Construction method of three-dimensional liver micro-tissue model and hepatotoxicity assessment application of three-dimensional liver micro-tissue model

Abstract

The invention discloses a construction method of a three-dimensional liver micro-tissue model and hepatotoxicity assessment application of the three-dimensional liver micro-tissue model, and belongs to the technical field of environmental science and engineering. The 3D liver micro-tissue model constructed by the invention consists of three cell lines, namely HepaRG (parenchymal hepatic cells), THP-1 (kupffer cells) and hTERT-HSC (hepatic stellate cells), so that a real liver environment can be better simulated, and the defects of a traditional model are overcome; particularly, the occurrence of hepatic fibrosis relates to complex interaction among a plurality of cell lines, however, most of the previous models for evaluating the potential of hepatic fibrosis by chemical substances are lack of consideration of interaction among multiple types of cells no matter whether hepatic parenchymal cell lines or hepatic stellate cell lines are used, and the 3D hepatic micro-tissue model takes the factor into account, so that the potential of hepatic fibrosis cannot be evaluated. Key hepatic fibrosis events caused by hepatic fibrosis substances can be copied, and a more suitable test platform is provided for evaluating the potential of chemical substance hepatic fibrosis.

Description

Technical Field

[0001] The present invention relates to the field of environmental science and engineering technology, and specifically to a method for constructing a three-dimensional liver microtissue model and its application in liver toxicity assessment. Background Art

[0002] Human liver diseases caused by environmental chemicals are receiving increasing attention. There are many types of chemicals, including dioxins, polychlorinated biphenyls, microcystis toxins, disinfection by-products, heavy metals, etc., which can enter the human body through the food chain or direct consumption and affect the liver. According to statistics, more than 50% of clinical cases of liver damage are caused by drugs, and in the past 20 years, industrial chemicals, biopesticides and other chemicals have also been proven to cause liver damage. A large number of chemicals are added every year, and the toxicity information of many chemicals is missing, and the harm to the human body is unknown. Therefore, it is urgent to quickly and accurately evaluate the hepatotoxicity of environmental chemicals.

[0003] Animal models can reflect the effects of chemical exposure at the tissue and individual levels, but there are species differences. For example, there are species differences in the cytochrome P450 metabolic process of rodents, and different sensitivities to liver toxic substances. Although the zebrafish model has advantages, the poisoning method leads to inaccurate determination of chemical absorption, which is different from the human body's absorption capacity and can easily misjudge the harm of chemicals to the human body.

[0004] Although the classic two-dimensional monolayer culture system is simple to operate and can be used for high-throughput toxicity testing, the biochemical reactions and interactions of the cells gradually disappear as the culture time increases, and it cannot accurately reflect the true toxicity under long-term repeated dose exposure in vivo. For example, when HepG2 and HepaRG cell lines are cultured in two dimensions, there is a large gap in phenotype and liver function compared with in vivo hepatocytes.

[0005] Three-dimensional (3D) in vitro cell culture models are an important advancement in simulating the physiological functions of the liver in vitro. They can better preserve the metabolic function of hepatocytes and reproduce the complex environment of multiple cell interactions in the liver. They have been applied in drug toxicity assessment and are gradually being introduced into the field of environmental toxicology. 3D hepatotoxicity testing models include sandwich culture, spheroid models, bioreactors, organ chips, and 3D bioprinting. However, each of these models has certain limitations. For example, the sandwich culture method may suffer from leakage, bile duct damage, and bile stasis during long-term culture; the bioreactor requires a huge amount of cells and is not suitable for high-throughput toxicity testing; and 3D bioprinting has problems such as poor compatibility between cells and biocompatible materials and low printing speed.

[0006] In vitro models cannot accurately simulate the in vivo liver environment: Although the classic two-dimensional monolayer culture system is easy to operate and can be used for high-throughput testing, there is a problem that cellular biochemical reactions and interactions disappear with culture time, and it cannot simulate the true toxic effects of long-term repeated dose exposure in vivo. In addition, the existing in vitro hepatocyte culture models that can be tested in high-throughput have their hepatocyte metabolism and detoxification functions significantly reduced in a short period of time. The culture system is simple and it is difficult to simulate the in vivo liver function. Summary of the Invention

