsphere formation time. The defined centrifugation speed and time range can avoid excessive centrifugal force that could damage the cells.

[0020] In step 3, the culture conditions of 37℃ and 5% CO2 simulate the physiological environment of the human body. The incubation time (7-20 days, preferably 9-14 days) allows the cells to fully aggregate and differentiate, forming mature three-dimensional microspheres.

[0021] In specific implementation schemes of some three-dimensional culture methods, the obtained three-dimensional hepatocyte microspheres should meet the following requirements: - The expression of the three-dimensional hepatocyte microspheres is selected from the following markers: HNF4α, ALB, CYP3A4, CYP2E1, nuclear receptor PXR and combinations thereof; - The average particle size of the three-dimensional hepatocyte microspheres is 600 μm to 700 μm; - The three-dimensional hepatocyte microspheres are intact and without any clumps falling off.

[0022] This is the quality standard for three-dimensional hepatocyte microspheres obtained using the method disclosed herein, ensuring that the microspheres possess the desired hepatocyte function and morphology. HNF4α, ALB, CYP3A4, CYP2E1, and the nuclear receptor PXR are all hepatocyte-specific markers, and their expression reflects the maturity and functional integrity of hepatocytes within the microspheres: HNF4α expression indicates that hepatocytes have completed differentiation; ALB expression indicates that hepatocytes have normal synthetic function; CYP3A4 and CYP2E1 expression indicates that hepatocytes have normal drug metabolism function; and PXR expression indicates that hepatocytes have normal metabolic regulation capacity. The expression of these markers can be confirmed using conventional methods, such as immunofluorescence staining, Western blot, and RT-PCR.

[0023] Regarding the average particle size (600-700 μm): This should not be interpreted as every microsphere in the microsphere population being within the 600-700 μm range. It is a mathematical distribution concept, representing the arithmetic mean obtained after detecting a certain number of microspheres using a specific measurement method. For example: Microsphere morphology images are acquired under an inverted microscope or fluorescence microscope. Image analysis software such as ImageJ is used to measure the particle size of a certain number of morphologically intact microspheres, recording the diameter of each microsphere and calculating the average particle size. Alternatively, dynamic light scattering (DLS) or a cell counter (such as Countess II) can be used to determine the microsphere particle size distribution to obtain the average particle size and particle size distribution range.

[0024] In some specific implementation schemes of three-dimensional culture methods, any of the aforementioned methods are performed using a 3D bioprinter. Using a 3D bioprinter to achieve three-dimensional culture of hepatocytes can further improve the precision and controllability of the culture. The 3D bioprinter can print HepaRG cells and the aforementioned three-dimensional culture medium according to a preset structure and density, constructing a structured three-dimensional culture system. This avoids the problems of uneven cell distribution and large differences in microsphere size in traditional inoculation methods, enabling the large-scale production of three-dimensional hepatocyte microspheres. This method is suitable for drug development scenarios where the size, morphology, and distribution of microspheres are critical (such as artificial liver construction and the establishment of high-precision drug screening models), and can significantly enhance the application value of three-dimensional hepatocyte microspheres.

[0025] Three-dimensional hepatocyte microspheres and their applications This disclosure also provides a three-dimensional hepatocyte microsphere, which is obtained by the method of this disclosure.

[0026] This disclosure also provides the use of the obtained three-dimensional hepatocyte microspheres in the manufacture of artificial livers. An artificial liver is a physical entity used to replace or assist liver function, and its core component is hepatocytes with normal liver function. The three-dimensional hepatocyte microspheres prepared in this disclosure possess mature hepatocyte functions, capable of synthesizing albumin, metabolizing drugs, and clearing toxins. They are also morphologically stable and can be mass-produced, serving as the core functional unit for constructing bio-artificial livers. By combining these three-dimensional hepatocyte microspheres with scaffold materials for artificial livers, an artificial liver device with complete liver function can be constructed for the treatment of diseases such as acute liver failure and chronic liver disease.

[0027] This disclosure also provides the use of three-dimensional hepatocyte microspheres in establishing drug screening models for liver disease treatment or in vitro simulated liver function models. Traditional drug screening models often use two-dimensional cultured hepatocytes, whose functions differ significantly from in vivo hepatocytes, resulting in low accuracy in drug screening. In vitro simulated liver function models require hepatocytes with intact liver function as the core. The three-dimensional hepatocyte microspheres of this disclosure have similar physiological functions and histological structures to in vivo hepatocytes, and can accurately simulate drug metabolism, toxic reactions, and pathological states of in vivo hepatocytes. Therefore, they can be used to establish efficient and accurate drug screening models for liver disease treatment, for screening drugs with anti-hepatitis effects and low hepatotoxicity, reducing the cost and risk of drug development. At the same time, they can also be used to establish in vitro simulated liver function models to study the pathogenesis of liver disease, the repair mechanism of liver function damage, etc., providing theoretical support for the diagnosis and treatment of liver disease.

