Liver organoid for traditional Chinese medicine screening and toxicity assessment and construction method and application thereof

Abstract

The invention belongs to a cell culture technology in the field of biomedicine, and particularly relates to a liver organoid for traditional Chinese medicine screening and toxicity assessment and a construction method and application thereof. The construction method comprises the following steps: carrying out plane multiplication culture on pluripotent stem cells, then digesting and subculturing, and repeating to obtain a cell culture medium; a pluripotent stem cell group which is uniform in cell morphology and free of spontaneous differentiation is obtained; performing two-dimensional plane directional induction on the pluripotent stem cell population to enable cells to synchronously and uniformly receive differentiation signals to obtain hepatogenic mother cells; culturing the obtained hepatic mastocytes by using a culture medium E under a three-dimensional suspension condition to promote the specificity and balling of the hepatic lineage; the culture medium E is an Advanced DMEM / F12 which comprises 1 to 5 [mu] M of RA, 2 to 10 [mu] M of Y-27632, 0.5 to 1 [mu] M of beta-nicotinamide mononucleotide, 5 to 10 [mu] M of senkyunolide A, B27 and N2; and then replacing the culture medium to further mature and functionalize the hepatocytes to obtain the liver organoid, and the obtained liver organoid has high stability, high uniformity, good survival rate and mature hepatocyte functions.

Description

Technical Field

[0001] This invention pertains to cell culture technology in the biomedical field, specifically relating to a liver organoid for screening and toxicity assessment of traditional Chinese medicine, its construction method, and its application. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] In the field of drug development and safety evaluation, especially for the study of complex traditional Chinese medicine compound formulas and monomers with unclear mechanisms of action, establishing an in vitro model that can highly simulate the structure and function of the human liver is of paramount importance. However, traditional in vitro hepatocyte models, such as hepatocellular carcinoma cell lines and primary hepatocytes, have significant limitations: hepatocellular carcinoma cell lines suffer from severe functional loss and have metabolic activity far lower than normal human hepatocytes; while primary hepatocytes are difficult to obtain, rapidly lose function during in vitro culture, and exhibit significant individual variability, making it difficult to meet the needs of standardized, large-scale drug screening.

[0004] The shortcomings of the above-mentioned models severely restrict the development of modern Chinese medicine. In terms of evaluating the efficacy of Chinese medicine, Chinese medicine often exerts its overall regulatory effect through multiple targets and pathways. However, the traditional two-dimensional monolayer hepatocyte model cannot reproduce the complex microenvironment and intercellular interactions of the liver, making it difficult to accurately reflect the synergistic, antagonistic, or transformation processes of Chinese medicine components and their metabolites in real liver tissue. This results in a huge gap between the screening results and the actual efficacy in vivo.

[0005] In the assessment of hepatotoxicity of traditional Chinese medicine (TCM), many TCMs exhibit delayed onset and metabolic dependence. Current models, lacking complete drug-metabolizing enzyme activity and bile duct excretion function, struggle to predict liver damage caused by TCMs and their metabolites, and cannot systematically assess their potential risks under long-term use.

[0006] Organoid technology represents a significant breakthrough in regenerative medicine and disease modeling research in recent years. It can highly simulate the tissue structure, cellular composition, and some physiological functions of the real liver in vitro, demonstrating enormous application potential in drug screening, toxicity assessment, disease modeling, and future cell therapy. However, existing technologies, especially liver organoids constructed based on direct three-dimensional differentiation of human pluripotent stem cells, generally suffer from large batch-to-batch variations, heterogeneous cell composition, and insufficient functional maturity. This high degree of heterogeneity significantly reduces the reliability and reproducibility of experimental data when used as a drug screening platform, failing to meet the stringent requirements for model stability and accuracy in traditional Chinese medicine research. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the purpose of this invention is to provide a liver organoid for screening and toxicity assessment of traditional Chinese medicine, its construction method, and its application. The construction method of this invention ensures that the obtained liver organoids possess high stability, high homogeneity, good survival rate, and mature hepatocyte function, providing a sensitive and reliable in vitro platform for the activity screening, toxicity assessment, and research on liver diseases such as metabolic-associated fatty liver disease (MAFLD) of traditional Chinese medicine.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a method for constructing liver organoids for screening and toxicity assessment of traditional Chinese medicine, comprising the following steps: Pluripotent stem cells were expanded and cultured in a plane, then digested and passaged, and the process was repeated to obtain a population of pluripotent stem cells with uniform cell morphology and no spontaneous differentiation. The pluripotent stem cell population was subjected to two-dimensional planar orientation induction to enable the cells to receive differentiation signals synchronously and uniformly, thereby obtaining hepatocytes. The obtained hepatocytes were cultured in three-dimensional suspension using medium E to promote hepatic lineage specialization and spheroidization. Medium E consisted of Advanced DMEM / F12 containing 1-5 μM RA, 2-10 μM Y-27632, 0.5-1 mM β-nicotinamide mononucleotide, 5-10 μM ligustilide A, B27 and N2. Then the culture medium is changed to allow the hepatocytes to further mature and function, thus obtaining the desired product.

[0009] Secondly, the present invention provides a liver organoid for screening and toxicity assessment of traditional Chinese medicine, obtained by the aforementioned construction method.

[0010] Thirdly, the present invention provides the application of the liver organoids used for screening and toxicity assessment of traditional Chinese medicine in the screening of the activity of traditional Chinese medicine, the assessment of the toxicity of traditional Chinese medicine, and the research on liver diseases.

[0011] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows: This invention replaces early three-dimensional suspension aggregation with two-dimensional monolayer culture in the early hepatic differentiation stage, completely avoiding apoptosis and necrosis of cells in the center of the aggregate due to limited diffusion of nutrients and oxygen. This not only significantly improves the survival rate of cells in the intermediate stage, but also provides better-condition and healthier "seed cells" for subsequent three-dimensional assembly, thus ensuring the overall quality and viability of the final organoid. At the same time, it overcomes the observation blind spots caused by the "black box" operation of traditional three-dimensional aggregate culture systems, enabling in-situ, non-destructive, real-time observation of the early hepatic differentiation stage (including endoderm induction and hepatic shaping).