[0007] The purpose of the present invention is to provide an innovative three-dimensional liver microtissue model, specifically to use three human cell lines, HepaRG, THP-1 and hTERT-HSC, to construct a 3D liver microtissue model through a micro-modeling method, so that the model can stably express toxicology-related genes and maintain a stable morphology within 14 days, thereby improving its physiological relevance to the human liver; comprehensively evaluate the gene expression and functional enrichment of the model at the transcriptome level, and by comparing the gene expression data with human liver tissue, HepG2 and HepaRG cells, clarify its application potential in reflecting the complex in vitro liver environment, evaluating the hepatotoxicity of chemicals and liver fibrosis; use this model in combination with transcriptomic testing to study the hepatotoxicity mechanism of environmental chemicals, provide data support for understanding the toxicity mechanism of chemicals to the human body, and then provide new methods and ideas for the hepatotoxicity assessment of chemicals, promote the development of the field of environmental toxicology, and solve the shortcomings of existing technologies in the hepatotoxicity assessment of environmental chemicals.

[0008] The present invention is achieved through the following technical solutions:

[0009] The present invention provides a method for constructing a three-dimensional liver microtissue model, comprising the following steps:

[0010] Cell preparation: three human cell lines are provided, including HepaRG hepatocytes, THP-1 monocytes and macrophages, and hTERT-HSC hepatic stellate cells.

[0011] Cell pretreatment: the HepaRG cells are revived, passaged, and differentiated for later use; the THP-1 cells are revived, passaged, and differentiated into macrophages; and the hTERT-HSC cells are revived and passaged;

[0012] For cell mixing, the three cell lines were mixed at a ratio of HepaRG:THP-1:hTERT-HSC = 1000:500:500;

[0013] Three-dimensional culture, the mixed cell suspension was added to In this system, after culturing in aggregation medium for 3 days, spherical three-dimensional liver microtissues with a diameter of 250-300 μm were formed, in which THP-1 and hTERT-HSC cells were located in the center of the spheroid and HepaRG cells surrounded the outer layer.

[0014] Furthermore, the differentiation medium contains 2% DMSO, 50 ng / mL HGF and 20 ng / mL OSM.

[0015] Furthermore, the THP-1 cells were differentiated by stimulating them with 100 ng / mL PMA for 24 hours, and the HepaRG cells were cultured in a differentiation medium containing 2% dimethyl sulfoxide (DMSO) for 14 days.

[0016] Furthermore, the aggregation culture medium comprises DMEM / F12 basal culture medium, 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 20 ng / mL epidermal growth factor (EGF) and 10 ng / mL fibroblast growth factor (FGF).

[0017] A method for constructing a three-dimensional liver microtissue model and application of the constructed three-dimensional liver microtissue model in the hepatotoxicity assessment of environmental chemicals include the following steps:

[0018] (a) The constructed 3D liver microtissues were exposed to the test chemicals and cultured for 14 days;

[0019] (b) detecting the morphological stability, gene expression profile, metabolic function indicators, and liver fibrosis-related markers of the three-dimensional liver microtissue at different time points during the exposure period;

[0020] (c) analyzing the hepatotoxicity and liver fibrosis potential of the chemical substances based on the test results.

[0021] Furthermore, the metabolic function indicators include albumin secretion, urea synthesis and cellular ATP level.

[0022] Furthermore, the liver fibrosis-related markers include gene or protein expression levels of αSMA, Col1α1, TNF-α and IL-6.

[0023] Furthermore, the gene expression profiling analysis includes:

[0024] (i) Detecting the expression of 1200 human genes using simplified transcriptome sequencing technology;

[0025] (ii) Time series clustering analysis was performed using the Mfuzz algorithm to identify differentially expressed genes;

[0026] (iii) The biological processes of differentially expressed genes were analyzed by GO function enrichment and KEGG pathway enrichment.

[0027] Furthermore, the chemical substances to be tested include TCDD, thioacetamide (TAA), benzopyrene (BaP) or 3,3',4,4',5-pentachlorobiphenyl (PCB126).

[0028] Furthermore, the morphological stability assessment includes: observing the diameter of the spheroids under a microscope on days 0, 3, 5, 7, 10, 12, and 14 of culture, and confirming that the diameter is maintained within the range of 250-300 μm.

