High-speed and large-scale preparation methods for liver organoids, and methods for screening drug efficacy and toxicity using them.

Abstract

This invention relates to a method for the rapid and large-scale preparation of liver organoids, and to methods for screening drugs related to liver diseases and screening drug in vitro toxicity using these methods. According to the above-described preparation method, liver organoids can be rapidly and massively prepared by continuously undergoing a three-dimensional differentiation process on the same microplate. The massively prepared liver organoids using the above-described method include hepatocytes (solid core part), cholangiocytes (cystic part), hepatic stellate cells, and Kupffer cells, possessing a cellular composition, structure, and function similar to actual liver tissue, and can stably proliferate in vitro. Furthermore, the in vitro drug toxicity screening method for liver organoids of this invention allows for the pre-screening of drugs that may have hepatotoxicity, shortening the time and cost of new drug development and improving its efficiency.

Description

Technical Field

[0001] This invention relates to a method for high-speed, large-scale preparation of liver organoids, a method for screening drugs for liver-related diseases, and a method for screening the in vitro toxicity of drugs. Background Technology

[0002] The liver is an organ with multiple functions, including metabolism, synthesis of various proteins such as cholesterol and bile acids, regulation and storage of glycine, and decomposition of toxic substances. Liver disease is one of the top five causes of death in South Korea, ranking second only to cancer among men in their 40s and 50s who are most active in society. In fact, South Korea has the highest mortality rate from liver diseases such as hepatitis, liver cancer, and cirrhosis among OECD countries, with annual medical expenses for liver disease treatment reaching 355 billion won, affecting approximately 1.66 million people.

[0003] The liver is a representative organ with intrinsic regenerative capacity in the body. Animal models or tumor-derived hepatocytes have been widely used in research on the mechanisms of liver diseases and the development of therapeutic drugs. However, due to genetic and physiological differences and limited functionality of human hepatocytes, in vivo-derived primary human hepatocytes (PHHs) have been evaluated as the most suitable model to date. However, in vivo-derived primary human hepatocytes have several limitations: they are difficult to culture in vitro, and their proliferative capacity and function are lost during in vitro culture, resulting in extremely limited usability. Therefore, there is a growing demand for the development of novel, patient-customized hepatocyte models based on human cells that possess stable in vitro proliferative capacity and high functionality.

[0004] To overcome the various limitations of in vivo-derived primary human hepatocytes, studies have been conducted using hepatocytes derived from pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). However, hepatocytes prepared using stem cell technology (differentiated hepatocytes and cross-differentiated hepatocytes derived from embryonic stem cells or induced pluripotent stem cells, etc.) have limited applicability due to their limited in vitro proliferation under reverse time and their functional differences from actual liver tissue.

[0005] Liver organoids derived from pluripotent stem cells (PSCs) have attracted significant attention as a promising alternative source. Organoids represent a novel stem cell differentiation technology that leverages the differentiation, self-renewal, and self-organization capabilities of stem cells. Through three-dimensional culture, they reproduce cellular composition and structure similar to those of organs in vivo. By simulating an environment similar to that of organs, they can be used for disease simulation studies and therapeutic drug screening for various diseases. Recently developed liver organoids have demonstrated stable long-term in vitro culture, exhibiting cellular composition, structure, and function similar to actual liver tissue. These organoids could be used for next-generation in vitro disease modeling of liver diseases and new drug development research.

[0006] However, to date, most liver organoid preparation technologies use both two-dimensional and three-dimensional differentiation, making it difficult to develop standardized technical methods and to prepare liver organoids in large quantities, which has fatal limitations.

[0007] Therefore, this invention optimizes the entire differentiation process of liver organoids, develops a method for preparing three-dimensional liver organoids, and, based on the above method, determines a method for high-speed and large-scale preparation of three-dimensional liver cell assemblies that can be continuously and without oscillation throughout the entire process on the same microplate, thus completing this invention.

[0008] Prior technology documents: Patent documents: (Patent document 1) KR 10-2107057. Summary of the Invention

[0009] Problems that need to be solved:

[0010] The technical problem to be achieved by the present invention is to provide a highly efficient, high-functionality, high-speed, and large-scale preparation method consisting only of differentiated three-dimensional liver cell aggregates, and a three-dimensional liver cell aggregate prepared by the above preparation method.

[0011] Another technical problem to be solved by the present invention is to provide a culture reagent for culturing the above-mentioned three-dimensional liver cell aggregates.

[0012] Another technical problem addressed by this invention is to provide the aforementioned three-dimensional liver cell aggregate. A method for screening drugs for liver-related diseases utilizing the aforementioned three-dimensional liver cell aggregate is provided, along with a method for providing information for the treatment of liver-related diseases and a method for screening the in vitro toxicity of drugs.

[0013] However, the technical problem to be solved by the present invention is not limited to the problem described herein, and other problems not mentioned herein can be clearly understood by those skilled in the art from the following description.

[0014] Solution to the problem:

[0015] To address the aforementioned problems, in a first aspect, the present invention provides a method for the high-speed, large-scale preparation of three-dimensional liver cell assemblies, comprising the following steps:

[0016] (1) The steps for forming an embryonic body from human stem cells;

[0017] (2) The step of inducing the embryo to differentiate into liver tissue and form a three-dimensional liver cell aggregate;

[0018] (3) The steps for further differentiation of the three-dimensional liver cell aggregate.

[0019] Among them, steps (1) to (3) are continuous non-oscillating cultures in the same microplate.

[0020] In another embodiment of the present invention, in step (1), the initial number of starting cells that can form an embryo body is 50 to 20,000, preferably 50 to 1,000, and more preferably 250.

[0021] In another embodiment of the invention, in step (2), a three-dimensional liver cell aggregate is formed over at least 20 to 30 days, approximately 20 days.

[0022] In another embodiment of the present invention, the stem cells isolated from humans include pluripotent stem cells (PSCs) and adult stem cells; pluripotent stem cells include human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs); adult stem cells include in vivo adult stem cells and adult stem cells prepared by cross-differentiation technology; hESCs are stem cells isolated or obtained from human embryos within 14 days of fertilization that have not undergone in vivo development.

[0023] In another embodiment of the invention, after forming a three-dimensional liver cell aggregate, further oscillation is performed, allowing for long-term culture.

[0024] In another embodiment of the invention, the microplate is a multi-microplate with notches, preferably a 96-well or 384-well microplate.

[0025] In another embodiment of the invention, the step of forming a three-dimensional liver cell aggregate includes an endoderm differentiation step or a hepatocyte differentiation step.