[0012] This invention can induce pluripotent stem cells into hepatocytes, and digest the hepatocytes for passage, which greatly reduces experimental costs, shortens experimental time, and reduces experimental differences between different batches. During the process of promoting liver lineage specialization and spheroidization by continuing to use culture medium E under three-dimensional suspension conditions, Y-27632, β-nicotinamide mononucleotide and ligustrazine lactone A were added to culture medium E. The three components worked synergistically to make the resulting cell aggregates more stable and uniform, and the bile duct cells in the obtained liver organoids were fully developed. This ensured that the prepared liver organoids could be used as a drug screening platform to guarantee the reliability and reproducibility of experimental data, thereby meeting the stringent requirements of model stability and accuracy in traditional Chinese medicine research.

[0013] Addressing the unique needs of traditional Chinese medicine (TCM) in terms of its multi-component, multi-target effects and the evaluation of metabolism-dependent hepatotoxicity, this invention aims to construct an in vitro model that highly simulates the real microenvironment and metabolic function of the human liver. This model will precisely support high-throughput screening of active components of TCM, systematic toxicity assessment, and research on their mechanisms of action in the prevention and treatment of metabolic-associated fatty liver disease (MAFLD). Using only ultra-low adsorption culture plates for cell aggregation, without relying on matrix gels to promote cell spheroidization, not only reduces experimental costs but also improves the sensitivity of organoids to drugs, making drug toxicity assessment and effective drug screening more precise and accurate. Attached Figure Description

[0014] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0015] Figure 1 This is the process of inducing pluripotent stem cells into liver-derived cells and bright-field images; Figure 2 This is the process of generating liver organoids by digesting liver cells and then seeding them into an ultra-low adsorption plate, along with bright-field images. Figure 3 These are bright field images of pellets formed using three different culture medium E formulations; Figure 4 The differences in RNA between pluripotent stem cells (iPSC), immature hepatoblasts (IH), and hepatoblastoma balls (HB) are as follows: Figure 5 This is the immunofluorescence verification of key proteins under a confocal microscope after liver organoid formation; Figure 6 These are bright-field images of liver organoids induced by free fatty acids (FFA). Figure 7 This was verified by liver organoid lipid droplet fluorescence (Bodipy 493 / 503) after induction by free fatty acids (FFA); Figure 8 These are the results of the detection of triglycerides (TG), trace amounts of reduced glutathione (GSH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in MAFLD liver organoids. Figure 9 It shows the proliferation process of the liver cells during the first passage to the blastoblasts and the bright-field image. Figure 10 It shows the proliferation process of liver cells being passaged to blast cells for the second time and the bright-field image. Figure 11 It shows the proliferation process and bright-field images of liver cells after cryopreservation and thawing. Figure 12 It is a bright-field image of liver cells aggregated into spheres after one passage; Figure 13 This is a comparative diagram of RNA expression in various organ spheres after pluripotent stem cells (iPSCs) and liver blast cells were digested into spheres and maintained in culture medium 1, culture medium 2, and culture medium 3 in an embodiment of the present invention. Figure 14 A comparative graph showing the drug toxicity sensitivity of liver organoids from different culture media sources; Figure 15 The figure shows a comparison of the experimental results of the effect of triptolide on the activity of liver organoids from different culture media. In the figure, a represents culture medium 1 and b represents culture medium 3. Detailed Implementation

[0016] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0017] In a first aspect, the present invention provides a method for constructing liver organoids for screening and toxicity assessment of traditional Chinese medicine, comprising the following steps: Pluripotent stem cells were expanded and cultured in a plane, then digested and passaged, and the process was repeated to obtain a population of pluripotent stem cells with uniform cell morphology and no spontaneous differentiation. The pluripotent stem cell population was subjected to two-dimensional planar orientation induction to enable the cells to receive differentiation signals synchronously and uniformly, thereby obtaining hepatocytes. The obtained hepatocytes were cultured in three-dimensional suspension using medium E to promote hepatic lineage specialization and spheroidization. Medium E consisted of Advanced DMEM / F12 containing 1-5 μM RA, 2-10 μM Y-27632, 0.5-1 mM β-nicotinamide mononucleotide, 5-10 μM ligustilide A, B27 and N2. Then the culture medium is changed to allow the hepatocytes to further mature and function, thus obtaining the desired product.

[0018] In this invention, pluripotent stem cells are passaged repeatedly for at least three generations, and microscopic observation confirms that the cells have uniform morphology and no spontaneous differentiation is observed. This ensures the consistency and stability of the starting cells in terms of pluripotency and state from the source, eliminating the influence of heterogeneity of starting cells between batches.

[0019] In the stage of directional induction of pluripotent stem cells into hepatocytes, three-dimensional suspension culture can lead to apoptosis or necrosis of central cells in the aggregates due to limited diffusion of nutrients and oxygen. Two-dimensional planar culture, on the other hand, has no diffusion restrictions, ensuring all cells receive sufficient nutrition and significantly improving cell survival rates in intermediate stages. Two-dimensional planar culture allows for in-situ, non-destructive, real-time observation of early hepatocyte differentiation stages (such as endoderm induction and hepatocyte shaping), overcoming the blind spots of three-dimensional "black box" operations that cannot observe the internal cell state. The uniformity of cell state in two-dimensional culture reduces the differences in experimental results between batches due to initial cell heterogeneity, facilitating standardized and large-scale applications. Cells cultured in two-dimensional planar culture are easier to digest and passage, and density adjustments are easier; experimental variables (such as inducing factor concentration and culture time) are more easily and precisely controlled, whereas the size and morphology of aggregates in three-dimensional suspension culture are difficult to control uniformly.

[0020] Y-27632 is a ROCK kinase inhibitor whose core function is to inhibit apoptosis and reduce cell death caused by changes in intercellular adhesion or environmental stress in the early stage of hepatocytes forming in three-dimensional suspension, significantly improving cell survival rate in the intermediate stage. β-Nicotinamide mononucleotide (NMN), as a precursor of NAD+, can rapidly increase intracellular NAD+ levels, enhance cellular energy metabolism activity, and strengthen intercellular adhesion, allowing cells to maintain vitality and aggregation stability under suspension pressure, avoiding aggregate loosening or core necrosis due to insufficient energy. Ligusticum lactone A targets the CLCC1 channel to precisely drive the functional maturation of bile duct cells, effectively upregulating the expression of bile duct-specific functional genes (such as CK19 and SOX9), compensating for the shortcomings of bile duct cell development in conventional organoids, and promoting the co-differentiation of the hepatobiliary lineage.

