A method for constructing liver organoids based on human ipscs and application thereof in drug hepatotoxicity evaluation

Abstract

The present application relates to a method for constructing a liver organoid based on human IPSC and its application in drug hepatotoxicity evaluation. Specifically, the present application relates to a method for inducing and differentiating iPSC in vitro to form a liver organoid, a liver organoid derived from iPSC cells and the application of the organoid in drug hepatotoxicity evaluation.

Description

Technical Field

[0001] This invention relates to the field of biomedicine. Specifically, this invention relates to a method for constructing liver organoids based on human iPSCs and their application in drug hepatotoxicity detection. Background Technology

[0002] Drug-induced liver injury (DILI) is a serious adverse drug reaction in clinical practice, significantly increasing the risk of failure in the clinical development of drugs and being one of the main reasons for the withdrawal of marketed drugs. Although hepatotoxicity is systematically assessed in the early stages of drug development through in vitro cell evaluation and animal experiments, traditional preclinical methods can only predict about 40-50% of clinical hepatotoxicity cases due to the limitations of existing models and interspecies differences. This insufficient predictive accuracy not only leads to huge losses in R&D investment and increases the risks and costs of new drug development, but also poses a potential threat to drug safety. Therefore, developing new models that can more accurately predict hepatotoxicity in humans has become a key technological bottleneck that urgently needs to be overcome in the field of drug development.

[0003] Currently, DILI detection models mainly include traditional two-dimensional cell culture models, animal models, and cutting-edge organoid and organ-on-a-chip models. Commonly used cell models such as HepG2, HepaRG, and primary human hepatocytes each have significant limitations: HepG2 cells lack key drug-metabolizing enzyme activity; HepaRG cells, while expressing cytochrome P450 enzymes, have a single cell type, mainly consisting of hepatocyte-like and bile duct-like cells, lacking non-parenchymal cells; and primary human hepatocytes, considered the "gold standard," rapidly dedifferentiate in two-dimensional culture, losing their characteristics, and are scarce and highly individualized, making large-scale screening difficult. More importantly, these traditional cell models cannot reproduce the three-dimensional microenvironment of the liver in vivo and the complex intercellular interactions. Although animal experiments have long been the cornerstone of safety evaluation, their predictive value for human responses is limited due to the inherent physiological and metabolic differences between species.

[0004] The development of organoid and organ-on-a-chip technologies has provided new tools for DILI (Digital Intestinal Organoid) detection. Organoids are three-dimensional cell cultures formed by in vitro culture of stem cells and other autologous cells, composed of multiple tissue- and organ-specific cells, and possessing key structural and functional characteristics of specific tissues and organs. Based on their cell origin, they are mainly divided into tissue-derived organoids (such as those directly cultured from adult stem cells in tissues or tumor stem cells in tumor tissues) and iPSC (induced pluripotent stem cell)-derived organoids (differentiated through directed induction by iPSCs). Since the successful cultivation of the first functional small intestinal organoid by the team of Dutch scientist Hans Clevers in 2009, organoid technology has developed rapidly and is now widely used in basic research (such as developmental biology and tumor biology), disease modeling, drug screening and toxicity prediction, personalized medicine (such as drug sensitivity testing), and regenerative medicine.

[0005] Numerous studies have utilized liver organoids for the detection and mechanistic exploration of drug-induced liver injury (DILI). However, existing models have significant limitations in cellular composition and spatial structure. Most reported liver organoids consist only of hepatocytes and bile duct epithelial cells, lacking non-parenchymal cells, such as hepatic stellate cells (HSCs) and Kupffer cells, which play a central role in the key pathological processes of DILI—immune stress and fibrosis. Although a few studies have constructed complex organoids containing non-parenchymal cells, they present all cells as homogeneous 3D spherical aggregates. This configuration does not conform to the actual microanatomical structure of the liver: under physiological conditions, Kupffer cells are anchored to the inner wall of the hepatic sinusoidal lumen as resident macrophages, rather than being scattered among parenchymal cells. Therefore, such models lack fidelity in the spatial localization and hierarchical structure of key cell types. Advanced models such as organ-on-a-chip simulate the cell arrangement of physiological spatial structures through compartmentalized culture chambers and have shown good results in DILI assays. However, their large-scale application is limited by the high cost of equipment, the need for primary liver, stellate, and Kupffer cells, the long purchase cycle, and the high cost.

[0006] Given the aforementioned limitations, there is an urgent need for a hepatotoxicity detection model that has a wide range of cell sources, controllable cycle costs, and high physiological relevance and predictive accuracy. This model is of great significance for improving the accuracy of drug safety evaluation and accelerating the drug development process. Summary of the Invention

[0007] This invention aims to provide a method for constructing liver organoids based on human iPSCs, addressing the problems of limited cell types and insufficient physiological relevance in existing models. Another objective of this invention is to apply the constructed organoids to drug hepatotoxicity evaluation, achieving efficient and accurate detection of multi-dimensional hepatotoxicity, including lipid accumulation, mitochondrial toxicity, cholestasis, and fibrosis.

[0008] To achieve the above objectives, the technical solution of the present invention is implemented as follows: This invention relates to a method for constructing liver organoids derived from iPSCs, comprising: 1) Targeted differentiation of iPSCs is achieved using a specific culture medium containing activin A, BMP4, and other components to obtain oriented endoderms; 2) Adjust the culture medium composition to continue differentiation into the foregut endoderm; 3) Foregut cells are dissociated into single cells, embedded in Matrix gel, and differentiated into liver progenitor organoids; 4) Provide specific factors to induce liver organoid maturation.