[0026] In another embodiment of the present invention, the above-mentioned endoderm differentiation step includes differentiation into any of the following endoderms: definitive endoderm (DE), foregut endoderm (FE), and posterior foregut endoderm (PFE).

[0027] In another embodiment of the invention, the step of forming a three-dimensional liver cell aggregate may involve adding Matrigel once or multiple times during the endoderm differentiation or hepatocyte differentiation steps. Preferably, Matrigel is added at a final concentration of 0.1-1% by volume during the foregut endoderm differentiation, retroforegut endoderm differentiation, and hepatocyte differentiation steps; more specifically, Matrigel is added to the culture medium rather than embedded (embedding refers to embedding organoids in Matrigel), and the aforementioned final concentration of 0.1-1% refers to a volume percentage concentration of Matrigel in the culture medium of 0.1-1%.

[0028] In another embodiment of the invention, the step of forming embryonic bodies is to add 20% KSR (serum substitute), 3% FBS (fetal bovine serum), 1% Glutamax (L-glutamine), 1% P / S (penicillin / streptomycin solution), 10 μM Y27632 (ROCK inhibitor) and 10 ng / mL bFGF (basic fibroblast growth factor) to DMEM / F12 medium.

[0029] In another embodiment of the invention, the shaping endoderm differentiation step involves adding 1% B27-VA (without vitamin A type B27 additive), 1% P / S, 1% NEAA (non-essential amino acids), 200 ng / mL activin A, 10 ng / mL BMP4 (bone morphogenetic protein 4), 10 μM LY294002 (PI3K inhibitor), and 3 μM CHIR99021 (GSK3 inhibitor) to RPMI1640 medium for 1 day of culture, followed by an additional 3 days of culture in RPMI1640 medium containing 1% B27-VA, 1% P / S, 1% NEAA, and 200 ng / mL activin A.

[0030] In another embodiment of the present invention, the foregut endoderm differentiation step is performed by adding B27-VA 1%, P / S 1%, NEAA 1%, Matrigel 0.5-1%, FGF4 (fibroblast growth factor 4) 500 ng / mL and 1 μM dorsomorphin to RPMI 1640 medium and culturing for 3 days.

[0031] In another embodiment of the present invention, the hindgut endoderm differentiation step is performed by adding 1% B27-VA, 1% P / S, 1% NEAA, 0.5-1% Matrigel, 500 ng / mL FGF4 and 2 μM retinoic acid to RPMI 1640 medium and culturing for 2 days.

[0032] In another embodiment of the present invention, the hepatoblast differentiation step is to add B27-VA 1%, P / S 1%, NEAA 1%, Matrigel 0.5-1%, BMP4 20ng / mL and bFGF 5ng / mL to RPMI 1640 medium and culture for 3 days.

[0033] In another embodiment of the invention, the step of forming a three-dimensional liver cell aggregate is to add 10% FBS, 1% NEAA, 1% Glutamax, 0.1% ITS (insulin-transferrin-selenium medium supplement), 10 μM Dexamethasone, 50 ng / mL HGF (hepatocyte growth factor), 20 ng / mL OSM (tumor suppressor M), 50 ng / mL EGF (epidermal growth factor), 0.5% Nicotinamide, and 4 ng / mL A83-01 (TGF-β inhibitor) to DMEM / F12 medium and culture for 6-10 days.

[0034] In another embodiment of the invention, the further differentiation step of the liver three-dimensional cell aggregate is carried out in expansion medium (EM), differentiation medium (DM) or a mixture thereof, and can be cultured in differentiation medium for 10-15 days.

[0035] In another embodiment of the invention, the maintenance culture medium (EM) is DMEM / F12 medium supplemented with B27 1%, N2 cell culture additive 1%, Glutamax 1%, HGF 25ng / mL, EGF 50ng / mL, N-Acetylcysteine ​​1mM, Gastrino 10nM, Forskolin 10μM, A83-01 5μM, Nicotinamide 10mM, OSM 10ng / mL, bFGF 10ng / mL and ITS 5μg / mL. Differentiation culture medium (DM) was prepared by adding B27 1%, N2 1%, Glutamax 1%, HGF 25ng / mL, EGF 50ng / mL, N-acetylcysteine ​​1mM, Gastrino 10nM, A83-01 0.5μM, DAPT (γ-secretase inhibitor) 10μM, and BMP7 (bone morphogenetic protein 7) 25ng / mL to DMEM / F12 medium.

[0036] In another embodiment of the invention, the shaping endoderm differentiation step is treated with 50 to 200 ng / mL activin A, preferably 200 ng / mL activin A.

[0037] In another embodiment of the invention, the foregut endoderm differentiation step is treated with 50 to 200 ng / mL noggin, preferably 200 ng / mL noggin or 1 μM dorsomorphin for 24 to 72 hours, preferably 72 hours.

[0038] In another embodiment of the invention, the hindgut endoderm differentiation step is treated with 0.5 to 2 μM retinoic acid (RA), preferably 2 μM retinoic acid, for 24 to 72 hours, preferably 48 hours.

[0039] In another embodiment of the invention, the hepatoblast differentiation step is performed with treatment of 5 to 50 ng / mL bFGF, preferably 5 ng / mL; and with treatment of 10 to 20 ng / mL BMP4, preferably 20 ng / mL.

[0040] In this document, “%” is used to indicate the concentration of a specific substance. Unless otherwise stated, solid / solid is (weight / weight)%, solid / liquid is (weight / volume)%, and liquid / liquid is (volume / volume)%.

[0041] In another embodiment of the present invention, during the shaping endoderm differentiation step, the expression of genes OCT4 and NANOG decreases, while the expression of genes SOX17, EOMES, CXCR4, and FOXA2 may increase. During the foregut endoderm differentiation step, the expression of gene SOX2 may increase, while the expression of gene CDX2 may be suppressed. The expression of genes HNF1B, HNF6, and ONECUT2 may increase during the hindgut endoderm differentiation step. During the aforementioned hepatoblast differentiation step, the expression of genes CK19, EPCAM, AFP, LGR5, ALB, TTR, SOX9, A1AT, and HNF4A may increase.

[0042] In a second aspect, the present invention provides a three-dimensional liver cell aggregate prepared using the high-speed, high-volume preparation method described in the first aspect above.