[0028] This disclosure also provides a method for establishing a drug screening model for liver disease treatment or an in vitro simulated liver function model, which includes the steps of any of the methods described above. The model constructed by this method more closely reflects the physiological state of hepatocytes in vivo, accurately reflecting the effects of drugs on hepatocytes and changes in liver function, and has higher accuracy and reliability compared to traditional two-dimensional models. Specifically, depending on the model's purpose (drug screening or liver function simulation), culture parameters (such as incubation time and cell density) can be adjusted to construct a targeted model, applicable to fields such as drug development and basic medical research. Attached Figure Description

[0029] Figure 1 The image shows a comparison of the bright-field morphology of HepaRG cells after being cultured for different days under different culture conditions in Example 1.

[0030] Figure 2 Example 2 demonstrates the expression levels of hepatocyte marker genes in HepaRG cells under different culture conditions, as detected by qPCR.

[0031] Figure 3Bright-field photographs of HepaRG microspheres prepared by high-throughput 3D printing technology in Example 3 are shown, demonstrating their uniform sphericity.

[0032] Figure 4 The image shows a fluorescence photograph of HepaRG microspheres cultured for 14 days after being stained with a live / dead dye, as shown in Example 3. The green fluorescence indicates surviving cells, demonstrating that the microspheres have good activity.

[0033] Figures 5A to 5C The results of drug hepatotoxicity testing (dose-response curves and IC50 values) using the 3D microsphere model of this disclosure are shown in Example 4. 3D Cell Viability Assay - Acetaminophen ( Figure 5A ), 3D cell viability assay - naphazoline ( Figure 5B ), 3D Cell Viability Experiment - Ambesentan ( Figure 5C ). Detailed Implementation

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. As used herein, the following terms have the following meanings.

[0035] As used in the specification and claims, the singular forms “a,” “an,” and “the” also include the plural forms, unless the context clearly specifies otherwise. Thus, for example, reference to “a cell” includes multiple cells, including mixtures thereof.

[0036] As used herein, the terms “comprising” or “containing” are intended to indicate that a composition and method includes the listed elements, but does not exclude other elements. When used to define compositions and methods, “consisting substantially of” means excluding other elements that are of any significance to the combination for the stated purpose. Thus, a composition consisting substantially of the elements defined herein does not exclude other materials or steps that do not materially affect the claimed essential and novel characteristics. “Containing” means excluding trace elements and substantial method steps that are other components. Embodiments defined by each of these transforming terms are within the scope of this disclosure.

[0037] As used in this article, the term "separated" means separated from cells and other components.

[0038] As used in this article, isolated cells are cells isolated from tissues or cells with dissimilar phenotypes or genotypes.

[0039] As described in this article, "culture medium" refers to the components required for cell growth, survival, and differentiation in vitro; it includes all common culture media used in the field of cell culture for cultivation and differentiation. Culture media used for liver microspheres and liver organoids are typically basal media containing carbon sources, nitrogen sources, and trace elements.

[0040] The term "Hepatocyte Maintenance Medium" (HMM) used in this article refers to a type of culture medium for in vitro cultured hepatocytes (including primary hepatocytes, hepatocyte lines, and differentiated hepatocyte-like cells). Its function is to maintain the survival, morphological integrity, and basic physiological functions of hepatocytes, and it can also serve as a base formulation for optimizing hepatocyte differentiation media. The base formulation typically uses Williams' Medium E, DMEM / Ham's F12 (1:1), etc., as a base, and adds essential nutrients for hepatocytes, including: amino acids, vitamins, glucose, and other basic nutrients; transferrin, insulin, and selenium to maintain cell metabolism and growth; bovine serum albumin (BSA) or human serum albumin (HSA) to provide osmotic stability and carrier function; and antioxidants (such as glutathione) to reduce oxidative stress damage during in vitro culture. Unlike hepatocyte differentiation media, the core objective of maintenance media is to maintain function rather than promote differentiation: strong differentiation inducers (such as DMSO or specific cytokine combinations) are not added.

[0041] As described in this article, “differentiation” refers to the phenomenon of the specific embodiment of cell structure or function during cell division, proliferation, and growth.

[0042] As described in this article, the term "passage" refers to the process of culturing a single cell or organoid in a healthy state for an extended period of time, and then transferring it to a new culture medium or continuing the culture. Each division of the cell population or change of the culture vessel / medium is called a passage.