[0009] On the other hand, the present invention relates to organoid identification and functional testing: detecting functional and specific cellular component markers of liver organoids, including albumin secretion level, urea secretion level, CYP enzyme activity detection and gene marker expression level detection.

[0010] This invention also provides a hepatotoxicity detection method: detecting the levels of lipid accumulation, oxidative stress, cholestasis, mitochondrial toxicity, fibrosis, and cell viability in liver organoids under different drug treatments.

[0011] Therefore, in a first aspect, the present invention provides a method for inducing differentiation of iPSCs in vitro to form liver organoids, the method comprising the following steps: i) Provide iPSC cells; ii) Culture the isolated iPSC cells provided in step i) in endoderm induction medium for 1 to 7 days, preferably 1 to 3 days, to obtain oriented endoderm; iii) The oriented endoderm obtained in step ii) is cultured in foregut induction medium for 1 to 7 days, preferably 1 to 3 days, to obtain foregut microspheres; iv) Dissociate the foregut microspheres obtained in step iii) into single cells, embed them in matrix gel, and culture them in liver progenitor organoid induction medium for 4 to 15 days, preferably 4 to 8 days, to obtain liver progenitor organs; and v) Culture the liver progenitor organoids obtained in step iv) in hepatocyte maturation medium for 7 to 25 days, preferably 7 to 14 days, to obtain liver organoids.

[0012] In a specific implementation, step v) of the method described in the first aspect of the present invention specifically includes: v-1) The liver progenitor organoids formed from the Matrigel were recovered, re-embedded in fresh Matrigel, and seeded in a dome shape into tissue culture plates. v-2) Continue culturing hepatocytes in a culture medium supplemented with hepatocyte growth factor, dexamethasone, oncostatin M, and penicillin / streptomycin for 7 to 14 days to obtain mature liver organoids.

[0013] In a specific implementation, step ii) of the method described in the first aspect of the present invention specifically includes: ii-1) Incubate for 1 day in RPMI 1640 medium containing Activin A and BMP4; ii-2) Incubate for 1 day in RPMI 1640 medium containing Activin A + 0.2% FBS; and ii-3) Incubate for 1 day in RPMI 1640 medium containing Activin A + 2% FBS; Preferably, the concentration of Activin A is 10-500 ng / ml, more preferably 10-400 ng / ml, 10-300 ng / ml, 10-200 ng / ml, and most preferably 100 ng / ml; The concentration of BMP4 is 10-100 ng / ml, preferably 50 ng / ml.

[0014] In a specific implementation, step iii) of the method described in the first aspect of the present invention specifically includes: iii-1) In a culture medium containing fibroblast growth factor (FGF) and a GSK3 inhibitor, directional differentiation of the endoderm into foregut microspheres is induced; preferably, iii-1) Culture oriented endoderms in Advanced DMEM / F12 medium containing B27, N2, FGF4, CHIR99021 and penicillin / streptomycin for 3 days.

[0015] In embodiments of the present invention, FGF is selected from any one or more of FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22 and FGF23.

[0016] In the specific implementation plan, FGF is FGF4.

[0017] In a specific implementation, the content of FGF (e.g., FGF4) can be from 10 ng / ml to 1000 ng / ml; preferably, the content of FGF (e.g., FGF4) is from 50 ng / ml to 900 ng / ml, 100 ng / ml to 800 ng / ml, 150 ng / ml to 700 ng / ml, or 200 ng / ml to 600 ng / ml; more preferably, the content of FGF (e.g., FGF4) is 500 ng / ml.

[0018] In the specific implementation plan, the GSK3 inhibitor is Chiron / CHIR99021.

[0019] In specific implementations, the GSK3 inhibitor can be administered in amounts of 1 μM to 100 μM, or 2 μM to 50 μM, or 3 μM to 25 μM. Preferably, the amount of GSK3 inhibitor is 3 μM.

[0020] In specific implementations, the iPSC cells provided by the present invention are mammalian-derived iPSC cells, such as, but not limited to, iPSC cells derived from humans, cattle, dogs, cats, sheep, goats, rats, or mice.

[0021] In a specific implementation, the iPSC cells provided by the present invention can be cultured and passaged in vitro to obtain a sufficient number of iPSC cells.

[0022] In a specific implementation, the interval between step i) and step ii) may include: Step ib) The isolated iPSC cells from step i) are single-celled.

[0023] In a specific implementation, steps iii) and iv) may optionally include: iii-b) Cryopreservation of foregut microspheres from step iii).

[0024] In a second aspect, the present invention provides a liver organoid derived from iPSC cells, which has a mixed morphology of cysts, clusters and fibroblast-like cells, with cysts measuring about 100-1000 μm in size, preferably about 500-1000 μm in size.

[0025] Thirdly, the present invention provides a method for assessing the extent of drug-induced liver damage in vitro, the method comprising the step of contacting the drug to be assessed with a liver organoid derived from iPSC cells provided by the present invention.