[0043] Thirdly, the present invention provides a culture reagent for culturing the three-dimensional liver cell aggregates described in the second aspect above. The culture reagent includes a recessed culture portion for culturing the three-dimensional liver cell aggregates and a covering portion covering the recessed culture portion. In a specific embodiment of the present invention, the recessed culture portion is, for example, a microplate body, and the covering portion covering the recessed culture portion is, for example, a corresponding microplate cap. Specifically, the recessed culture portion and the covering portion covering the recessed culture portion can be capped microplates for culturing organoids, preferably 96-well or 384-well capped microplates.

[0044] In one embodiment of the invention, the culture reagent may include a culture medium for culturing the aforementioned three-dimensional liver cell aggregates. According to a specific embodiment of the invention, the culture medium includes one or more culture media used in the method of the first aspect described above.

[0045] In one embodiment of the present invention, the culture reagent may include a preservation solution.

[0046] Fourthly, the present invention provides a drug screening method for liver-related diseases, comprising the following steps:

[0047] (1) The step of preparing and culturing a three-dimensional liver cell aggregate from cells derived from patients with liver-related diseases using the method described in the first aspect;

[0048] (2) The step of contacting the candidate substance with the above-mentioned three-dimensional liver cell aggregate;

[0049] (3) The step of determining the drug for treating liver-related diseases based on the situation of the three-dimensional liver cell aggregates that the candidate substance has been in contact with in step (2).

[0050] In one embodiment of the present invention, in the drug screening method of the fourth aspect, step (3) includes detecting cell viability, oxygen consumption rate (OCR) and measuring the expression level of biomarkers of liver-related diseases, and thereby determining, screening and evaluating drugs for treating liver-related diseases.

[0051] In another embodiment of the invention, liver-related diseases include at least one of the following: hepatitis virus, simple steatohepatitis, non-alcoholic fatty liver disease, liver inflammation, non-alcoholic steatohepatitis (NASH), cholestatic liver disease, liver fibrosis, cirrhosis, liver failure, and liver cancer.

[0052] Fifthly, the present invention provides a method for screening the in vitro toxicity of drugs using a three-dimensional liver cell aggregate, comprising the following steps:

[0053] (1) In any one or more steps of the high-speed mass production method for liver three-dimensional cell assemblies described in the first aspect, the candidate material is contacted with the three-dimensional cell assemblies;

[0054] (2) For the three-dimensional liver cell aggregates in the above steps, compare the reactions caused by the presence and absence of candidate substances;

[0055] (3) Identify whether cells in the above-mentioned three-dimensional liver cell assembly are dead.

[0056] In one embodiment of the present invention, in the screening method of the fifth aspect, the cells in step (3) include, but are not limited to, one or more of the following: hepatocytes, cholangiocytes, hepatic stellate cells, and Kupffer cells.

[0057] The effects of the invention:

[0058] This invention relates to a method for the rapid and large-scale preparation of liver organoids, a method for screening drugs for liver-related diseases using these organoids, and a method for screening the in vitro toxicity of these drugs. According to the above preparation method, liver organoids can be rapidly and massively prepared through a three-dimensional differentiation process on the same microplate. The massively prepared liver organoids contain hepatocytes, cholangiocytes, hepatic stellate cells, and Kupffer cells, exhibiting cellular composition, structure, and function similar to actual liver tissue. Furthermore, the in vitro toxicity screening method for liver organoids used in this invention can pre-screen drugs with hepatotoxicity, thereby shortening the time and cost of new drug development and improving its efficiency. Attached Figure Description

[0059] Figure 1 The present invention describes a method for preparing liver organoids consisting solely of a three-dimensional differentiation process. Unlike traditional methods that differentiate liver cells from human stem cells in a two-dimensional manner, the method for preparing liver organoids of the present invention includes processes such as embryonic body (EB) formation, shaped endoderm (DE) differentiation, foregut endoderm (FE) differentiation, post-foregut endoderm (PFE) differentiation, hepatocyte (HB) differentiation, and hepatocyte maturation. All of these processes are completed through three-dimensional culture, and the method demonstrates the process of high-speed and large-scale preparation of liver organoids through continuous non-oscillating culture.

[0060] Figure 2 This indicates the differentiation steps of the endoderm, and different initial cell numbers and activin A concentrations lead to the expression of SOX17, EOMES, and CXCR4 genes. Figure 2 In this context, hESCs refers to embryonic stem cells.

[0061] Figure 3 This indicates the expression of SOX2 and CDX2 genes after treatment with different concentrations of compounds such as noggin (NOG) or dorsomorphin (DOR) for 24, 48, and 72 hours during the foregut endoderm differentiation step. Figure 3 In this context, CHIR refers to CHIR99021 (a GSK3 inhibitor); IWP2 refers to Wnt / β-catenin inhibitor.

[0062] Figure 4 This indicates the expression of HNF1B, HNF6, and ONECUT2 genes after treatment with different concentrations of retinoic acid (RA) for 24, 48, and 72 hours during the hindgut-foregut endoderm differentiation stage. SB431542: TGF-β / Smad inhibitor.

[0063] Figure 5This indicates the expression of CK19, EPCAM, AFP, LGR5, ALB, and TTR genes treated with different concentrations of bFGF and BMP4 during the hepatoblast differentiation process.

[0064] Figure 6 This indicates the expression of specific genes in the liver organoids prepared under the optimized differentiation conditions of this invention at the undifferentiated step, the defined endoderm (DE) differentiation step, the foregut endoderm (FE) differentiation step, the post-foregut endoderm (PFE) differentiation step, and the hepatocyte (HB) differentiation step. Figure 6 In this context, hESC stands for embryonic stem cells.

[0065] Figure 7 This indicates that, in addition to embryonic stem cell (hESC) line H1, other embryonic stem cell lines H9 and induced stem cell lines hiPSC line 1 and hiPSC line 2 were used to verify reproducibility results under optimized differentiation conditions.

[0066] Figure 8 This indicates the preparation efficiency of liver organoids prepared under the optimized differentiation conditions in this invention, as well as the preparation ratio of liver organoids with different morphologies. Figure 8 In this context, LOs refer to liver organoids; Cyst LOs refer to cystic liver organoids; and Solid LOs refer to solid liver organoids.

[0067] Figure 9 Gene expression was compared between liver organoids including cysts and solids (Cyst), liver organoids composed only of solids (Solid), and embryonic stem cells (hESCs).

[0068] Figure 10 This indicates the subculture results of liver organoids including cysts and solids (hereinafter referred to as "cyst liver organoids") (CystLOs) and liver organoids consisting only of solids (Solid LOs).