[0043] As used in this article, 3D hepatocyte spheroids refer to spherical, three-dimensional aggregates of hepatocytes (including primary hepatocytes, hepatocyte lines such as HepaRG and HepG2, or hepatocyte-like cells differentiated from stem cells) that spontaneously aggregate under specific in vitro culture conditions. The cells do not grow in a single layer adhering to the culture surface; instead, they form a spherical structure through multi-layered stacking. Direct contact is established between cells through tight junctions and gap junctions, while extracellular matrix (ECM) components are deposited within the spheroids. Compared to two-dimensional (2D) adherent hepatocytes, hepatocytes in 3D spheroids can better maintain or even enhance core functions, such as high expression of drug-metabolizing enzymes and responses to drugs and toxins that more closely resemble in vivo physiological states. Typically, techniques such as ultra-low adsorption culture plates (to prevent cell adhesion), hanging drop culture, spin culture, or 3D bioprinting, combined with specialized 3D culture media, are needed to promote the spontaneous aggregation of cells into uniformly sized spheroids.

[0044] HepaRG cells: a cell line derived from human liver cancer that can differentiate into hepatocyte-like cells and bile duct epithelial cell-like cells with mature liver function.

[0045] Ultra-low adsorption support: The surface is treated (such as with a hydrophilic coating) to reduce the adsorption force between cells and the support surface, preventing cells from adhering to the wall and thus promoting the spontaneous aggregation of cells in the culture system to form three-dimensional aggregates (microspheres). Commonly used materials include ultra-low adsorption plates and culture dishes, which are readily available on the market.

[0046] Table 1. Information on the main reagents and equipment used in the examples Example 1: 3D Differentiation Culture of HepaRG Cells This example compares the effects of different culture media on the 3D differentiation of HepaRG cells. The experimental groups are as follows: Culture medium 1: RPMI-1640 + 100 U / ml penicillin and 0.1 mg / ml streptomycin were used as the basal culture medium (culture medium 1). Culture medium 2: Add 2.5 μM CHIR99021 (culture medium 2) to culture medium 1. Culture medium 3 (the culture medium disclosed herein): Lonza hepatocyte culture medium was used with its specific additives (without EGF), and supplemented with 10 ng / mL HGF, 0.1 μM dexamethasone, 20 ng / mL hemostatin M, 100 U / ml penicillin, and 0.1 mg / ml streptomycin.

[0047] HepaRG cells were seeded at a density of 400 cells per well in 96-well ultra-low adsorption plates. Cell aggregation was promoted by centrifugation at 150 × g for 5 minutes. The cells were then cultured in three different culture media, with fresh culture medium replaced every 2 days.

[0048] The results are as follows Figure 1 As shown: Cells using culture medium 1 and culture medium 2 showed obvious death (microspheres turned black and disintegrated) after 10 to 13 days of culture; while cells using culture medium 3 of this disclosure formed 3D microspheres with long survival time, compact structure and high density, and maintained good morphology throughout the entire culture cycle.

[0049] Example 2: Identification of Liver Microglobule Function To evaluate the differentiation effect, undifferentiated HepaRG cells, DMSO-induced 2D differentiated cells, 3D microspheres using culture medium 2, and 3D microspheres using culture medium 3 of this disclosure were collected, and the expression levels of key genes in hepatocytes were detected by qPCR.

[0050] like Figure 2 As shown, compared with other groups, the 3D microspheres differentiated using the culture medium disclosed in this paper showed the highest levels of mRNA expression of hepatocyte markers (HNF4α, ALB), drug-metabolizing enzymes (CYP3A4, CYP2E1), and nuclear receptor PXR, indicating that they had the highest hepatocyte maturity.

[0051] Example 3: High-throughput preparation and activity verification of liver microspheres To verify the feasibility of high-throughput preparation, a high-throughput 3D bioprinter was used to precisely print HepaRG cells mixed with matrix gel into a 96-well ultra-low adsorption plate.

[0052] like Figure 3 As shown, the microspheres prepared by this method are uniform in size and have a high single-sphere rate (approximately 90%). After culturing the microspheres for 14 days, staining for live and dead cells was performed, and fluorescence images were obtained (…). Figure 4 The results showed that the vast majority of cells exhibited green fluorescence (live cells), demonstrating that the microspheres maintained high activity after long-term culture.

[0053] Example 4: Application of drug hepatotoxicity detection Matured 3D microspheres were exposed to different concentrations of known hepatotoxic drugs: acetaminophen (APAP), nefazodone (Nef), and ambexin (Amb) for 7 days. Cell viability was assessed using the 3D CellTiter-Glo® (3D-CTG) assay.