[0026] Compared with existing technologies, the liver organoid construction and hepatotoxicity detection method described in this invention have the following advantages: iPSCs are widely available and can be expanded and passaged, providing a highly accessible and low-cost source for organoids. Organoids include both parenchymal and non-parenchymal cells, with comprehensive lineages and functions, providing highly physiologically relevant models and supporting multidimensional detection of hepatotoxicity types and mechanistic studies. The foregut stage can be cryopreserved, and cryopreservation and thawing do not affect subsequent differentiation, effectively shortening the culture cycle. Single cells in the foregut stage are then embedded, a process compatible with high-throughput plating systems, which helps improve throughput and data stability.

[0027] Compared with the method in CN201780065706.5, the method in this application has the following different technical effects: By adjusting and optimizing the formulation of liver organoid differentiation / maturation culture medium factors and the culture time of different steps, liver organoids with special morphological structures are obtained, exhibiting a mixed morphology of 3D microspheres and 2D fibroblast-like cells. The 3D microspheres contain hepatocytes, bile duct cells, and stellate cells, while the 2D fibroblast-like cells are Kupffer cells. This morphology more closely resembles the actual physiological structure. Kupffer cells, as liver-resident macrophages, are normally positioned to attach to the hepatic sinusoidal endothelium, separated from hepatocytes but capable of interaction, rather than being mixed in with the spherical structure. This more physiologically relevant structure can better simulate the physiological functions of the real liver and can be used for hepatotoxicity detection, especially for cholestasis and fibrosis detection.

[0028] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings and tables used in the embodiments of the present invention will be briefly introduced below. The accompanying drawings and tables described below are only used to explain some embodiments of the present invention. Attached Figure Description

[0029] Figure 1 This is a flowchart of the method for constructing liver organoids derived from iPSCs in Embodiment 1 of the present invention.

[0030] Figure 2 These are bright-field images of different stages of liver organoid differentiation in Embodiment 2 of the present invention.

[0031] Figure 3 This is the result of qPCR expression detection of liver organoids in Example 2 of the present invention.

[0032] Figure 4 The images show the immunofluorescence detection results of liver parenchymal cell and stellate cell markers in Example 2 of this invention. From left to right, they are HNF4a, A-SMA, and honest staining images.

[0033] Figure 5 The results are the functional detection results of liver organoids in Example 2 of this invention, including albumin secretion, urea secretion, and CYP enzyme activity.

[0034] Figure 6 This describes the effect of drug treatment on the levels of AST, ALB, GSH, and LDH in Example 3 of this invention.

[0035] Figure 7 The results of high-content analysis predicting the lipid accumulation toxicity of the drug in Example 3 of this invention are as follows: A. Lipidtox staining images of organoids treated with different concentrations of non-aluridine (FIAU): blue indicates staining with the nuclear dye Hoechst 3332; white indicates staining with the lipid dye Lipidtox. All images are dual-channel merged images.

[0036] B. Per-well multi-field LIPIDTOX positive percentage statistics C. Fluorescence intensity ratio of LIPIDTOX to Hoechst 33342 per well in multi-field view It is evident that the degree of lipid accumulation increases in a concentration-dependent manner.

[0037] Figure 8 This is the high-content analysis used in Example 3 of the present invention to predict the mitochondrial toxicity and cytotoxicity of the drug.

[0038] Staining images of organoids treated with acetaminophen (APAP) and nefazodone (Nef): Blue indicates staining with the nuclear dye Hoechst 3332; orange indicates staining with the mitochondrial membrane potential dye tetramethylrhodamine methyl ester (TMRM); red indicates staining with the cytotoxic dye Celltox. Figure 9 In Example 3 of this invention, high-content analysis was used to predict the cholestasis toxicity of the drug: the bile acid transport activity was detected using the bile acid tracer probe CLF. The left image is CLF fluorescence channel imaging (green), and the right image is fluorescence superimposed bright field imaging.

[0039] Figure 10 This is a chromogenic staining image of paraffin sections of organoids in Embodiment 3 of the present invention. The left side is the control group, and the right side is the drug-treated group. The drug is methotrexate (MTX). The staining images of the two regions on the sections are shown respectively.

[0040] Figure 11 This is a diagram of drug toxicity detection results based on 3D-CTG in Embodiment 3 of the present invention. Detailed Implementation

[0041] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0042] Culture and passage of induced pluripotent stem cells (iPSCs) The induced pluripotent stem cells used in this article can be obtained from a variety of sources, including, but not limited to, iPSCs derived by transfecting certain stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts. Common sources of iPSCs are described in Chinese patent application CN201780065705.3, the contents of which are incorporated herein by reference.

[0043] In the specific implementation plan, the human-induced pluripotent stem cells used in this paper were purchased from Wuhan Saiss Biotechnology Co., Ltd., catalog number SC-DRY0100.

[0044] After obtaining iPSCs, they can be cultured and passaged in complete medium mTeSR and culture plates coated with matrix gel to obtain sufficient iPSCs for subsequent steps.

[0045] Specifically, iPSC cells were suspended in a complete culture medium containing ROCKi+P / S and the cells were plated on a matrix gel-coated culture plate.

[0046] The matrix adhesive used in this article may be fibrinogen, laminin, hyalin or a complex thereof.

[0047] After 1 to 7 days of culture and passage, sufficient iPSC cells can be obtained for the following steps.

[0048] Inducing iPSC cells to differentiate into directed endoderm iPSC cells can be induced to differentiate into the endodermis using any method known in the art, as described in detail in Chinese patent application CN201780065705.3, the contents of which are incorporated herein by reference.