[0069] Figure 11 This indicates confirmation of stable in vitro proliferation of cystic liver organoids.

[0070] Figure 12 This indicates the results of viability verification upon freezing and thawing of cystic liver organoids.

[0071] Figure 13 The expression of ALB, SOX9, A1AT, HNF4A, EPCAM, and LGR5 genes in cyst liver organoids was shown. Figure 13In the formula, DAPI stands for 4',6-diamidinyl-2-phenylindole; ZO-1 stands for zonula occludens1; and Actin stands for actin.

[0072] Figure 14 This indicates the gene expression level of cyst liver organoids matured in differentiation culture medium (DM) via qPCR. Figure 14 In this context, DM stands for differentiation medium, and EM stands for expansion medium.

[0073] Figure 15 This indicates the gene expression of matured cyst liver organoids in differentiation culture medium (DM) using immunohistochemical staining. Figure 15 In the original text, DAPI stands for 4',6-diamidinyl-2-phenylindole; ZO-1 stands for zonulaoccludens1.

[0074] Figure 16 The results of indocyanine green uptake assay and PAS staining (periodic acid-Schiff staining) were used to determine the exogenous compound metabolism and glycogen storage capacity of mature cystic liver organoids. Figure 16 In this context, DM stands for differentiation culture medium, and EM stands for maintenance culture medium.

[0075] Figure 17 The results of ELISA analysis were used to determine the CYP3A4 activity, total bile acid production, albumin (ALB) secretion, and urea secretion in mature cystic liver organoids.

[0076] Figure 18 This indicates the expression of specific genes in the capillary bile membrane, such as MRP2 and BSEP, in mature cystic liver organoids. Figure 18 In the Chinese text, DAPI stands for 4',6-diamidinyl-2-phenylindole; Actin is a myoprotein.

[0077] Figure 19 This represents the morphological results of liver organoids prepared by three-dimensional continuous culture on 96-well plates during the foregut endoderm (FE) and hepatocyte (HB) differentiation steps.

[0078] Figure 20 This represents the results of morphological analysis of liver organoids prepared by three-dimensional continuous culture in 96-well plates with different starting cell numbers.

[0079] Figure 21The results show the effect of different starting cell numbers on the efficiency of liver organoid preparation by three-dimensional continuous culture in 96-well plates.

[0080] Figure 22 This indicates the effect of matrix gel on liver organoids prepared by three-dimensional continuous culture in 96-well plates.

[0081] Figure 23 This indicates the morphological differences in liver organoids prepared by three-dimensional continuous culture on 96-well plates, depending on the concentration of the matrix gel.

[0082] Figure 24 According to Example 2.1, the morphology and gene expression of liver organoids were observed when a large number of prepared cyst liver organoids were cultured in maintenance medium (EM), differentiation medium (DM), and a 1:1 mixture of EM and DM.

[0083] Figure 25 The expression of ALB, A1AT, TTR, HNF4A, AFP, SOX9, and CK19 genes in the mature, mass-produced liver organoids (M Liver) prepared according to Example 2.2, the mature cyst liver organoids (T Liver) prepared according to Example 1.3, and the HepG2 hepatocyte cell line was compared. Figure 25 In this context, hESC stands for embryonic stem cell; T Liver for traditional liver organoid; and M Liver for micro liver organoid.

[0084] Figure 26 The cell composition of matured, mass-produced liver organoids was analyzed, and cell-specific genes were observed in hepatocytes (solid core part, hepatocytes) and cystic part (cholangiocytes).

[0085] Figure 27 This represents the results of immunohistochemical staining performed to confirm the presence of hepatic stellate cells in mature, mass-produced liver organoids. Figure 27 In the formula, DAPI: 4',6-diamidinyl-2-phenylindole; α-SMA: α-smooth muscle actin.

[0086] Figure 28 This indicates the morphological changes of two-dimensional (2D) differentiated hepatocytes and cystic liver organoids during hepatotoxic drug treatment.

[0087] Figure 29This indicates the toxic reactions caused by hepatotoxic drugs in 2D differentiated hepatocytes and cystic liver organoids. The results were validated at the molecular biological level by TUNEL (terminal deoxynucleotidyltransferase dUTP nick end labeling) staining.

[0088] Figure 30 The responses of 2D differentiated hepatocytes (2D heps) and cystic liver organoids (LOs) to low, moderate, and high doses of hepatotoxic drugs were compared. Detailed Implementation

[0089] Existing liver organoid preparation technologies face several technical challenges, including: 1) the random and spontaneous preparation of uncontrolled two-dimensional and three-dimensional differentiation processes for liver organoids; 2) the transfer from the initial 96-well plates to other culture methods such as 6cm dishes; 3) the use of a matrix gel embedding process; 4) the use of the high-valent differentiation promoter R-spondin; 5) low differentiation efficiency and reproducibility; and 6) the lack of large-scale preparation systems, making it difficult to develop standardized preparation systems.

[0090] To address these technical challenges, standardize the liver organoid preparation process, and enable high-speed, large-scale preparation, this invention develops a liver organoid preparation technology consisting solely of a three-dimensional differentiation process. This technology then enables non-oscillating culture on microplates, achieving near 100% efficiency in the high-speed, large-scale preparation of liver organoids.

[0091] More specifically, the entire differentiation process from human pluripotent stem cells to liver organoids, including embryonic body (EB) formation, definitive endoderm (DE) differentiation, foregut endoderm (FE) differentiation, posterior foregut endoderm (PFE) differentiation, and liver organoid maturation, was optimized at each differentiation step. Based on this, a three-dimensional differentiation process was continuously performed on the same microplate to produce large quantities of liver organoids, ultimately completing this invention. Figure 1 ).

[0092] In this invention, a "three-dimensional cell aggregate" can be an organoid. The term "organoid" in this invention refers to a three-dimensional cell aggregate formed through self-regeneration and self-organization by pluripotent stem cells, such as adult stem cells, embryonic stem cells, and induced blastocysts, derived from human liver tissue or prepared through various stem cell technologies such as cross-differentiation. Unlike two-dimensional culture, three-dimensional cell culture allows cells to grow in any direction in vitro, and these organoids can mimic the interactions of organs within a living organism, enabling applications such as drug development for treating diseases. Specifically, organoids constructed from patient tissues can be used for disease modeling based on the patient's genetic information and for drug screening through repeated trials.