[0021] Y-27632 reduces cell death, while NMN enhances energy metabolism and cell adhesion, jointly addressing the issues of cell survival and aggregation stability in the early stages of suspension spheroidization. Ligusticum lactone A guides the maturation of bile duct cells, working synergistically with the former two to ensure that the aggregates not only have stable morphology but also possess a fully functional hepatobiliary lineage structure, avoiding organoid functional defects caused by a single lineage. Ultimately, this achieves synergistic regulation of "reduced death → enhanced aggregation → directed differentiation," effectively solving common problems in three-dimensional suspension culture such as core necrosis, high heterogeneity, and insufficient function.

[0022] The technical solution of this invention forms spherical structures with regular shape, intact edges, and uniform size. There is very little cell shedding at the bottom of the well plate, avoiding the problems of inconsistent size and loose structure commonly found in three-dimensional culture. The spheroid cells are in a synchronized state with high differentiation uniformity. The expression of pluripotency genes (Nanog, OCT4) is significantly reduced, while the expression of liver differentiation marker genes (CXCR4, SOX17) is significantly upregulated, laying a synchronized foundation for subsequent organoid maturation. The expression of bile duct cell maturation-related genes (CK19, SOX9) is significantly increased, and the expression of intercellular adhesion genes (CDH1) and energy metabolism genes (PGC-1α) is enhanced, giving the spheroid organoids more complete functional potential.

[0023] Two-dimensionally induced liver-derived stem cells can be passaged to increase yield or cryopreserved in a standardized manner to establish a cell bank, greatly improving the process's planarability, reproducibility, and scalability potential, and avoiding batch-to-batch variations caused by de novo induction in each experiment. The ultra-low adsorption plate eliminates the need for matrix gel, forcing cells to spontaneously aggregate into regularly shaped and uniformly sized spheres, reducing heterogeneity caused by matrix gel and improving drug sensitivity. Furthermore, its synergistic effect with culture medium E effectively addresses the common problems of large batch-to-batch variations, heterogeneous cell composition, and insufficient functional maturity in liver organoids constructed based on direct three-dimensional differentiation of human pluripotent stem cells.

[0024] In some embodiments, the culture medium E is Advanced DMEM / F12 comprising 2-4 μM RA, 5-10 μM Y-27632, 0.7-1 mM β-nicotinamide mononucleotide, 7-10 μM ligustilide A, B27 (50×) and N2 (100×).

[0025] B27 and N2 are both serum-free additives commonly used in cell culture. B27 contains various vitamins, hormones (such as insulin), antioxidants (such as vitamin E), and growth factors; it supports cell survival by inhibiting apoptosis of hepatocytes during differentiation and spheroidization, thus improving cell survival rate; it provides key signals for the directed differentiation of hepatocytes (such as endoderm → hepatocyte → mature hepatocytes), maintains specific liver functions (such as metabolism and secretion); and reduces cell damage caused by oxidative stress in three-dimensional suspension culture.

[0026] N2 additive contains insulin, transferrin, sodium selenate, progesterone, etc. Insulin promotes glucose uptake and energy metabolism, providing energy support for hepatocyte spheroidization and organoid maturation; transferrin maintains iron homeostasis and supports cell division; sodium selenate enhances intercellular adhesion and helps stabilize three-dimensional suspension spheroidization; and it inhibits spontaneous cell differentiation, maintaining the lineage specificity of hepatocytes.

[0027] RA (Retinoic Acid) is a key differentiation regulatory molecule in the construction of liver organoids in this invention. In the three-dimensional suspension spheroidization stage (culture medium E) and the subsequent maturation stage (culture medium F), RA acts as a core signaling factor to guide the liver to differentiate into blast cells into mature hepatocytes or bile duct cell lineages, promote the transformation of cells from the blast cell stage to functionally mature hepatocytes, and enhance the liver-specific function of organoids.

[0028] Preferably, the culture medium E is Advanced DMEM / F12 comprising 2 μM RA, 10 μM Y-27632, 1 mM β-nicotinamide mononucleotide, 10 μM ligustilide A, B27 and N2.

[0029] Preferably, the obtained liver cells are digested, inoculated into ultra-low adsorption plates, and cultured stably in medium E for 0.5-1.5 days.

[0030] The ultra-low adsorption plate provides a three-dimensional spheroidization environment for hepatocytes through its physical surface properties. Its surface has no cell adsorption sites, which forces hepatocytes to spontaneously aggregate into spheres in a suspended state, avoiding adherent growth and mimicking the three-dimensional structure of the liver in vivo. Cell spheroidization can be achieved without the use of matrix gel, reducing the source of heterogeneity and lowering reagent costs. In addition, without matrix gel interference, drugs can directly contact organoid cells, improving the accuracy of traditional Chinese medicine activity screening and toxicity assessment.

[0031] The ultra-low adsorption plate promotes cell aggregation, while the Y-27632 and NMN in culture medium E ensure the survival and stable adhesion of aggregated cells, preventing spheroid disintegration or core necrosis. The ultra-low adsorption plate constructs a three-dimensional spherical structure, and the RA and ligustrazine A in culture medium E guide the cells inside the spheroids to differentiate into a hepatobiliary lineage, forming fully functional organoids. The ultra-low adsorption plate ensures uniform spheroid size, and culture medium E maintains consistent cell differentiation status. The two work together to reduce experimental heterogeneity, while the absence of matrix gel interference enhances the sensitivity of drug screening, meeting the needs of traditional Chinese medicine research for standardized and precise models.

[0032] In a further optimized manner, the obtained liver cells were digested, inoculated into an ultra-low adsorption plate, and cultured stably in medium E for 1 day.

[0033] In some embodiments, pluripotent stem cells are seeded onto a culture surface covered with pluripotent stem cell working solution and subjected to planar expansion culture using mTeSR Plus medium; when the cell confluence reaches 70%-85%, they are digested and passaged.

[0034] The working solution (such as a reagent containing extracellular matrix components) simulates the microenvironment for stem cell survival in vivo, promoting the adherent growth of pluripotent stem cells; maintaining the undifferentiated state of stem cells, preventing spontaneous differentiation of cells during expansion, and ensuring the stability of the pluripotency of the starting cells.

[0035] mTeSR Plus is a serum-free, chemically defined culture medium specifically for pluripotent stem cells. It can precisely provide the nutritional factors and signaling molecules required for stem cell proliferation. It promotes rapid cell expansion to obtain a sufficient number of starting cells, while strictly maintaining the pluripotency and homogeneity of stem cells, avoiding differentiation or functional abnormalities.