[0029] The present invention has the following beneficial effects:

[0030] 1. The present invention can simulate the real liver environment. The constructed 3D liver microtissue model is composed of three cell lines, HepaRG, THP-1 and hTERT-HSC, in a specific proportion, which can better simulate the real liver environment. Compared with the 2D monolayer cultured HepG2 and HepaRG cell lines, its gene expression is more similar to that of the human liver. In terms of the assessment of liver fibrosis potential, the genes involved in the liver fibrosis pathway expressed are more comprehensive, with 95 genes, 34 of which were not detected in the 2D monolayer cultured HepaRG cell line. This shows that the model has significant advantages in reflecting the complex in vitro liver environment and predicting the liver fibrosis potential of chemical substances.

[0031] 2. The model is highly stable. During the 14-day culture process, the 3D liver microtissue morphology is stable, and the spheroid diameter ranges from 250-300μm without significant changes. From the gene expression level, it stably expresses genes related to toxicological pathways, and the expression levels of genes related to responding to external stimuli and maintaining cell homeostasis are high. Through Pearson correlation analysis, the similarity of gene expression at each time point is more than 85%, indicating that gene expression is at a stable level, providing a reliable basis for the subsequent use of this model for hepatotoxicity testing of environmental chemicals.

[0032] 3. It can clarify the characteristics of gene expression and biological pathways. Transcriptome analysis of 3D liver microtissues revealed that its gene expression and biological pathways showed clear characteristics and patterns. 866 genes were expressed simultaneously at 7 time points, and these genes were enriched in biological processes such as cell circulation and metabolism; 38 genes with high expression levels were enriched in biological processes such as detoxification and maintaining homeostasis. The genes were further divided into 8 clusters to analyze their dynamic changes, and it was found that the changing trends of genes in different clusters were related to specific biological processes, which helps to deeply understand the physiological processes of liver cells and the mechanisms by which chemicals affect them.

[0033] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a diagram of the liver microtissue preparation process;

[0035] Figure 2 For the technology roadmap;

[0036] Figure 3 This is a list of 363 genes in liver microtissue;

[0037] Figure 4 The morphology of liver microtissues during 14 days of culture. DETAILED DESCRIPTION

[0038] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0039] See also Figure 1-2 The present invention provides a technical solution: a method for constructing a three-dimensional liver microtissue model, comprising the following steps:

[0040] For cell preparation, we provide three human cell lines, including HepaRG hepatocytes, THP-1 monocyte-macrophages, and hTERT-HSC hepatic stellate cells.

[0041] Cell pretreatment: the HepaRG cells are revived, passaged, and differentiated for later use; the THP-1 cells are revived, passaged, and differentiated into macrophages; and the hTERT-HSC cells are revived and passaged for culture.

[0042] The cells were mixed at a ratio of HepaRG:THP-1:hTERT-HSC = 1000:500:500.

[0043] Three-dimensional culture, the mixed cell suspension was added to In this system, after culturing in aggregation medium for 3 days, spherical three-dimensional liver microtissues with a diameter of 250-300 μm were formed, in which THP-1 and hTERT-HSC cells were located in the center of the spheroid and HepaRG cells surrounded the outer layer.

[0044] The differentiation medium contained 2% DMSO, 50 ng / mL HGF and 20 ng / mL OSM. The THP-1 cells were differentiated by stimulating THP-1 cells with 100 ng / mL PMA for 24 hours. The HepaRG cells were cultured in a differentiation medium containing 2% dimethyl sulfoxide (DMSO) for 14 days. The aggregation medium contained DMEM / F12 basal medium, 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 20 ng / mL epidermal growth factor (EGF) and 10 ng / mL fibroblast growth factor (FGF).

[0045] Construction of a three-dimensional liver microtissue model: Three cell lines were selected: undifferentiated HepaRG (primary human liver cancer cells), THP-1 (monocyte macrophages), and hTERT-HSC (human hepatic stellate cells). The HepaRG cell line is ready for use after a series of standard processes such as recovery, passaging, and differentiation; the THP-1 cell line needs to be recovered, passaged, and differentiated under specific conditions; the hTERT-HSC cell line is also recovered and passaged for subsequent experiments. By accurately calculating the amount of cells used, the three differentiated cells are mixed in a specific ratio (each liver microtissue consists of 1000 HepaRG, 500 THP-1, and 500 hTERT-HSC). Utilization

[0046] The system is constructed through the steps of preliminary preparation, mold preparation, cell mixing, etc., and a spherical liver microtissue with a diameter of about 300μm is formed after culturing in the aggregation culture medium for 3 days. The center of the spheroid is clustered with THP-1 and hTERT-HSC, and surrounded by HepaRG cells. The 3D liver microtissue model is constructed using three human cell lines: HepaRG, THP-1 and hTERT-HSC. Through specific cell culture, processing and mixing methods, in the 3D Petri Spheroid liver microtissues with specific structures are cultured in the system to simulate the real liver environment.