[0093] In this invention, the term "stem cell" refers to a cell that has the ability to self-replicate and differentiate into two or more cells, and can be classified into pluripotent stem cells, multipotent stem cells, and other types.

[0094] In this invention, the term "hESC" refers to stem cells isolated or obtained from human embryos within 14 days of fertilization that have not undergone in vivo development.

[0095] In this invention, the term "culture medium" refers to a medium for the proliferation, survival, and differentiation support of liver organoids in vitro, including conventional media suitable for liver organoid culture and differentiation used in this field. The type of culture medium and culture conditions can be appropriately selected depending on the cell type. Specifically, the culture medium typically includes a minimum cell culture medium (CCMM) containing carbon sources, nitrogen sources, and trace elements. Minimal cell culture media include, but are not limited to, DMEM (Dulbecco's Modified Eagle's Medium), F-10, F-12, DMEM / F12, Advanced DMEM / F12, α-MEM (α-Minimal essential Medium), IMDM (Iscove's Modified Dulbecco), BME (BasalMedium Eagle), RPMI 1640, etc.

[0096] The terminology used in the embodiments is for illustrative purposes only and should not be construed as limiting. Singular expressions are to be understood to include both singular and plural meanings unless there is a clear difference in meaning within the text. In the terminology of this specification, "comprising" or "having" should be understood to indicate the presence of features, numbers, steps, operations, components, parts, or combinations thereof marked on the specification, rather than precluding the possibility of the presence or addition of one or more other features or numbers, steps, operations, components, parts, or combinations thereof.

[0097] Unless otherwise defined, all terms used herein, including technical or scientific terms, shall have the same meaning as commonly understood by one of ordinary knowledge in the art to which the embodiments pertain. Terms commonly used, such as those defined beforehand, shall be interpreted as having a consistent meaning in the related art and shall not be ideally or excessively interpreted in a formal sense unless expressly defined herein.

[0098] Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. However, various modifications can be made to the embodiments, and therefore the scope of the patent application is not limited or restricted by these embodiments. All modifications and equivalent substitutions to the embodiments should be understood to be included within the scope of the claims.

[0099] Furthermore, when describing the embodiments with reference to the accompanying drawings, the same components will be given the same reference numerals regardless of the graphic symbols used, and repeated descriptions will be omitted. When describing embodiments, detailed descriptions of related technologies will be omitted if it is believed that such detailed descriptions would unnecessarily obscure the summary of the embodiments.

[0100] Example

[0101] Example 1. Liver organoid preparation technology consisting solely of three-dimensional differentiation

[0102] 1.1 Optimizing the conditions for each differentiation step in the preparation of liver organoids

[0103] The optimized results of differentiation conditions for each differentiation step in the complete differentiation process from human stem cells to liver organoids, namely embryonic body (EB) formation, definitive endoderm (DE) differentiation, foregut endoderm (FE) differentiation, posterior foregut endoderm (PFE) differentiation, and liver organoid maturation, are as follows.

[0104] To optimize embryonic body (EB) formation and endoderm (DE) differentiation conditions, and to induce differentiation of various initial cell numbers (1×10⁻⁶), the following methods were employed. 3 5×10 3 and 1×10 4The concentrations of activin A (0, 50, 100, 200 ng / mL) were measured, and the expression of DE-specific genes SOX17, EOMES, and CXCR4 was confirmed under each condition. The results showed that the initial cell number for DE differentiation was 1 × 10⁻⁶ cells. 4 The highest expression level was observed when treated with 200 ng / mL activin A, indicating the highest differentiation efficiency. Figure 2 Based on this, the endodermal differentiation step involves adding 1% B27-VA (without vitamin A type B27 additive), 1% P / S, 1% NEAA (non-essential amino acids), 200 ng / mL activin A, 10 ng / mL BMP4 (bone morphogenetic protein 4), 10 μM LY294002 (PI3K inhibitor), and 3 μM CHIR99021 (GSK3 inhibitor) to RPMI1640 medium for 1 day of culture, followed by an additional 3 days of culture in RPMI1640 medium containing 1% B27-VA, 1% P / S, 1% NEAA, and 200 ng / mL activin A.

[0105] To determine the optimal conditions for foregut endoderm (FE) differentiation, noggin, a mid-hindgut differentiation factor BMP inhibitor, was used at different concentrations (50, 100, and 200 ng / mL) for 24, 48, and 72 hours. Under these conditions, the expression levels of the foregut endoderm genes CDX2 and SOX2 were confirmed. The results showed that treatment with 200 ng / mL noggin for 72 hours resulted in the highest SOX2 expression and the most suppressed CDX2 expression. Furthermore, when the small molecule compound dorsomorphin was used instead of noggin, it induced a higher SOX2 expression level, confirming that dorsomorphin can replace noggin during foregut endoderm (FE) differentiation. Treatment with 1 μM dorsomorphin showed the highest expression level. Figure 3 Based on this, the foregut endoderm differentiation step involves adding 1% B27-VA, 1% P / S, 1% NEAA, 0.5-1% Matrigel, 500 ng / mL FGF4 (fibroblast growth factor 4), and 1 μM dorsomorphin to RPMI 1640 medium and culturing for 3 days.

[0106] To optimize the differentiation conditions of the foregut endoderm (PFE), retinoic acid (RA) was used as an important factor for PFE differentiation. Treatment with different concentrations (0.5, 1, and 2 μM) for 24, 48, and 72 hours was conducted, and the expression of PFE genes NF1B, NF6, and ONECUT2 was confirmed under each condition. The results showed that treatment with 2 μM RA for 48 hours resulted in the highest expression level and the highest differentiation efficiency for PFE differentiation. Figure 4 Based on this, the hindgut endoderm differentiation step involves adding 1% B27-VA, 1% P / S, 1% NEAA, 0.5-1% Matrigel, 500 ng / mL FGF4 and 2 μM retinoic acid to RPMI 1640 medium and culturing for 2 days.

[0107] To optimize hepatoblast (HB) differentiation conditions, cells were treated with different concentrations of basic fibroblast growth factor (bFGF) (0, 5, 50 ng / mL) and bone morphogenetic protein 4 (BMP4) (0, 10, 20 ng / mL). The expression of CK19, EPCAM, AFP, LGR5, ALB, and TTR genes was determined under each condition. The results showed that treatment with 5 ng / mL bFGF and 20 ng / mL BMP4 resulted in the highest expression levels, indicating the highest differentiation efficiency. Figure 5 Based on this, the hepatoblast differentiation step involves adding B27-VA 1%, P / S 1%, NEAA 1%, Matrigel 0.5-1%, BMP4 20 ng / mL and bFGF 5 ng / mL to RPMI 1640 medium and culturing for 3 days.