[0049] Specifically, single cells of iPSCs were isolated using a single-cell digestion solution (Wuhan Saiss Biotechnology Co., Ltd., RC-012) and induced to differentiate in an endoderm induction medium according to the following steps: a) On day 1, cultured in RPMI 1640 medium containing Activin A and BMP4; b) On the second day, cultured in RPMI 1640 medium containing Activin A + 0.2% FBS; and c) On the third day, culture in RPMI 1640 medium containing Activin A + 2% FBS.

[0050] The concentration of Activin A is 10-500 ng / ml, preferably 10-400 ng / ml, 10-300 ng / ml, 10-200 ng / ml, and most preferably 100 ng / ml; the concentration of BMP4 is 10-100 ng / ml, preferably 50 ng / ml.

[0051] Inducing directional differentiation of endoderm into foregut microspheres Directed endoderm differentiation into foregut microspheres was induced in Advanced DMEM / F12 medium containing FGF pathway activators (e.g., fibroblast growth factor) and GSK3 inhibitors (e.g., CHIR99021).

[0052] Fibroblast growth factor (FGF) is a family of growth factors involved in angiogenesis, wound healing, and embryonic development. FGF is a heparin-binding protein, and interaction with heparan sulfate proteoglycans associated with the cell surface has been shown to be essential for FGF signal transduction. Suitable FGF pathway activators will be readily understood by those skilled in the art. Exemplary FGF pathway activators include, but are not limited to, one or more molecules selected from the group consisting of: FGF1, FGF2, FGF3, FGF4, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF23.

[0053] In a specific implementation, the content of fibroblast growth factor can be from 10 ng / ml to 1000 ng / ml; preferably, the content of fibroblast growth factor is from 50 ng / ml to 900 ng / ml, 100 ng / ml to 800 ng / ml, 150 ng / ml to 700 ng / ml, or 200 ng / ml to 600 ng / ml; more preferably, the content of fibroblast growth factor is 500 ng / ml.

[0054] Those skilled in the art will readily understand suitable GSK3 inhibitors. Exemplary GSK3 inhibitors include, but are not limited to, Chiron / CHIR99021, which inhibits GSK3β. Those skilled in the art will recognize suitable GSK3 inhibitors for carrying out the disclosed methods. GSK3 inhibitors can be administered in amounts from 1 μM to 100 μM, or 2 μM to 50 μM, or 3 μM to 25 μM. Preferably, the amount of GSK3 inhibitor is 3 μM. Those skilled in the art will readily understand appropriate amounts and durations.

[0055] For example, d) On the fourth day, oriented endoderm was cultured in Advanced DMEM / F12 medium containing B27, N2, FGF4, CHIR99021 and penicillin / streptomycin for 3 days, with the medium being changed daily, to obtain foregut microspheres.

[0056] Inducing foregut microspheres to differentiate into hepatic progenitor organoids in matrix gel Cell dissociation and matrigel embedding e) On the seventh day, the foregut microspheres were subjected to cell dissociation by adding digestive enzymes (Accutase) to form a single-cell suspension; the centrifuged cells were resuspended in matrix gel at a concentration of 1×10⁻⁶. 5 The cells were seeded into 24-well culture plates in a dome-shaped manner at a ratio of 1 cell / 50 μL of matrix gel.

[0057] The matrix gel is a three-dimensional extracellular matrix gel rich in laminin, type IV collagen and nestin, used to support three-dimensional cell culture and organoid formation.

[0058] After the matrix gel solidifies, liver precursor induction medium is added for culturing.

[0059] Liver precursor stage culture (hepatic progenitor organoid culture) f) On days seven to ten, after the matrix gel has solidified, liver precursor induction medium is added for culturing.

[0060] The culture medium is Advanced DMEM / F12, supplemented with the following components: B27, N2, GlutaMAX, FGF2, VEGF, EGF, CHIR99021, A83-01, ascorbic acid, and penicillin / streptomycin. A83-01 and ascorbic acid should be prepared fresh before use.

[0061] g) On day eleven, the culture medium was changed, and culture continued until day fourteen. The medium was based on Advanced DMEM / F12, supplemented with B27, N2, GlutaMAX, and retinoic acid. Culture was continued under these conditions to promote further differentiation of organoids into the liver lineage.

[0062] Organoid re-embedding and maturation culture h) On day 15, the formed organoids were recovered from the matrix gel, re-embedded in fresh matrix gel, and seeded in dome-shaped 24-well culture plates treated with tissue culture (TC). Maturation medium was then added for long-term culture.

[0063] The mature culture medium is a hepatocyte culture medium supplemented with additives such as transferrin, ascorbic acid, insulin, hydrocortisone and bovine serum albumin (fatty acid-free), and further supplemented with hepatocyte growth factor, dexamethasone and oncostatin M.

[0064] The culture medium is changed every 2–3 days during the culture period, and the culture is continued for about 10 days to obtain functionally mature liver organoids.

[0065] Features of the liver organoid of the present invention In some embodiments, the liver organoids obtained according to the method of the present invention exhibit characteristics highly similar to human livers in terms of morphology, expression of molecular markers, and functional levels.

[0066] In terms of morphology, continuous observation of organoids under a bright-field microscope revealed that on day 7, microspheres of the foregut endoderm formed; on day 15, hepatic progenitor cell organoids formed, exhibiting a mixed morphology of microspheres and fibroblast-like cells; on day 22, the organoids further matured, showing a mixed distribution of cystic structures, clustered structures, and fibroblast-like cells, with the cystic structures having a diameter of approximately 100–300 μm; by day 29, the organoids continued to grow, reaching a diameter of over 500 μm, demonstrating stable three-dimensional growth capability.