[0036] In some embodiments, when performing two-dimensional planar orientation induction on a pluripotent stem cell population, the first day is cultured in medium A containing Activin A and BMP4 for 4-7 hours, followed by culture in medium B containing Activin A and BMP4 for the remaining time. Then, culture in medium C containing Activin A and B27 for 2-3 days; Then culture the cells in medium D containing FGF4 and CHIR99021 for 2-3 days to promote differentiation of hepatocytes into blast cells.

[0037] Day 1: Culture medium A → Culture medium B. Activin A activates the Smad2 / 3 signaling pathway, inducing pluripotent stem cells to differentiate into the endoderm. BMP4 activates the Smad1 / 5 / 8 signaling pathway, synergistically enhancing endoderm differentiation efficiency and inhibiting paradifferentiation towards the mesoderm / ectoderm direction, in conjunction with Activin A. Culture medium A (RPMI 1640 + insulin-free B27): initially stimulates cells to enter the differentiation state; the absence of insulin avoids interference with early endoderm induction. Culture medium B (25% mTeSR Plus + 75% RB): adjusts the nutritional basis, maintaining cell survival while allowing cells to smoothly transition to the continuous differentiation state, ensuring the synchronicity of endoderm differentiation.

[0038] In culture medium stage C, Activin A continues to activate the Smad2 / 3 pathway, consolidating the differentiation state of the fixed endoderm and reducing cell migration to other lineages; B27 supplement provides vitamins, hormones and other nutrients to support the survival and maturation of endoderm cells and prevent cell apoptosis during differentiation; after this stage, the cells have differentiated into a highly homogeneous fixed endoderm and have the potential to differentiate into the liver lineage.

[0039] During culture medium D phase, FGF4 (fibroblast growth factor 4) activates the FGFR signaling pathway, which is a key factor guiding the differentiation of the fixed endoderm into the liver and into the blastocyst, and promotes the expression of liver lineage-specific genes (such as HNF4α and FOXA2); CHIR99021 (GSK3β inhibitor) activates the Wnt / β-catenin signaling pathway, which synergistically enhances the liver differentiation efficiency with FGF4 and inhibits the differentiation of bile ducts or other cell lineages.

[0040] In two-dimensional planar culture, all cells uniformly receive FGF4 and CHIR99021 signals, ensuring a high degree of consistency in the morphology and differentiation state of hepatocytes towards blast cells.

[0041] Preferably, when performing two-dimensional planar directional induction on a pluripotent stem cell population, the culture is first performed on the first day using medium A containing Activin A and BMP4 for 5-6 hours, and then cultured for the remaining time using medium B containing Activin A and BMP4. Then, cultured in medium C containing Activin A and B27 for 2 days; The cells were then cultured in medium D containing FGF4 and CHIR99021 for 2 days to promote differentiation of hepatocytes into blast cells.

[0042] Preferably, culture medium A is RPMI 1640 containing 50-100 ng / mL Activin A, 20-60 ng / mL BMP4, and 1-4% B27 (without insulin); Culture medium B is a mixed culture medium containing 50-100 ng / mL Activin A and 20-60 ng / mL BMP4 of mTeSR Plus and RB, with a volume ratio of mTeSR Plus to RB of 1:2-4; Culture medium C is a mixed culture medium containing 50-100 ng / mL Activin A of mTeSR Plus and RB, with a volume ratio of mTeSR Plus to RB of 1:2-4; Culture medium D: Advanced DMEM / F12 containing 500 ng / mL FGF4, 3 μM CHIR99021, B27 and N2.

[0043] Further preferably, culture medium A is RPMI 1640 containing 100 ng / mL Activin A, 50 ng / mL BMP4, and 2% B27 (without insulin); Culture medium B was 25% mTeSR Plus + 75% RB (RB: RPMI 1640 containing 2% B27) containing 100 ng / mL Activin A and 50 ng / mL BMP4. Culture medium C consisted of 25% mTeSR Plus + 75% RB containing 100 ng / mL Activin A; Culture medium D: Advanced DMEM / F12 containing 500 ng / mL FGF4, 3 μM CHIR99021, B27 and N2.

[0044] In some embodiments, after culture in culture medium E is completed, culture medium E is replaced with culture medium F for 1-3 days; then culture medium F is updated and culture continues for 1-3 days; then culture medium F is replaced with culture medium G for 1-3 days; and culture medium G is continuously updated and culture continues until liver organoids are obtained.

[0045] Preferably, the culture medium F is Advanced DMEM / F12 containing 2-4 μM RA, 0.7-1 mM nicotinamide mononucleotide, 7-10 μM ligustilide A, B27 and N2; Culture medium G was Williams' E (Gibco, A1217601) medium containing primary hepatocyte maintenance culture additive (Gibco, CM4000), 1-4% B27, 0.5-1.51% N2, 0.7-1mM nicotinamide mononucleotide, 7-10μM ligustilide A, 5-10ng / mL OSM and 5-10ng / mL HGF.

[0046] Culture medium F is a crucial stage in the transition from liver to blast cells to mature liver cells. Its core function is to promote the transformation of cells from the blast cell stage to functionally mature liver cells and enhance the liver-specific function of organoids. RA (retinoic acid) in the culture medium acts as a core signaling factor, guiding the directional differentiation of liver to blast cells into mature liver cells or bile duct cells. Nicotinamide mononucleotide (NMN) and ligustrolactone A work synergistically to maintain cell viability and differentiation direction, while B27+N2 provides nutritional support to ensure cell survival and initial maturation in a three-dimensional suspension state.

[0047] Culture medium G is the core stage for the functional maturation and long-term maintenance of liver organoids. Its role is to promote the complete maturation of hepatocytes and maintain their physiological functions. It contains primary hepatocyte maintenance additives, OSM (antitumor suppressant M), HGF (hepatocyte growth factor), and other components, which can enhance the metabolic activity of hepatocytes (such as the expression of drug-metabolizing enzymes) and bile secretion function. The continuous addition of NMN and ligustrolactone A maintains cellular energy metabolism and the maturation state of bile duct cells, supports the long-term stable culture of organoids, and meets the application needs of traditional Chinese medicine screening, toxicity assessment, etc.

[0048] Further preferred, the culture medium F is Advanced DMEM / F12 containing 2 μM RA, nicotinamide mononucleotide (1 mM), ligustilide A (10 μM), B27 and N2; Culture medium G was Williams' E (Gibco, A1217601) medium containing primary hepatocyte maintenance culture additive (Gibco, CM4000), 2% B27, 1% N2, nicotinamide mononucleotide (1mM), ligustilide A (10μM), 10ng / mL OSM and 10ng / mL HGF.