[0047] 1. Evaluation of benzopyrene (BaP) hepatotoxicity:

[0048] Model preparation: HepaRG, THP-1 and hTERT-HSC cells were recovered, passaged and differentiated, and then mixed at a ratio of 1000 HepaRG, 500 THP-1 and 500 hTERT-HSC per liver microtissue. The system was cultured in aggregation medium for 3 days to form 3D liver microtissue spheroids.

[0049] Experimental Grouping: Multiple BaP concentration gradients (1.98 nM, 19.82 nM, 198.16 nM, 1981.64 nM, 3963.28 nM, and 19816.4 nM) were set up, with DMSO as the solvent control and a blank control group. Liver microtissue samples were collected at different time points during the 14-day exposure period.

[0050] Effect:

[0051] 1: BaP produced dose-dependent cytotoxicity. 19.82nM and 3.96nM BaP significantly reduced the viability of liver microtissue cells, and 1.98nM BaP inhibited cell survival by approximately 50%. 19.82nM, 3.96nM, and 1.98nM BaP significantly reduced albumin release from liver microtissues.

[0052] 2: BaP caused the upregulation of cytochrome enzyme CYP1A1 gene expression and significantly upregulated AHR gene expression; the upregulation of liver fibrosis markers αSMA and Col1α1 gene expression was verified by immunohistochemical analysis results, indicating that BaP has the potential to induce liver fibrosis.

[0053] 3: After 14 days of BaP exposure, 363 genes in liver microtissues were identified as differentially expressed genes, and the foldchange values were significantly positively or negatively correlated with the exposure concentration (P-value < 0.05). Among them, 180 differentially expressed genes were upregulated, and 183 genes were downregulated. Upregulated differentially expressed genes were enriched in retinol metabolism, drug metabolism, xenobiotic metabolism regulated by P450 enzymes, the p53 signaling pathway, and pathways involved in various carcinogenesis processes; downregulated differentially expressed genes were enriched in pathways such as osteoclast differentiation regulated by immune signaling pathways, regulation of lipolysis in adipocytes, and ubiquitin-mediated protein degradation. These results indicate that BaP can affect the normal function of liver microtissues through multiple pathways, and this model comprehensively and deeply reveals the mechanism of BaP's toxic effects.

[0054] A method for constructing a three-dimensional liver microtissue model and application of the constructed three-dimensional liver microtissue model in the hepatotoxicity assessment of environmental chemicals include the following steps:

[0055] (a) The constructed 3D liver microtissues were exposed to the test chemicals and cultured for 14 days;

[0056] (b) detecting the morphological stability, gene expression profile, metabolic function indicators, and liver fibrosis-related markers of the three-dimensional liver microtissue at different time points during the exposure period;

[0057] (c) analyzing the hepatotoxicity and liver fibrosis potential of the chemical substances based on the test results.

[0058] Metabolic function indicators include albumin secretion, urea synthesis, and cellular ATP levels. Liver fibrosis-related markers include the gene or protein expression levels of αSMA, Col1α1, TNF-α, and IL-6.

[0059] Gene expression profiling includes:

[0060] (i) Detecting the expression of 1200 human genes using simplified transcriptome sequencing technology;

[0061] (ii) Time series clustering analysis was performed using the Mfuzz algorithm to identify differentially expressed genes;

[0062] (iii) The biological processes of differentially expressed genes were analyzed by GO function enrichment and KEGG pathway enrichment.