[0067] Regarding molecular marker expression, qPCR results showed that the liver organoid simultaneously expressed multiple hepatocyte and non-parenchymal cell-related marker genes. Among them, hepatocyte markers HNF4A and ALB were significantly expressed; cholangiocellular markers CK19 and SOX9 were also detected; and the expression of stellate cell marker α-SMA and Kupffer cell markers CD14 and CD68 was also detected. Furthermore, the organoid expressed multiple liver-related cytochrome P450 enzyme genes (including CYP3A4, CYP2B6, and CYP2E1) and liver transport protein genes MRP2 and NTCP, indicating that the organoid possesses liver-like characteristics with coexistence of multiple cell types.

[0068] Immunofluorescence assays further confirmed the presence of significant HNF4A-positive and α-SMA-positive cells in the organoids, indicating that hepatocytes and stellate cells were successfully co-cultured and stably existed in a three-dimensional structure.

[0069] At the functional level, the liver organoids exhibited typical liver metabolic and secretory functions. Functional assays showed that the organoids could continuously secrete albumin and urea, with albumin secretion levels of 200–700 ng / mL / day / well and urea production levels of 0.3–0.5 mg / dL / day / well; CYP3A4 enzyme activity with a high signal-to-noise ratio (greater than 10) was also detected. These results indicate that the liver organoids constructed in this embodiment are highly similar to human liver tissue in terms of structural composition, molecular expression, and functional characteristics.

[0070] Application of liver organoids obtained by the method of this invention in drug toxicity evaluation The liver organoids constructed in this invention can be used for multidimensional toxicity evaluation of drug-induced liver injury (DILI) in vitro, and can comprehensively reflect the toxic effects of drugs on the liver at the biochemical level, organelle function level, tissue structure level and overall cell viability level.

[0071] In terms of biochemical indicator detection, by treating organoids with drugs and detecting changes in indicators such as aspartate aminotransferase (AST), albumin (ALB), glutathione (GSH), and lactate dehydrogenase (LDH), the effects of drugs on hepatocyte synthetic capacity, redox homeostasis, and the degree of cell damage can be effectively reflected. Experimental results showed that treatment with classic hepatotoxic drugs could induce a decrease in ALB secretion, a reduction in GSH levels, and changes in LDH levels, indicating that the organoids can sensitively respond to drug-induced hepatocyte damage.

[0072] In terms of evaluating specific toxicity types, the liver organoids can be combined with high-content imaging technology to achieve in vitro reproduction of various hepatotoxicity mechanisms. Detection of lipid accumulation within the organoids using lipid fluorescent dyes can effectively evaluate drug-induced fatty liver injury; combined detection of mitochondrial membrane potential dyes and cytotoxic dyes can reflect drug-induced mitochondrial dysfunction and cytotoxicity; detection of bile acid distribution within the organoid lumen using bile acid transport probes can be used to evaluate drug-induced cholestasis toxicity; furthermore, analysis of fibrosis-related structural changes in the organoids using histological staining methods can be used to predict the risk of drug-induced liver fibrosis.

[0073] In terms of overall toxicity prediction, the liver organoids can also be used for drug hepatotoxicity screening based on cell viability. The inhibitory effects of different drugs on organoid viability under conditions above clinical exposure levels were evaluated using a three-dimensional cell viability assay. The results showed good agreement with the clinical DILI risk stratification of known drugs, indicating that the organoid model has high predictive accuracy.

[0074] In summary, the liver organoids of the present invention can evaluate the hepatotoxic effects of drugs in vitro at multiple levels and at multiple endpoints, and are applicable to drug safety screening, hepatotoxicity mechanism research, and risk classification of candidate compounds in the process of new drug development.

[0075] the term: Unless otherwise stated, the terminology should be understood in accordance with the common usage of those skilled in the art in the relevant field.

[0076] As used herein, the term “about” in the context of numerical values ​​means within an acceptable range of error for a particular value as determined by a person skilled in the art, which will depend in part on how the value is measured or determined, for example, the limitations of the measurement system. For example, according to practice in the art, “about” may mean within a standard deviation of 1 or greater than 1. Alternatively, “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term may mean within an order of magnitude, preferably within 5 times the value.

[0077] As used in this article, “induced pluripotent stem cells” (iPSCs) refer to a type of pluripotent stem cells artificially derived from non-pluripotent cells, usually adult cells.

[0078] As used herein, the term "organoid" refers to a tissue culture that forms a three-dimensional assembly that at least partially mimics the structure and / or function of an organ, such as a human organ. Organoids can be generated from pluripotent stem cells in, for example, a three-dimensional (3D) environment. One such 3D environment used for organoids is a spherical 3D environment. Organoids can be further considered as miniaturized and simplified forms of organs.

[0079] As used in this article, the term "single-celled" generally refers to the process of separating a group of cells (such as tissue blocks, callus, or cell clusters) that were originally growing together, connected to each other, or mixed together, through physical, chemical, or enzymatic means to obtain a dispersed, independent single cell.

[0080] Cells that have undergone "single-cell" treatment are usually referred to as "single-cell suspensions" or are in a "single-cell state".