[0049] Secondly, the present invention provides a liver organoid for screening and toxicity assessment of traditional Chinese medicine, obtained by the aforementioned construction method.

[0050] Thirdly, the present invention provides the application of the liver organoids used for screening and toxicity assessment of traditional Chinese medicine in the screening of the activity of traditional Chinese medicine, the assessment of the toxicity of traditional Chinese medicine, and the research on liver diseases.

[0051] The present invention will be further described below with reference to the embodiments.

[0052] Example 1 The following steps are used to culture metabolically related fatty liver organoids: Inducing liver organoids begins with culturing pluripotent stem cells. Pluripotent stem cells frozen in liquid nitrogen are retrieved and seeded in six-well plates coated with pluripotent stem cell working medium (Sybe Biosciences). The medium is changed daily. The medium used can be PGM1 and / or mTeSR1 and / or mTeSR Plus. When the cell density reaches 80%, the cells are digested using pluripotent stem cell digestion solution (Sybe Biosciences) and seeded at a density of 200,000 cells / well in six-well plates coated with pluripotent stem cell working medium (Sybe Biosciences). The medium is changed daily. After three stable passages, the morphology of the pluripotent stem cells is observed to determine the formation of undifferentiated cells. Figure 1 If the Day 0 state is reached, the next step of organoid induction can proceed.

[0053] The culture medium kit used for culturing and constructing liver organoids includes culture medium A, culture medium B, culture medium C, culture medium D, culture medium E, and culture medium F; Culture medium A: RPMI 1640 containing 100 ng / mL Activin A, 50 ng / mL BMP4, and 2% B27 (without insulin); Culture medium B: 25% mTeSR Plus + 75% RB containing 100 ng / mL Activin A and 50 ng / mL BMP4 (RB: RPMI 1640 containing 2% B27). Culture medium C: 25% mTeSR Plus + 75% RB containing 100 ng / mL Activin A; Culture medium D: Advanced DMEM / F12 containing 500 ng / mL FGF4, 3 μM CHIR99021, B27 and N2; Culture medium E: Advanced DMEM / F12 containing 2 μM RA, 10 μM Y-27632, nicotinamide mononucleotide (1 mM), ligustilide A (10 μM), B27 and N2; Culture medium F: Advanced DMEM / F12 containing 2 μM RA, nicotinamide mononucleotide (1 mM), ligustilide A (10 μM), B27 and N2; Culture medium G: Williams 'E' (Gibco, A1217601) medium containing primary hepatocyte maintenance culture additive (Gibco, CM4000), 2% B27, 1% N2, nicotinamide mononucleotide (1mM), ligustilide A (10μM), 10 ng / mL OSM and 10 ng / mL HGF.

[0054] The specific training process is as follows: Day 0: After stabilizing and passaged 3 times, pluripotent stem cells were seeded in a six-well plate with a pluripotent stem cell substrate solution and stabilized for one day. Day 1: Discard the old culture medium and replace it with culture medium A for 5 hours of incubation. Then discard culture medium A and replace it with culture medium B for 19 hours of incubation, until the desired results are achieved. Figure 1 The status of Day 1; Day 2: Discard culture medium B and replace it with culture medium C. Incubate for 2 days to achieve the desired results. Figure 1 The status of Day 3; Day 4: Discard culture medium C and replace it with culture medium D. Incubate for 2 days to achieve the desired results. Figure 1 The status of Day 6; Day 6: Liver-derived stem cells obtained after continuous incubation in media A, B, C, and D were digested with pluripotent stem cell digestion solution (Sybe Biosciences) and seeded at a cell density of 10,000 cells / well in 96-well ultra-low adsorption plates for 48 hours. The resulting organoids were in the following state: Figure 2 As shown in Day 8, the culture medium used was medium E. The remaining cells can be used for passage.

[0055] Day 8: Discard culture medium E and replace with culture medium F for 2 days of incubation. The resulting organoids are in the following state: Figure 2 As shown in Day 10; Day 10: Discard culture medium F and replace with fresh culture medium F for 2 days of incubation. The resulting organoids are in the following state: Figure 2 As shown in Day 12; Day 12: Discard culture medium F and replace it with culture medium G for incubation for 2 days; Days 14-30: Discard culture medium G and replace with new culture medium G, changing the medium daily to obtain metabolically related fatty liver organoids. By day 20, the organoids were in the following state: Figure 2 As shown in Day 20.

[0056] After digesting the liver cells into blastocysts and resuspending them into blastocysts, we evaluated the blastocyst formation using three different culture medium E formulations, with the culture methods described above.

[0057] Group 1: The EG culture medium does not contain nicotinamide mononucleotide or ligustrazine lactone A; Culture medium group 2: EG culture medium with added nicotinamide mononucleotide (0.5mM) + ligustrazine lactone A (1μM); Culture medium group 3: EG culture medium with added nicotinamide mononucleotide (1mM) + ligustrazine lactone A (10μM); Nicotinamide mononucleotide (NMN) enhances overall cellular energy metabolism and activity by rapidly increasing NAD+ levels, making cells more vigorous during division, migration, and aggregation. This is crucial for the energy homeostasis and resilience of hepatocytes under suspension pressure and lacking matrix gel protection. Synergistically working with Y-27632 (which reduces apoptosis), it promotes more stable and homogeneous cell aggregates through three dimensions: reducing cell death, enhancing dynamism, and providing signaling.

[0058] SenA, through its unique CLCC1 targeting action, precisely drives the functional maturation of bile duct cells and effectively upregulates bile duct-specific functional genes, thereby compensating for the functional shortcomings of underdeveloped bile duct cells in conventional organoids.

[0059] To overcome the industry-wide challenge of uneven hepatic progenitor cell spheroid size and easy core necrosis in matrix-free suspension culture, this invention significantly improves spheroid quality by optimizing the culture medium formulation during the initial stage of three-dimensional assembly. The key optimization lies in the addition of nicotinamide mononucleotide (1 mM) and ligustilide A (10 μM) to the culture medium. Figure 3As shown, using culture media 1 and 2 resulted in poor spheroidization: the spheroids had irregular edges, varied in size, and exhibited significant cell detachment at the bottom of the wells. This indicates unstable cell aggregation, a high apoptosis rate, and a tendency to form loosely structured, unevenly sized aggregates, which are highly susceptible to core necrosis during subsequent culture. In contrast, using the optimized culture media 3, liver cells formed regular, well-defined, and uniformly sized spherical structures with minimal cell detachment. This demonstrates that the optimized formulation of this invention effectively promotes stable cell adhesion and uniform aggregation, laying the foundation for the formation of uniformly sized, densely structured spheroids from the outset, thereby avoiding size dispersion and internal defects caused by poor initial aggregation.