[0108] Based on the optimized differentiation conditions described above, liver organoids were prepared, and gene expression at each step was confirmed by qPCR and immunohistostaining. The results showed that during the undifferentiated step, the defined endoderm (DE) differentiation step, the foregut endoderm (FE) differentiation step, the post-foregut endoderm (PFE) differentiation step, and the hepatocyte (HB) differentiation step, the specific genes for each step were correctly activated or inactivated. Figure 6 ).

[0109] To verify the reproducibility of the optimized differentiation conditions in this invention, liver organoids were prepared using embryonic stem cell line H1 hESC, as well as other embryonic stem cell lines H9 hESC and induced basal stem cell lines hiPSC cell line 1 and hiPSC cell line 2. The same result was confirmed in other cell lines, indicating that the optimized differentiation conditions of this invention are reproducible. Figure 7 ).

[0110] 1.2 Characteristics of liver organoids prepared under optimized differentiation conditions

[0111] When using the optimized differentiation conditions of this invention, liver organoids were prepared with 100% efficiency. 90% of these liver organoids were cystic liver organoids (Cyst LOs) centered on hepatocytes (solid core part, hepatocytes) and surrounded by numerous cystic cholangiocytes (cystic part, cholangiocytes), while 10% were liver organoids composed solely of hepatocytes (Solid LOs). Figure 8 ).

[0112] There is no significant difference in gene expression between liver organoids in the form of Cyst encapsulation (hereinafter referred to as "Cyst liver organoids") and liver organoids composed only of solid. Figure 9 However, liver organoids composed solely of solid material are difficult to proliferate during subculture. Figure 10 Conversely, Cyst liver organoids can stably proliferate in vitro during physical or chemical subculture and remain stable for more than 6 months (~10 generations). Figure 11 Therefore, Cyst liver organoids were used in subsequent experiments.

[0113] After more than three repeated freeze-thaw cycles, the Cyst liver organoids did not lose their in vitro proliferation capacity and remained stable. Figure 12 Furthermore, immunohistochemical staining confirmed that the prepared liver organoids expressed genes such as ALB, SOX9, A1AT, HNF4A, EPCAM, and LGR5. Figure 13 In other words, liver organoids prepared under the above-mentioned optimized differentiation conditions exhibit typical liver organoid structural characteristics, can stably proliferate in vitro, and are beneficial for large-scale drug screening and new drug development research that require a large number of liver organoids.

[0114] 1.3 Maturation of liver organoids

[0115] The prepared Cyst liver organoids were induced to mature in differentiation medium (DM) for one week. Gene expression levels were compared with those of immature Cyst liver organoids cultured in maintenance medium (EM) using qPCR and immunohistochemical staining. Compared with immature Cyst liver organoids derived from EM, the mature Cyst liver organoids derived from DM showed higher expression levels of various hepatocyte, bile duct cell, and prehepatic cell-specific genes. Figure 14 and 15 ).

[0116] To verify the in vitro functionality of mature Cyst liver organoids, ICG uptake analysis (exogenous substance metabolism) and PAS staining (glycogen storage) were performed. ELISA analysis confirmed CYP3A4 activity, total bile acid production, albumin production, and urea secretion. Results showed that mature Cyst liver organoids exhibited improved exogenous substance metabolism and glycogen storage capacity compared to immature Cyst liver organoids. Figure 16 ), CYP3A4 activity, total bile acid production, albumin and urea secretion capacity were also improved. Figure 17 The above results indicate that the Cyst liver organoid of the present invention is functionally mature and has sufficient in vitro functionality.

[0117] Bile production is a primary function of the liver. Bile produced by hepatocytes is transported to the bile ducts via thin-walled tubular structures called bile capillaries. The mature Cyst liver organoids of this invention exhibit marker genes for the bile capillary membrane, such as MRP2 and BSEP, indicating that the mature Cyst liver organoids of this invention are structurally similar to actual liver tissue. Figure 18 ).

[0118] Example 2. Mass production technology of continuously three-dimensionally differentiated liver organoids on the same microplate

[0119] 2.1 Mass Production Method of Liver Organoids

[0120] Example 1 started with an initial 96-well plate, but began with the shaping endoderm (DE) step, transferred to a 6 cm culture dish, and had to use a Matrigel drop embedding process, which incorporates the limitations of existing organoid technologies.

[0121] To establish a unified, full-cycle differentiation technology for organoid preparation and drug screening, it is necessary to develop a continuous culture method that eliminates the need for transfer and shaking culture from multi-well plates (such as 96-well plates) to other culture dishes. Furthermore, matrix gel embedding on 96-well plates is practically difficult to achieve, thus requiring the development of methods to omit or replace it. Recently, some researchers have reported methods for preparing various organ-specific organoids using self-made microplates. Utilizing existing commercially available microplate organoid preparation technologies suitable for high-throughput drug screening and high-content imaging is essential for commercialization.

[0122] Therefore, this invention develops a method for preparing liver organoids by continuous culture on a 96-well plate initially inoculated with cells, without the need for shaking culture. The stem cells used in this embodiment are embryonic stem cell line H1.

[0123] Embryonic stem cells (hESCs) isolated into single cells were seeded in 96-well plates with an initial cell number of 1 × 10⁶ cells. 2 2.5×10 2 1×10 3 2.5×10 3 and 5×10 3 In Example 1, differentiation was induced stepwise under optimized conditions. The morphology of the aggregates at different initial cell numbers was observed during the foregut endoderm (FE) and hepatocyte (HB) differentiation steps. The results showed that, although size differences existed, spherical aggregates were confirmed to form in all cases. Figure 19 ).

[0124] However, in the liver organoid formation process, the morphology varies greatly depending on the number of initial cells. If 1×10⁻⁶ cells are used... 3 More than one starting cell, and very few common liver organoids with Cyst morphology (1×10⁻⁶). 3 ) or not formed at all (2.5×10 3 5×10 3 Additionally, with an initial cell number of 1×10⁻⁶... 2 2.5×10 2 In this case, a typical differentiation state of liver organoids appeared ( Figure 20 ).

[0125] In terms of the efficiency of liver organoid preparation, the starting cell number was 2.5 × 10⁻⁶. 2 At this stage, the efficiency of liver organoid preparation was 100%, and liver organoids with typical Cyst morphology were observed in all wells of the 96-well plate. On the other hand, the efficiency was relatively low under other conditions, at 5 × 10⁻⁶. 3 Under these conditions, liver organoids that did not form a typical morphology ( Figure 21 ).