[0081] Methods for “single-cell” cell formation are well known to those skilled in the art, including but not limited to: enzymatic digestion, mechanical methods, chemical induction / culture medium induction, micromanipulation / microfluidic separation, etc.

[0082] Example 1. Induction of liver organoid differentiation In this embodiment, iPSCs were cultured and expanded, and then induced to differentiate into liver organoids. The specific experimental procedures are as follows: 1.1 iPSC culture and passage (1) Preheat the complete culture medium and the culture plate coated with matrix gel. Remove the cryovial from the liquid nitrogen, thaw it quickly at 37°C, gently shake the cryovial, and remove it when only a small frozen mass remains. Wipe the surface with 75% alcohol for disinfection.

[0083] (2) Use a pipette to gently transfer the frozen solution to a 15 mL centrifuge tube containing 5-6 mL of preheated complete culture medium, and centrifuge at 300 g for 5 min at room temperature. Aspirate the supernatant, slowly add complete culture medium (3 mL for a 6 cm dish), add ROCKi+P / S, and gently tap the bottom of the centrifuge tube with your finger to detach the cell clusters from the bottom and disperse them. Gently transfer the mixture to the coated culture plate.

[0084] (3) Incubate at 37°C and change the culture medium daily (the culture medium needs to be preheated).

[0085] (4) When the colonies become larger, the centers become denser and brighter (in contrast to the edges), and adjacent colonies begin to merge, subculture can be performed. Preheat calcium- and magnesium-free PBS solution, digestion solution, and complete culture medium at 37°C. Aspirate the supernatant, add preheated PBS solution, and wash once. Add 1 mL of digestion solution to each well of a 6-well plate, 2 mL to a 6 cm dish, and 3 mL to a 10 cm dish, and incubate at 37°C for 1-2 minutes. Gently tap the culture plate with your finger. When you observe under a microscope that most of the clone edges begin to detach from the bottom of the culture plate and most of the cells inside the clone detach in clusters, add an appropriate amount of complete culture medium (twice the volume of digestion solution) to stop digestion. Transfer to a centrifuge tube and centrifuge at 300g for 5 minutes. Resuspend in complete culture medium and subculture into a new culture plate at a ratio of 1:6 to 1:20. Incubate at 37°C, changing the culture medium daily. The culture medium needs to be preheated.

[0086] 1.2 Differentiation (1) Single-cell inoculation was performed one day before differentiation: Prepare and preheat the single-cell inoculation complete culture medium. Remove the cell plate, aspirate the culture medium, and wash once with calcium- and magnesium-free PBS buffer. Add single-cell digestion solution to completely cover the bottom of the plate, incubate in an incubator for 5-10 min, observe under a microscope that the clones detach from the bottom of the plate, the cells inside the clones separate from each other, and observe with the naked eye that the cell colonies become opaque and white, indicating that the cell digestion time is ideal. Use a pipette to fan-shaped blow on the bottom of the culture plate to detach the attached cell colonies, gently and slowly blow and aspirate to mix, transfer to a 15 mL centrifuge tube, and centrifuge at 200 g for 5 minutes. Discard the supernatant, resuspend the cells in single-cell inoculation complete culture medium, gently blow and mix, and seed 0.2 M per well of a 48-well plate into the coated culture plate. Mix horizontally in a cross shape, place at room temperature for 10-15 minutes, and then place the cells in a 37℃ constant temperature CO2 cell culture incubator; (2) On the first day of differentiation, the medium was changed to endoderm induction medium: RPMI 1640 medium + 100 ng / ml activin A + 50 ng / ml BMP4; FBS was thawed and used on the second day. (3) On the second day, the medium was changed to RPMI 1640 medium + 100 ng / ml Activin A + 0.2% FBS; (4) On the third day, the medium was changed to RPMI 1640 medium + 100 ng / ml activin A and 2% FBS; (5) Foregut induction begins on the fourth day. The medium is changed to Advanced DMEM / F12 + B27 + N2 + 500 ng / ml fibroblast growth factor (FGF4) + 3 μm CHIR99021 + 1% penicillin / streptomycin. The medium is changed daily, and the same changes are made on the fifth and sixth days. (6) On the seventh day, cell dissociation and embedding were performed: the culture medium was aspirated, the cells were washed once with 4 ml of calcium- and magnesium-free DPBS, 2 ml of digestive enzyme (accutase, the amount is suitable for 6 cm dishes) was added, and the cells were incubated at 37°C for 2-5 min until the single cells began to round out. The cells were gently pipetted and transferred to 15 ml tubes, and gently pipetted to disperse them. The remaining cells were rinsed with 8 ml of preheated culture medium (Advanced DMEM / F12) and transferred to tubes. 20 μl of the cells were counted. The remaining cells were centrifuged at 200 g for 4 min, resuspended in matrix gel, and seeded at 50 μl per 100,000 cells in dome-shaped plates. The culture medium was Advanced DMEM / F12 + 1 × B27 + 1 × N2 + 1 × GlutaMaX + 5 ng / ml FGF2 + 10 ng / mL VEGF + 20 ng / mL EGF + 3 μM CHIR99021 + 0.5 μM A83-01 + 50 μg / mL ascorbic acid + 1% penicillin / streptomycin), where A83-01 and ascorbic acid should be prepared and used immediately. Change the solution on day 9 as above; (7) On the eleventh day, the medium was changed to Advanced DMEM / F12 + 1 × B27 + 1 × N2 + 1 × GlutaMaX + 2 μM retinoic acid + 1% penicillin / streptomycin. The medium was changed again on the thirteenth day. (8) On day 15, organoids were collected and re-embedded in matrix gel, then added in dome form to 24-well culture plates treated with tissue culture (TC) and cultured using mature medium. The medium formulation was: hepatocyte growth medium (HCM; Lonza, CC-3198) + complementary additives except EGF + 10 ng / mL hepatocyte growth factor (HGF) + 0.1 μM dexamethasone + 20 ng / mL oncostatin M (OSM) + 1% penicillin / streptomycin. The medium was changed every 2-3 days and cultured for another 10 days. After that, organoids were collected for subsequent analysis or continued culture.