[0060] Furthermore, such as Figure 4 The qPCR experiments shown demonstrate that the expression of pluripotency genes (Nanog, OCT4, SOX2) in the "hepatocyte spheroids" formed under these optimized conditions was significantly reduced, while the expression of hepatocyte differentiation marker genes (CXCR4, SOX17, FOXA2) was significantly upregulated. More importantly, compared to non-spheroidized hepatocyte spheroids, the spheroidized cells exhibited more optimized expression profiles for key genes. This indicates that the spheroidization conditions provided by this invention not only achieve physical homogenization but also simultaneously guide cells into a more synchronized and stable pre-differentiation state. A cell spheroid with a uniform initial state and synchronized differentiation is the biological guarantee that it will uniformly receive nutrient signals during subsequent extended culture and avoid core necrosis due to differences in internal cell states.

[0061] Subsequent qPCR results further confirmed that the expression levels of key genes in culture medium group 3 were significantly higher than those in the other three groups: Regarding genes related to bile duct cell maturation, the relative mRNA expression levels of CK19 (a bile duct epithelial cell-specific marker) and SOX9 (a key transcription factor for bile duct progenitor cell differentiation) in culture medium group 3 were significantly higher than those in the pluripotent stem cell group, culture medium group 1, and culture medium group 2. Figure 13 The results suggest that high-dose NMN combined with ligustrazine lactone A can more efficiently promote the differentiation of pluripotent stem cells into bile duct cells. Regarding cell connectivity-related genes, the expression level of CDH1 (a core molecule mediating intercellular adhesion) was significantly increased in group 3 of the culture medium, indicating stronger intercellular adhesion and better structural stability in this group of organoid cells. Regarding energy metabolism-related genes, the expression level of PGC-1α (a key regulator of mitochondrial biosynthesis) was significantly higher in group 3 of the culture medium than in other groups, suggesting that high-dose combination can effectively optimize the energy metabolism efficiency of organoid cells.

[0062] This invention, through specific culture medium optimization, simultaneously overcomes two major challenges in glue-free suspension culture: uneven physical aggregation and asynchronous biological states, thereby obtaining liver progenitor cell spheres with uniform size, intact structure, and synchronized differentiation. Building upon this, the system achieves dual functional enhancement by introducing a synergistic combination of nicotinamide mononucleotide (NMN) and ligustrazine lactone A (SenA): NMN effectively enhances the metabolic activity and adhesion stability of cells in suspension by increasing intracellular NAD+ levels; SenA specifically upregulates the expression of mature functional genes in bile duct cells (such as CFTR and CK19), precisely promoting the co-differentiation of the hepatobiliary lineage. This provides a high-quality starting point for subsequent cultivation of high-quality mature liver organoids with complete cellular function and no core necrosis, possessing excellent structural integrity, metabolic activity, and lineage-directed differentiation capabilities.

[0063] Immunofluorescence assays were performed on liver organoids from the later stages of the induction process, such as... Figure 5 As shown, liver organoids in the later stages of the induction process exhibited high expression of EPCAM, ALB, and HNF4a.

[0064] Liver organoids (using culture medium 3) derived from liver cells after spherogenesis were further induced to form metabolically related fatty liver organoids. Through specific metabolic stress induction, the transformation to the MAFLD pathological phenotype was successfully achieved. The specific implementation steps are as follows: The method for preparing free fatty acids involves saponifying oleic acid and palmitic acid with NaOH and then dissolving them in a PBS solution containing fatty acid-free bovine serum protein. This yields a clear and transparent stock solution of oleic acid and palmitic acid, which improves the stability of the solution. In this invention, the molar ratio of oleic acid to palmitic acid is 2:1 in the culture medium, and the final concentration of free fatty acids is 500 μM.

[0065] A mixture of free fatty acids (oleic acid and palmitic acid in a molar ratio of 2:1) was used to stimulate liver organoids for 24 hours, with a final free fatty acid concentration of 500 μM in culture medium G.

[0066] like Figure 6 As shown, under bright field conditions, obvious lipid droplet formation can be observed on liver organoids. Figure 7 As shown, Bodipy 493 / 503 lipid droplet fluorescence staining of metabolically related fatty liver organoids clearly revealed that, compared with normal liver organoids, the lipid droplet accumulation in liver organoids induced by free fatty acids was significantly increased. Figure 8As shown, the levels of triglycerides (TG), trace amounts of reduced glutathione (GSH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in MAFLD liver organoids (Mod) were significantly higher than those in normal liver organoids (Control). This invention successfully constructed an in vitro liver organoid model that systematically simulates the core pathological features of metabolic-associated fatty liver disease (MAFLD) through free fatty acid induction. This model not only morphologically confirmed significant hepatocyte steatosis (lipid droplet accumulation) (bright-field and Bodipy staining), but also fully reproduced the progressive pathological process of MAFLD from lipid accumulation, metabolic stress and oxidative damage to final hepatocyte damage through biochemical indicators (significantly elevated TG, GSH, ALT, and AST), thus providing a highly biomimetic in vitro platform for disease mechanism research and drug screening.

[0067] To evaluate the performance of hepatocytes derived from induced pluripotent stem cells in stable passage and cryopreservation, a validation experiment was conducted. The specific passage steps are as follows: One day before subculturing, add 1.5 mL of bottom-laying working solution (Cellapy, CA3003100) to each well of a six-well plate; 12 hours later, aspirate the original culture medium, rinse the bottom of the wells once with digestion solution (Cellapy, CA1023100), add 1 mL of digestion solution to each well, and place in an incubator for digestion for 4 minutes.

[0068] After observing the cell state under a microscope, add 1 mL of Advanced DMEM / F12 or RPMI 1640 to each well to stop digestion. Gently pipette the bottom of the well to detach the cells, collect the cell suspension into a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes.