[0063] The model was applied to the hepatotoxicity testing of environmental chemicals, including TCDD, thioacetamide (TAA), benzo[a]pyrene (BaP), or 3,3',4,4',5-pentachlorobiphenyl (PCB126). Using typical environmental chemicals such as TCDD as the study target, liver microtissues were exposed to a culture medium containing a specific concentration of TCDD (1.0 ng / mL) for 14 days. Samples were collected at different time points (3, 5, 7, 10, 12, and 14 days) for transcriptome analysis, analyzing differentially expressed genes and enriched biological processes. The results showed that the number of differentially expressed genes induced by TCDD varied cyclically over time, and the gene enrichment at different exposure time points reflected the toxic effects of TCDD, ranging from detoxification to interference with cell fate and DNA replication. The model was also used to test the hepatotoxicity and liver fibrosis potential of four typical environmental chemicals: thioacetamide (TAA), benzo[a]pyrene (BaP), 3,3',4,4',5-pentachlorobiphenyl (PCB126), and TCDD. Evaluation was conducted based on biological activity (ATP test, albumin detection test), molecular markers (AhR, CYP1A1, αSMA and col1α1), histological changes (Sirius red staining) and immunohistochemistry. It was found that TAA and BaP had the potential to induce liver fibrosis, while PCB126 and TCDD had no detectable liver fibrosis potential under the set parameters.

[0064] Morphological stability assessment included observing the spheroid diameter under a microscope on culture days 0, 3, 5, 7, 10, 12, and 14 and confirming that it remained within the range of 250-300 μm.

[0065] During the 14-day culture period, the morphological changes of liver microtissues were monitored regularly (days 0, 3, 5, 7, 10, 12, and 14). Figure 4Figures a, b, c, d, e, f, and g correspond to liver microtissue morphology observed on days 0, 3, 5, 7, 10, 12, and 14 after preparation, respectively. Figure h shows the morphology of the liver microtissue under confocal microscopy after 14 days of culture. Green represents HepaRG, red represents THP-1, and blue represents hepatic stellate cells (HSCs). The results show that the liver microtissue morphology is stable during culture, with the diameter of the spheroids maintained between 250 and 300 μm. Gene expression analysis of the liver microtissues was performed using a simplified transcriptome analysis, encompassing RNA extraction, library construction, and sequencing, counting and classifying 1200 human genes. Gene expression was assessed using the Mfuzz transcriptome time series processing method, and GO function enrichment and KEGG pathway enrichment analyses were performed. Results showed that the 3D liver microtissues stably expressed genes associated with toxicological pathways over the 14 days of culture. Genes associated with responses to external stimuli and maintenance of cellular homeostasis were highly expressed, demonstrating an overall stable state. At the same time, the gene expression data of 3D liver microtissues were compared with those of human liver tissue, HepG2 and HepaRG cells, and it was found that the gene expression of 3D liver microtissues was more similar to that of human liver, and could summarize the key events of liver fibrosis.

[0066] 2. Evaluation of Thioacetamide (TAA) Hepatotoxicity:

[0067] Model construction: HepaRG, THP-1 and hTERT-HSC cells were recovered, passaged and differentiated, and then mixed at a ratio of 1000 HepaRG, 500 THP-1 and 500 hTERT-HSC per liver microtissue. The system was cultured in aggregation medium for three days to form 3D liver microtissue spheroids. Experimental groups were divided into groups with different TAA concentrations (0.5mM, 1.0mM, 2.0mM, 4.0mM, and 8.0mM). Culture medium without TAA served as a blank control group, and a solvent control group was also established (if solvent was required for TAA dissolution). During the 14-day exposure period, liver microtissue samples were collected regularly (on days 3, 7, 10, and 14) for subsequent testing.

[0068] Effect:

[0069] 1: As TAA concentration increased, the activity of liver microtissue cells gradually decreased, with a significant decrease at concentrations of 4.0mM and 8.0mM. Microscopic observation revealed significant changes in the morphology of liver microtissues in the high-concentration TAA treatment group, with loose connections between cells and signs of cell necrosis.

[0070] 2. TAAs significantly upregulated the expression of αSMA and Col1α1 genes in liver microtissues in a concentration-dependent manner. Western blotting and immunohistochemistry further confirmed the elevated expression of αSMA and Col1α1 proteins, indicating that TAAs can induce liver fibrosis. Furthermore, the expression of genes associated with inflammatory factors, such as TNF-α and IL-6, was significantly upregulated, suggesting that inflammation is involved in TAA-induced liver injury.

[0071] 3. Detection of liver microtissue indicators related to liver metabolic function, such as albumin synthesis and urea synthesis capacity. The results showed that as TAA concentration increased, albumin and urea synthesis decreased significantly, indicating that TAA severely inhibited the metabolic function of liver microtissue, affecting the liver's normal detoxification and substance synthesis functions.