[0126] Unlike traditional methods of matrix gel embedding, the method used for large-scale preparation via continuous non-oscillating culture of stem cells involves directly adding matrix gel to the culture medium. Figure 1 The foregut endoderm (FE), hindate endoderm (PFE), and hepatoblast (HB) cells were treated with a final concentration of 1% Matrigel. The Matrigel concentration refers to the final volume percentage of Matrigel in the culture medium. Compared to the untreated population, Matrigel treatment significantly improved liver organoid growth rate and Cyst formation efficiency in all differentiation stages. Figure 22 ).

[0127] To confirm the effectiveness of Matrigel in three-dimensional continuous non-oscillating culture, different concentrations of Matrigel (0%, 0.5%, 1%, 2.5%, and 5%) were used to treat foregut endoderm (FE), hindbrain endoderm (PFE), and hepatocytes (HB) at different stages, and their effects were compared. The results showed that no Cyst-shaped liver organoids were observed to form when there was no Matrigel treatment or high concentrations (2.5% and 5%), while low concentrations (0.5% and 1%) effectively formed liver organoids with Cyst morphology. Figure 23 Furthermore, in the case of 0.5% or 1% treatment, the formation of Cyst-shaped liver organoids was also observed in the hepatoblast (HB) step, confirming that adding low concentrations of matrix gel directly to the culture medium is more effective in generating Cyst-shaped liver organoids. Figure 23 ).

[0128] 2.2 Optimize the culture conditions for large-scale preparation of liver organoids

[0129] In Example 2.1, to optimize the culture conditions for the mass-produced liver organoids, they were cultured in maintenance medium (EM), differentiation medium (DM), and a 1:1 mixture of EM and DM. The results showed that in the liver organoids cultured in differentiation medium (DM) (hereinafter referred to as "matured mass-produced liver organoids"), most specific genes were highly expressed. Figure 24 The above results indicate that functional maturation can be achieved by culturing large quantities of liver organoids in differentiation medium (DM).

[0130] 2.3 Characteristics of large-scale preparation of liver organoids after maturation

[0131] The gene expression of the matured, mass-produced liver organoids (micro liver organoids, M Liver) in Example 2.2 was compared with that of the matured Cyst liver organoids (traditional liver organoids, T Liver) in Example 1.3 and the HepG2 hepatocyte cell line (a 2D hepatocellular carcinoma cell line, usually used as a control for functional comparison with 3D liver organoids). The results confirmed that most specific genes were expressed at higher levels in the matured, mass-produced liver organoids of Example 2.2. Figure 25 This indicates that the mature and mass-produced liver organoids of this invention are superior to liver cell lines and liver organoids prepared by existing technologies.

[0132] To understand the cellular composition of matured, mass-produced liver organoids, immunohistochemical staining analysis was performed. Morphological analysis confirmed that these liver organoids possessed both solid (solid core part, hepatocytes) and cystic (cystic part, cholangiocytes) cells. Actual immunohistochemical staining results confirmed that the hepatocyte portion of the matured, mass-produced liver organoids expressed ALB and taurocholate cotransporter (NTCP), while the cholangiocyte portion did not express ALB or NTCP. Figure 26 Furthermore, immunohistochemical staining was used to verify the presence of non-solid cells—hepatic stellate cells and Kupffer cells—in matured, mass-produced liver organoids. The results showed that in mass-produced liver organoids differentiated for 20-40 days, hepatic stellate cell-specific genes α-smooth muscle actin (α-SMA), WT1, and vimentin were expressed, and the expression of the Kupffer cell-specific gene CD68 was confirmed. Figure 27 The above results indicate that liver organoids prepared in large quantities through maturation can have a cellular composition similar to that of actual liver tissue (hepatocytes, bile duct cells, hepatic stellate cells, Cooper cells, etc.) during a short-term differentiation process.

[0133] Example 3. In vitro toxicity screening technology based on liver organoids

[0134] Basic in vitro toxicity risk studies were conducted using the liver organoids of this invention. To this end, the toxicity of low-frequency hepatotoxic drugs thioridazine hydrochloride and chloroquine diphosphate, as well as drugs withdrawn from the market due to hepatotoxicity, such as cyclosporin A, nefazodone, rac-perhexiline maleate, sulindac, troglitazone, and tamoxifen, were compared and analyzed in two-dimensional (2D) differentiated hepatocytes and Cyst liver organoids of Example 1.2 of this invention. After treatment with six drugs (nefazodone, thioridazine HCl, Rac-perhexiline maleate, chloroquine diphosphate, troglitazone, and tamoxifen), cell death in 2D differentiated hepatocytes and a reduction in the size of Cyst liver organoids were observed. No morphological changes were observed in the cyclosporin A and sulindac treatment groups. Figure 28 ).

[0135] To validate the toxic effects of each drug at the molecular biological level, TUNEL (terminal deoxynucleotidyl transferase dUTP nickend labeling) staining was performed. The results showed that most drugs exhibited more sensitive toxic responses in Cyst liver organoids than in 2D differentiated hepatocytes. Figure 29 Cyclosporin A showed more sensitive toxicity in liver organoids at all concentrations. Nefazodone, thioridazine HCl, Rac-Perhexiline maleate, sulindac, and chloroquine diphosphate showed more sensitive toxicity in liver organoids than in two-dimensionally differentiated hepatocytes at both low and high concentrations.

[0136] Furthermore, no toxicity was observed when treated with rosiglitazone and buspirone, which are structurally similar to troglitazone and nefazodone but non-toxic. This indicates that Cyst liver organoids can accurately predict the toxicity level of drugs. Figure 28 and Figure 29 ).

[0137] The results confirmed that Cyst liver organoids exhibited more accurate and sensitive toxicity responses than two-dimensionally differentiated hepatocytes. Figure 30 The above results demonstrate that not only the Cyst liver organoids of this invention, but also mature, mass-produced liver organoids with a cell composition similar to that of actual liver tissue, can serve as in vitro models for evaluating the toxicity of drug candidates, thus proving their applicability.

[0138] As described above, although the embodiments are illustrated with limited figures, various technical modifications and variations can be applied based on the above description by those skilled in the art. For example, appropriate results can also be obtained by performing the techniques and methods in different orders, combining or integrating the system, structure, equipment, circuitry, and other elements in a different form than the method, or by replacing or substituting them with other components or articles.