[0069] After centrifugation, discard the supernatant and simultaneously aspirate the supernatant from the working solution in the six-well plate. Resuspend the cells in Advanced DMEM / F12 medium containing 2 μM RA, 2% B27, and 1% N2. Add 2 mL of medium to each well and seed the cells at a 1:5 ratio into the pre-planted wells. The specific cryopreservation steps are as follows: Aspirate the original medium, rinse the bottom of the wells once with digestion solution (Cellapy, CA1023100), add 1 mL of digestion solution to each well, and incubate for 4 minutes.

[0070] Add 1 mL of Advanced DMEM / F12 to each well to stop digestion. Gently pipette the cells to detach from the bottom of the well. Collect the cell suspension into a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. After centrifugation, discard the supernatant and resuspend the cells in 1 mL of cryopreservation buffer (Stem Cell, 07930). Transfer the cells from the 15 mL centrifuge tube to cryovials. Place the cryovials in a gradient cryopreservation box and store the box at -80°C. The next day, transfer the cryovials to liquid nitrogen for long-term storage.

[0071] Based on observations of cell states after passage and cryopreservation, the results are as follows: Figure 9 , Figure 10 and Figure 11 As shown, the results indicate that hepatocytes can maintain their original state after the first passage; however, after the second passage, the cells exhibit significant loss of function and slowed proliferation, and are difficult to restore to their initial hepatocyte state. Furthermore, the cell state after cryopreservation and thawing is similar to that after the second passage, indicating that hepatocytes induced under the current system are not suitable for cryopreservation-thawing.

[0072] The specific procedures for drug toxicity sensitivity testing are as follows: Group 1: Medium EG without nicotinamide mononucleotide and ligustrazine lactone A; the corresponding medium G for Group 1 is Williams 'E (Gibco, A1217601) medium containing primary hepatocyte maintenance culture additive (Gibco, CM4000), 2% B27, 1% N2, 10 ng / mL OSM and 10 ng / mL HGF.

[0073] Culture medium group 3: EG culture medium with added nicotinamide mononucleotide (1mM) + ligustrazine lactone A (10μM); The corresponding culture medium G for the three culture media groups was Williams' E (Gibco, A1217601) medium containing primary hepatocyte maintenance culture additive (Gibco, CM4000), 2% B27, 1% N2, nicotinamide mononucleotide (1mM), ligustilide A (10μM), 10ng / mL OSM and 10ng / mL HGF.

[0074] I. Drug administration stimulation: Liver organoids were induced using culture medium 1 and culture medium 3, respectively. After the organoids matured, the liver organoids induced by culture medium 1 were divided into three groups: Culture medium 1 normal control group: The induced liver organoids were added to the corresponding culture medium G of culture medium 1 group and cultured for another 24 hours.

[0075] Culture medium 1 + triptolide-1μM group: The induced liver organoids were added to the corresponding culture medium G of culture medium 1 group, and 1μM triptolide was added to culture medium G. The culture was continued for 24h.

[0076] Culture medium 1 + triptolide-10μM group: The induced liver organoids were added to the corresponding culture medium G, and 10μM triptolide was added to culture medium G. The culture was continued for 24h.

[0077] The liver organoids induced by culture medium 3 were divided into two groups: Culture medium group 3: The induced liver organoids were added to the corresponding culture medium G of culture medium group 3 and cultured for another 24 hours.

[0078] Culture medium 3 + triptolide-1μM group: The induced liver organoids were added to the corresponding culture medium G of culture medium 3, and 1μM triptolide was added to culture medium G. The culture was continued for 24h.

[0079] II. Fluorescent staining: Prepare the staining mixture: Annexin V Binding Buffer (1×): Annexin V Binding Buffer (10×): DDW = 1:9. Prepare the staining mixture: Annexin V Binding Buffer (1×): FITC: PI = 40:1:1. Discard the old culture medium, add 100 μL of PBS to each well, aspirate the organoids into the used ultra-low adsorption plate, add 20 μL of the prepared staining mixture to each well, and incubate at room temperature in the dark for 20 min. After incubation, observe under a confocal microscope.

[0080] Figure 14 The image shows the apoptosis and necrosis of liver organoids from culture media 1 and 3 under the action of triptolide, as detected by Annexin V-PI double staining.

[0081] Drug toxicity evaluation results showed that after treatment with 1 μM triptolide, the apoptosis level of liver organoids derived from culture medium 1 was not significantly different from that of the control group; only when the drug concentration was increased to 10 μM did a large number of apoptotic cells appear in these organoids. In stark contrast, only 1 μM triptolide could induce significant apoptosis in liver organoids derived from culture medium 3. These results indicate that liver organoids constructed from culture medium 3 are more sensitive to triptolide stimulation.

[0082] The experiment on the effect of triptolide on liver organoid activity was conducted as follows: I. Drug administration stimulation: Liver organoids were induced using culture medium group 1 and culture medium group 3, respectively. After the organoids matured, the liver organoids induced by culture medium group 1 were divided into a normal group and a drug-treated group: Culture medium 1 normal control group: The induced liver organoids were added to the corresponding culture medium G of culture medium 1 group and cultured for another 24 hours.

[0083] Culture medium 1 + triptolide (0.1μM, 0.5μM, 1μM, 5μM, 10μM) groups: The induced liver organoids were added to the corresponding culture medium G of culture medium 1 group. Different concentrations of triptolide were added to culture medium G, and the culture was continued for 24h.

[0084] The liver organoids induced by culture medium 3 were divided into two groups: Culture medium group 3: The induced liver organoids were added to the corresponding culture medium G of culture medium group 3 and cultured for another 24 hours.

[0085] Culture medium 3 + triptolide (0.1μM, 0.5μM, 1μM, 5μM, 10μM): The induced liver organoids were added to the corresponding culture medium G of culture medium 3, and different concentrations of triptolide were added to culture medium G. The culture was continued for 24h.

[0086] The ATP release in organoids before and after treatment with triptolide was detected using the CellTiter-Glo® 3D Cell Viability Assay kit (Promega, G9681). Figure 15 Figures a and b show the effects of triptolide on liver organoid activity by detecting the relative ATP content of organoids using the CellTiter-Glo® 3DCell Viability Assay Kit (Promega, G9681).

[0087] The results of gradient concentration triptolide stimulation experiments showed a significant difference in the response thresholds of the two types of liver organoids: organoids from medium 1 only showed a significant decrease after treatment with 5 μM triptolide; while organoids from medium 3 showed a significant decrease in indicators even at a low concentration of 0.1 μM. This difference directly reflects that the liver organoids constructed from medium 3 are significantly more sensitive to triptolide stimulation than the former.