[0072] The preferred embodiments of the present invention disclosed above are intended only to help illustrate the present invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the present invention to the specific embodiments described. Obviously, many modifications and variations are possible based on the content of this specification. These embodiments are selected and described in detail in this specification to better explain the principles and practical applications of the present invention, thereby enabling those skilled in the art to better understand and utilize the present invention. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for constructing a three-dimensional liver microtissue model, characterized in that: The following steps are involved: Cell preparation: three human cell lines are provided, including HepaRG hepatocytes, THP-1 monocytes and macrophages, and hTERT-HSC hepatic stellate cells. Cell pretreatment: the HepaRG cells are revived, passaged, and differentiated for later use; the THP-1 cells are revived, passaged, and differentiated into macrophages; and the hTERT-HSC cells are revived and passaged; For cell mixing, the three cell lines were mixed at a ratio of HepaRG:THP-1:hTERT-HSC = 1000:500:500; Three-dimensional culture, the mixed cell suspension was added to In this system, after culturing in aggregation medium for 3 days, spherical three-dimensional liver microtissues with a diameter of 250-300 μm were formed, in which THP-1 and hTERT-HSC cells were located in the center of the spheroid and HepaRG cells surrounded the outer layer.

2. The method for constructing a three-dimensional liver microtissue model according to claim 1, characterized in that: The differentiation medium contains 2% dimethyl sulfoxide (DMSO), 50 ng / mL hepatocyte growth factor (HGF), and 20 ng / mL interleukin-6-like cytokine (OSM).

3. The method for constructing a three-dimensional liver microtissue model according to claim 1, wherein: The THP-1 cells were differentiated by stimulating them with 100 ng / mL PMA (phorbol 12-myristate 13-acetate) for 24 hours, and the HepaRG cells were cultured in a differentiation medium containing 2% DMSO for 14 days.

4. The method for constructing a three-dimensional liver microtissue model according to claim 1, characterized in that: The aggregation medium contains DMEM / F12 basal medium, 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 20 ng / mL epidermal growth factor (EGF) and 10 ng / mL fibroblast growth factor (FGF).

5. An application of a three-dimensional liver microtissue model constructed by the method for constructing a three-dimensional liver microtissue model according to any one of claims 1 to 4 in the assessment of hepatotoxicity of environmental chemicals, characterized in that: The following steps are involved: (a) exposing the three-dimensional liver microtissue constructed by the method for constructing a three-dimensional liver microtissue model according to any one of claims 1 to 4 to a chemical substance to be tested and culturing the mixture for 14 days; (b) detecting the morphological stability, gene expression profile, metabolic function indicators, and liver fibrosis-related markers of the three-dimensional liver microtissue at different time points during the exposure period; (c) analyzing the hepatotoxicity and liver fibrosis potential of the chemical substances based on the test results.

6. Use of a three-dimensional liver microtissue model according to claim 5 in the assessment of hepatotoxicity of environmental chemicals, characterized in that: The metabolic function indicators include albumin secretion, urea synthesis and cellular ATP level.

7. Use of a three-dimensional liver microtissue model according to claim 5 in the assessment of hepatotoxicity of environmental chemicals, characterized in that: The liver fibrosis-related markers include gene or protein expression levels of αSMA, Col1α1, TNF-α and IL-6.

8. Use of a three-dimensional liver microtissue model in the hepatotoxicity assessment of environmental chemicals according to claim 5, characterized in that: The gene expression profiling analysis includes: (i) Detecting the expression of 1200 human genes using simplified transcriptome sequencing technology; (ii) Time series clustering analysis was performed using the Mfuzz algorithm to identify differentially expressed genes; (iii) The biological processes of differentially expressed genes were analyzed by GO function enrichment and KEGG pathway enrichment.

9. Use of a three-dimensional liver microtissue model in the hepatotoxicity assessment of environmental chemicals according to claim 5, characterized in that: The chemical substances to be tested include TCDD, thioacetamide (TAA), benzopyrene (BaP) or 3,3',4,4',5-pentachlorobiphenyl (PCB126).

10. Use of a three-dimensional liver microtissue model according to claim 5 in the assessment of hepatotoxicity of environmental chemicals, characterized in that: The morphological stability assessment includes: observing the spheroid diameter under a microscope on culture days 0, 3, 5, 7, 10, 12, and 14, and confirming that the diameter is maintained within the range of 250-300 μm.

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