[0087] It should be noted that when organoids differentiate to the foregut microsphere stage, they can be cryopreserved. After testing, thawing and continuing downstream differentiation after cryopreservation does not affect the function of the final liver organoid.

[0088] The reagents required for liver organoid culture in Example 1 are shown in Table 1.

[0089] Table 1 Example 2. Identification of liver organoids (1) Bright-field observation: The liver organoids prepared in this embodiment were tracked and observed. The morphology was observed under an optical microscope. It was observed that the foregut endoderm microspheres formed on day 7, the liver progenitor cell organoids formed on day 15, and the liver organoids were mixed with microspheres and fibroblast-like cells. The liver organoids matured on day 22, and were mixed with cysts, clusters and fibroblast-like cells. The size of the cysts was about 100-300 μm. The liver organoids grew further on day 29, and the diameter could reach more than 500 μm. The results are shown in the figure. Figure 2 .

[0090] (2) qPCR detection: In this embodiment, marker genes of hepatocytes, non-parenchymal cells, transporters, and cytochrome enzymes were detected, including hepatocyte markers HNF4A and ALB; cholangiocarcinoma markers CK19 and SOX9; stellate cells A-SMA and Kupffer cells CD14, CD68, CYP enzymes CYP3A4, CYP2B6, CYP2E1, and transporter proteins MRP2 and NTCP, etc. The detection results are shown in […]. Figure 3 .

[0091] (3) Immunofluorescence detection: In this embodiment, the liver parenchymal cell marker HNF4A and the stellate cell marker α-SMA were detected, and the results are as follows: Figure 4 It showed obvious expression of HNF4a and α-SMA.

[0092] (4) Liver organoid function detection: In this embodiment, the albumin level, urea level, and CYP3A4 enzyme activity of the liver organoids were detected. The results showed that the obtained liver organoids had the ability to secrete albumin and urea and had active CYP3A4 enzyme. Two wells were used for each indicator. The albumin level was 200-700 ng / ml / day / well, the urea level was 0.3-0.5 mg / dL / day / well, and the signal-to-noise ratio of CYP3A4 enzyme activity detection was greater than 10, indicating that the liver organoids constructed in this invention have functions similar to those of human liver. See the results below. Figure 5 .

[0093] Example 3. DILI test based on liver organoids 1. Drug treatment and control group setup: Add DMSO control and the compound to the culture medium and expose for 3-4 days as needed for the experiment. See Table 2 for the compounds used and their treatments.

[0094] 2. Biochemical-based DILI detection The effects of drug treatment on AST, ALB, GSH, and LDH levels were detected according to the kit instructions. Results showed that APAP treatment decreased ALB secretion, reduced GSH levels, and increased LDH. Nef treatment also decreased ALB secretion, reduced GSH levels, and decreased LDH. (See attached results). Figure 6 This indicates that biochemical experiments can detect the effects of drugs on the damage and synthetic capacity of hepatocytes.

[0095] (1) Lipid accumulation toxicity test The degree of lipid accumulation at the organoid level was detected using Lipidtox dye and a high-content device. The results showed that the degree of lipid accumulation in organoids increased with the increase of drug FIAU concentration, indicating that the high-content Lipidtox-based assay can reproduce the lipid accumulation toxicity of the drug.

[0096] (2) Detection of mitochondrial toxicity and cytotoxicity TMRM, Celltox dye, and high-content devices were used to detect organoid-level mitochondrial toxicity and cytotoxicity. TMRM characterizes normal mitochondria by measuring mitochondrial membrane potential. The results showed that the number of normal mitochondria decreased at the organoid level after APAP and Nef treatment, while the cytotoxicity levels were not significantly different. This indicates that TMRM-based detection with high content can reproduce the mitochondrial toxicity of the drugs APAP and Nef.

[0097] (3) Cholestasis detection The bile acid dye CLF and a high-content device were used to detect the bile transport function of the drug. The results showed that under normal conditions, CLF can be effectively transported into the lumen of the organoid. However, after Nef treatment, the fluorescence intensity in the lumen decreased, indicating that bile transport was blocked.

[0098] (4) Liver fibrosis detection Organoids were treated with the drug MTX, and the degree of liver fibrosis was analyzed by masson staining of drug-treated and control organoids. The results showed that the thickness of the green dye at the edge (epithelial cell-like arrangement) of the organoids increased after MTX treatment, representing an increased degree of fibrosis. This demonstrates that human liver organoids (HLOs) can effectively predict drug-induced fibrosis. Results are shown below. Figure 10 .