[0088] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing liver organoids for screening and toxicity assessment of traditional Chinese medicine, characterized in that: Includes the following steps: Pluripotent stem cells were expanded and cultured in a plane, then digested and passaged, and the process was repeated to obtain a population of pluripotent stem cells with uniform cell morphology and no spontaneous differentiation. The pluripotent stem cell population was subjected to two-dimensional planar orientation induction to enable the cells to receive differentiation signals synchronously and uniformly, thereby obtaining hepatocytes. The obtained hepatocytes were cultured in three-dimensional suspension using medium E to promote hepatic lineage specialization and spheroidization. Medium E consisted of Advanced DMEM / F12 containing 1-5 μM RA, 2-10 μM Y-27632, 0.5-1 mM β-nicotinamide mononucleotide, 5-10 μM ligustilide A, B27 and N2. Then the culture medium is changed to allow the hepatocytes to further mature and function, thus obtaining the desired product.

2. The method for constructing liver organoids for screening and toxicity assessment of traditional Chinese medicine according to claim 1, characterized in that: Culture medium E is Advanced DMEM / F12 containing 2-4 μM RA, 5-10 μM Y-27632, 0.7-1 mM β-nicotinamide mononucleotide, 7-10 μM ligustrazine lactone A, B27 and N2; Preferably, the culture medium E is Advanced DMEM / F12 comprising 2 μM RA, 10 μM Y-27632, 1 mM β-nicotinamide mononucleotide, 10 μM ligustilide A, B27 and N2.

3. The method for constructing liver organoids for screening and toxicity assessment of traditional Chinese medicine according to claim 1, characterized in that: The obtained liver cells were digested and seeded in ultra-low adsorption plates, and cultured stably in medium E for 0.5-1.5 days. Preferably, the obtained liver cells are digested, inoculated into an ultra-low adsorption plate, and cultured stably in medium E for 1 day.

4. The method for constructing liver organoids for screening and toxicity assessment of traditional Chinese medicine according to claim 1, characterized in that: Pluripotent stem cells were seeded onto a culture surface covered with pluripotent stem cell working solution and expanded in planar culture using mTeSR Plus medium. When the cell confluence reached 70%-85%, the cells were digested and passaged.

5. The method for constructing liver organoids for screening and toxicity assessment of traditional Chinese medicine according to claim 1, characterized in that: When performing two-dimensional planar orientation induction on pluripotent stem cell populations, the culture was first carried out in medium A containing Activin A and BMP4 for 4-7 hours on the first day, and then cultured in medium B containing Activin A and BMP4 for the remaining time. Then, culture in medium C containing Activin A and B27 for 2-3 days; Then culture in medium D containing FGF4 and CHIR99021 for 2-3 days to promote hepatocyte differentiation into blastoblasts; Preferably, when performing two-dimensional planar directional induction on a pluripotent stem cell population, the culture is first performed on the first day using medium A containing Activin A and BMP4 for 5-6 hours, and then cultured for the remaining time using medium B containing Activin A and BMP4. Then, cultured in medium C containing Activin A and B27 for 2 days; The cells were then cultured in medium D containing FGF4 and CHIR99021 for 2 days to promote differentiation of hepatocytes into blast cells. Preferably, culture medium A is RPMI 1640 containing 50-100 ng / mL Activin A, 20-60 ng / mL BMP4, and 1-4% B27; Culture medium B is a mixed culture medium containing 50-100 ng / mL Activin A and 20-60 ng / mL BMP4 of mTeSR Plus and RB, with a volume ratio of mTeSR Plus to RB of 1:2-4; Culture medium C is a mixed culture medium containing 50-100 ng / mL Activin A of mTeSR Plus and RB, with a volume ratio of mTeSR Plus to RB of 1:2-4; Culture medium D: Advanced DMEM / F12 containing 500 ng / mL FGF4, 3 μM CHIR99021, B27 and N2; Preferably, culture medium A is RPMI 1640 containing 100 ng / mL Activin A, 50 ng / mL BMP4, and 2% B27; Culture medium B consisted of 25% mTeSR Plus + 75% RB containing 100 ng / mL Activin A and 50 ng / mL BMP4, with RB containing 2% B27 in RPMI 1640. Culture medium C consisted of 25% mTeSR Plus + 75% RB containing 100 ng / mL Activin A; Culture medium D: Advanced DMEM / F12 containing 500 ng / mL FGF4, 3 μM CHIR99021, B27 and N2.

6. The method for constructing liver organoids for screening and toxicity assessment of traditional Chinese medicine according to claim 1, characterized in that: After culturing in culture medium E, replace culture medium E with culture medium F and culture for 1-3 days; then replace culture medium F and continue culturing for 1-3 days; then replace culture medium F with culture medium G and culture for 1-3 days; and continuously replace culture medium G and continue culturing until liver organoids are obtained.

7. The method for constructing liver organoids for screening and toxicity assessment of traditional Chinese medicine according to claim 6, characterized in that: Culture medium F was Advanced DMEM / F12 containing 2-4 μM RA, 0.7-1 mM nicotinamide mononucleotide, 7-10 μM ligustilide A, B27 and N2; Culture medium G was Williams' E medium containing primary hepatocyte maintenance culture additive, 1-4% B27, 0.5-1.51% N2, 0.7-1mM nicotinamide mononucleotide, 7-10μM ligustilide A, 5-10ng / mL OSM and 5-10ng / mL HGF.

8. The method for constructing liver organoids for screening and toxicity assessment of traditional Chinese medicine according to claim 7, characterized in that: Culture medium F was AdvancedDMEM / F12 containing 2 μM RA, 1 mM nicotinamide mononucleotide, 10 μM ligustilide A, B27 and N2; Culture medium G was Williams' E medium containing primary hepatocyte maintenance culture additive, 2% B27, 1% N2, 1mM nicotinamide mononucleotide, ligustrazine lactone A, 10 ng / mL OSM and 10 ng / mL HGF.

9. A liver organoid for screening and toxicity assessment of traditional Chinese medicine, characterized in that: Obtained by the construction method described in any one of claims 1-8.

10. The application of the liver organoids described in claim 9 for screening and toxicity assessment of traditional Chinese medicine in the screening of the activity of traditional Chinese medicine, the assessment of the toxicity of traditional Chinese medicine, and the study of liver diseases.

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