[0139] Therefore, other embodiments and contents equivalent to the scope of this patent application are all within the scope of the claims of this invention.

Claims

1. A method for high-speed, large-scale preparation of three-dimensional liver cell assemblies, characterized in that, Includes the following steps: (1) The step of forming embryonic bodies from human stem cells is to add 20% serum substitute, 3% FBS, 1% L-glutamine, 1% penicillin / streptomycin solution, 10 μM ROCK inhibitor and 10 ng / mL basic fibroblast growth factor to DMEM / F12 medium; wherein the initial number of starting cells is 100 to 250. (2) The step of inducing differentiation from the embryonic body into liver tissue and forming a three-dimensional liver cell aggregate within 20 to 30 days; the step of forming a three-dimensional liver cell aggregate is to add FBS 10%, NEAA 1%, L-glutamine 1%, insulin-transferrin-selenium medium supplement 0.1%, dexamethasone 10 μM, hepatocyte growth factor 50 ng / mL, tumor suppressin M 20 ng / mL, epidermal growth factor 50 ng / mL, nicotinamide 0.5% and A83-01 4 ng / mL to DMEM / F12 medium and culture for 6 to 10 days; (3) The step of further differentiation of the three-dimensional liver cell aggregate is carried out in a differentiation culture medium for 10-15 days; wherein the differentiation culture medium is DMEM / F12 medium with the following added: B27 1%, N2 1%, L-glutamine 1%, hepatocyte growth factor 25 ng / mL, epidermal growth factor 50 ng / mL, N-acetylcysteine ​​1mM, gastrin 10 nM, A83-01 0.5 μM, γ-secretase inhibitor 10 μM and bone morphogenetic protein 7 25 ng / mL; Among them, steps (1) to (3) are continuous non-oscillating culture on the same microplate. During continuous non-oscillating culture, the matrix gel is directly added to the culture medium, and the matrix gel has a volume percentage concentration of 0.5% or 1%. The microporous plate is a multi-microporous plate with notches, and the multi-microporous plate with notches is a 96-well plate; The stem cells are hESC or hiPSC, and the hESC is a stem cell isolated or obtained from a human embryo within 14 days of fertilization that has not undergone in vivo development.

2. The method according to claim 1, characterized in that, In step (1), the serum substitute is KSR.

3. The method according to claim 1, characterized in that, In step (1), the ROCK inhibitor is Y27632.

4. The method according to claim 1, characterized in that, In step (3), the γ-secretase inhibitor is DAPT.

5. The method according to claim 1, characterized in that, After the three-dimensional liver cell aggregate is formed, it can be further cultured by shaking, and can be cultured for a long time.

6. The method according to claim 1, characterized in that, The steps for forming a three-dimensional liver cell aggregate include an endoderm differentiation step or a hepatocyte differentiation step.

7. The method according to claim 6, characterized in that, The endoderm differentiation step is selected from any one or more endoderm differentiations of the fixed endoderm, foregut endoderm, and hinforegut endoderm. The defined endoderm differentiation step involves adding 1% vitamin A type B27 additive (removed), 1% penicillin / streptomycin solution, 1% non-essential amino acids, 200 ng / mL activin A, 410 ng / mL bone morphogenetic protein, 10 μM PI3K inhibitor, and 3 μM GSK3 inhibitor to RPMI1640 medium for 1 day of culture, followed by an additional 3 days of culture in RPMI1640 medium containing 1% vitamin A type B27 additive (removed), 1% penicillin / streptomycin solution, 1% non-essential amino acids, and 200 ng / mL activin A. The foregut endoderm differentiation step involves adding 1% of vitamin A type B27-removed additive, 1% penicillin / streptomycin solution, 1% non-essential amino acids, 0.5% or 1% of matrix gel volume percentage, 4500 ng / mL of fibroblast growth factor, and 1 μM of dihydrodeoxymorphine to RPMI 1640 medium and culturing for 3 days. The hindgut endoderm differentiation step involves adding 1% of vitamin A type B27-removed additive, 1% penicillin / streptomycin solution, 1% non-essential amino acids, 0.5% or 1% of matrix gel volume percentage, 4500 ng / mL of fibroblast growth factor, and 2 μM of retinoic acid to RPMI 1640 medium and culturing for 2 days. The hepatoblast differentiation step involved adding 1% vitamin A type B27 additive (without P / S), 1% non-essential amino acids, 0.5% or 1% matrix gel volume percentage, 420 ng / mL bone morphogenetic protein, and 5 ng / mL basic fibroblast growth factor to RPMI 1640 medium and culturing for 3 days.

8. The method according to claim 7, characterized in that, The PI3K inhibitor is LY294002.

9. The method according to claim 7, characterized in that, The GSK3 inhibitor is CHIR99021.

10. The method according to claim 6, characterized in that, In the step of forming the three-dimensional liver cell aggregate, during the endoderm differentiation or hepatocyte differentiation step, matrix gel is added once or multiple times at a volume ratio of 0.5% or 1%.

11. A drug screening method for liver-related diseases, characterized in that, Includes the following steps: (1) The step of preparing a three-dimensional liver cell aggregate from cells derived from patients with liver-related diseases using the method according to claim 1; (2) The step of contacting the candidate substance with the three-dimensional liver cell aggregate; (3) The steps of screening and evaluating drugs for liver-related diseases based on the contact of candidate substances with the three-dimensional liver cell aggregate.

12. The method according to claim 11, characterized in that, In step (3), drugs for liver-related diseases are screened or evaluated by detecting cell survival rate, oxygen consumption rate or expression levels of biomarkers for liver-related diseases.

13. The method according to claim 11, characterized in that, The liver-related diseases are selected from at least one of the following: simple steatohepatitis, non-alcoholic fatty liver disease, liver inflammation, cholestatic liver disease, liver fibrosis, cirrhosis, liver failure, and liver cancer.

14. A method for screening the in vitro toxicity of drugs, characterized in that, Includes the following steps: (1) In step (3) of the method of claim 1, the step of contacting the candidate material with the mature three-dimensional cell assembly; (2) A step of comparing the reactions caused by the presence or absence of candidate substances in the three-dimensional liver cell aggregate in the above steps; (3) The step of identifying whether the cells in the three-dimensional liver cell assembly are dead.

15. The method according to claim 14, characterized in that, In step (3), the cells are selected from any one or more of hepatocytes, bile duct cells, hepatic stellate cells, and Cooper cells.

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