[0054] The results showed that nefazodone had an IC50 value less than 10 μM, exhibiting strong hepatotoxicity; acetaminophen had an IC50 of approximately 500 μM, indicating moderate toxicity; and ambesentan had an IC50 greater than 50 μM, indicating low toxicity. These results are highly consistent with the clinical hepatotoxicity risk of these three drugs, demonstrating the reliability and application value of the published model in predicting drug hepatotoxicity. Figures 5A to 5C ).

[0055] The technical solution disclosed herein has the following features: DMSO-free: Completely avoids the potential toxicity of DMSO and its interference with experiments, making the test results more reliable.

[0056] High functional maturity: The 3D microsphere model constructed in this disclosure has significantly higher expression levels of key hepatocyte markers (HNF4α, ALB), drug-metabolizing enzymes (CYP3A4, CYP2E1) and nuclear receptor (PXR) than the traditional 2D model, and is closer to the functional state of hepatocytes in vivo.

[0057] Suitable for high-throughput screening: The method disclosed herein is highly compatible with standardized formats such as multi-well plates and is easy to automate, which greatly improves the efficiency of large-scale hepatotoxicity screening in the early stages of drug development.

[0058] High predictive accuracy: The IC50 values ​​obtained by testing known hepatotoxic drugs (such as nefazodone and acetaminophen) using the model disclosed in this publication are highly consistent with the clinical risk of hepatotoxicity, demonstrating its good predictive value.

[0059] Compared to existing technologies (such as CN111004770B), the method disclosed herein uses fewer components and does not involve staged culture. While not limited to a specific theory, it can be interpreted as stimulating hepatocytes to differentiate bidirectionally to obtain liver microspheres containing hepatocytes and bile duct cells. However, existing technologies obtain liver organoids from multiple lineages through the temporal and staged development of pluripotent stem cells. The cells used in this disclosure are HepaRG cells, which possess only bidirectional differentiation potential for hepatocytes and bile duct cells, effectively avoiding interference from other lineage cell components. Therefore, from the perspectives of cell source, differentiation principle, and culture results, CN111004770B and this disclosure do not belong to the same type of method.

Claims

1. A three-dimensional culture medium comprising or consisting of the following: Basic culture medium 5 ng / mL to 50 ng / mL HGF 0.1μM to 1μM dexamethasone 5 ng / mL to 50 ng / mL of tumor suppressor M 10 U / ml to 1000 U / ml penicillin, and Streptomycin from 0.01 mg / ml to 1 mg / ml; The cell culture medium does not contain EGF; in: The basal culture medium is a hepatocyte maintenance culture medium, which is selected from: Lonza HCM medium, Gibco hepatoZYME-SFM medium, and Sciencell HM medium.

2. The three-dimensional culture medium according to claim 1, comprising or consisting of the following: Basic culture medium 10 ng / mL HGF, 0.1 μM dexamethasone, 20 ng / mL of tumor suppressor M 100U / ml penicillin, and 0.1 mg / ml streptomycin.

3. A method for three-dimensional culture of hepatocytes, comprising the following steps: Contact HepaRG cells with the three-dimensional culture medium as described in claim 1 or 2.

4. The method according to claim 3, comprising the steps of: 1) The HepaRG cells are seeded on an ultra-low adsorption support (preferably an ultra-low adsorption plate) at a density of 200 to 1600 cells / well (preferably 400 cells / well); 2) Optionally, the HepaRG cells are centrifuged at 100g to 200g for 3 to 5 minutes; 3) Incubate HepaRG cells in the three-dimensional culture medium at 37°C and 5% CO2 for 7 to 20 days, preferably 9 to 14 days; 4) Obtain three-dimensional hepatocyte microspheres; Preferably, the three-dimensional culture medium is replaced every two days.

5. The method according to claim 4, wherein: The expression of the three-dimensional hepatocyte microspheres was selected from the following markers: HNF4α, ALB, CYP3A4, CYP2E1, nuclear receptor PXR and combinations thereof; The average particle size of the three-dimensional hepatocyte microspheres is 600 μm to 700 μm; The three-dimensional hepatocyte microspheres were intact and without any clumps falling off.

6. The method according to claim 3, wherein it is performed using a 3D bioprinter.

7. A three-dimensional hepatocyte microsphere obtained by the method described in any one of claims 3 to 6.

8. Use of the three-dimensional hepatocyte microspheres of claim 7 in the manufacture of an artificial liver.

9. The use of the three-dimensional hepatocyte microspheres according to claim 7 in establishing a screening model for liver disease treatment drugs or an in vitro simulated liver function model.

10. A method for establishing a drug screening model for liver disease treatment or an in vitro simulated liver function model, comprising the method described in any one of claims 3 to 6.

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