[0099] (5) Drug toxicity evaluation based on cell viability The hepatotoxicity of the drugs troglitazone, pioglitazone, and cyclosporin A was detected using 3D-CTG at concentrations above 10 Cmax (the known Cmax values ​​for troglitazone are 6.08 μM, pioglitazone is 1.5 μM, and cyclosporin A is 0.77 μM). Results showed that 100 μM troglitazone inhibited organoid viability by over 99%, 100 μM pioglitazone by approximately 11%, and 10 μM cyclosporin A by approximately 29%. The FDA classifies these three drugs as high-risk, low-risk, and low-risk in the DILI category, respectively. The 3D-CTG results are largely consistent with these findings, indicating that these liver organoids can effectively predict drug hepatotoxicity at the cell viability level.

[0100] This invention establishes an efficient and stable method for constructing liver organoids derived from human iPSCs and systematically verifies its application value in multidimensional evaluation of drug hepatotoxicity. This model overcomes the limitations of traditional models, providing a more reliable and physiologically accurate platform for early drug safety evaluation.

[0101] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

[0102] The compound information required for DILI detection in Example 3 includes compound name, detection concentration, time, and detection index, as shown in Table 2.

[0103] Table 2 The reagent information required for organoid identification and DILI detection in Examples 2 and 3 of this invention, including reagent name, manufacturer and catalog number, is shown in Table 3.

[0104] Table 3

Claims

1. A method for inducing differentiation of iPSCs in vitro to form liver organoids, the method comprising the following steps: i) Provide iPSC cells; ii) Culture the isolated iPSC cells provided in step i) in endoderm induction medium for 1 to 7 days, preferably 1 to 3 days, to obtain oriented endoderm; iii) The oriented endoderm obtained in step ii) is cultured in foregut induction medium for 1 to 7 days, preferably 1 to 3 days, to obtain foregut microspheres; iv) Dissociate the foregut microspheres obtained in step iii) into single cells, embed them in matrix gel, and culture them in liver progenitor organoid induction medium for 4 to 15 days, preferably 4 to 8 days, to obtain liver progenitor organs; and v) Culture the liver progenitor organoids obtained in step iv) in hepatocyte maturation medium for 7 to 25 days, preferably 7 to 14 days, to obtain liver organoids.

2. The method according to claim 1, wherein: Step iv) includes: iv-1) Cell dissociation of foregut microspheres was performed by adding digestive enzymes to form a single-cell suspension; iv-2) Resuspend the centrifuged single cells in matrix gel; iv-3) Add Advanced DMEM / F12 medium containing B27, N2, GlutaMAX, FGF2, VEGF, EGF, CHIR99021, A83-01, ascorbic acid, and penicillin / streptomycin to the solidified matrix gel and incubate for 3-5 days; and iv-3) Replace the medium with Advanced DMEM / F12 medium containing B27, N2, GlutaMAX and retinoic acid, and continue culturing for 3-5 days.

3. The method according to claim 1, wherein: Step v) includes: v-1) The liver progenitor organoids formed from the Matrigel were recovered, re-embedded in fresh Matrigel, and seeded in a dome shape into tissue culture plates. v-2) Continue culturing hepatocytes in a culture medium supplemented with hepatocyte growth factor, dexamethasone, oncostatin M, and penicillin / streptomycin for 7 to 14 days to obtain mature liver organoids.

4. The method according to claim 1, wherein: Step ii) includes: ii-1) Incubate for 1 day in RPMI 1640 medium containing Activin A and BMP4; ii-2) Incubate for 1 day in RPMI 1640 medium containing Activin A + 0.2% FBS; and ii-3) Incubate for 1 day in RPMI 1640 medium containing Activin A + 2% FBS; Preferably, the concentration of Activin A is 10-500 ng / ml, more preferably 10-400 ng / ml, 10-300 ng / ml, 10-200 ng / ml, and most preferably 100 ng / ml; The concentration of BMP4 is 10-100 ng / ml, preferably 50 ng / ml.

5. The method according to claim 1, wherein: Step iii) includes: iii-1) In a culture medium containing fibroblast growth factor (FGF) and a GSK3 inhibitor, directional differentiation of the endoderm into foregut microspheres is induced; preferably, iii-1) Culture oriented endoderms in Advanced DMEM / F12 medium containing B27, N2, FGF4, CHIR99021 and penicillin / streptomycin for 3 days.

6. The method according to any one of claims 1-5, wherein The iPSC cells are human iPSCs.

7. The method according to any one of claims 1-5, wherein Optionally included between steps iii) and iv): Cryopreservation of foregut microspheres (step iii).

8. A liver organoid derived from iPSC cells, which exhibits a mixed morphology of sac-like, cluster-like, and fibroblast-like cells, with sac-like size of approximately 100-1000 μm, preferably, the sac-like size being approximately 500-1000 μm; Optionally, the liver organoid derived from iPSC cells is obtained by the method described in any one of claims 1-7.

9. The liver organoid derived from iPSC cells according to claim 8, wherein... The liver organoids expressed hepatocyte markers HNF4A and ALB; bile duct cell markers CK19 and SOX9; stellate cell marker α-SMA; and Kupffer cell markers CD14 and CD68. The liver organoids expressed the cytochrome P450 enzyme genes CYP3A4, CYP2B6, and CYP2E1, as well as the liver transporter protein genes MRP2 and NTCP; and It continuously secretes albumin and urea.

10. A method for assessing the extent of drug-induced liver damage in vitro, the method comprising the step of contacting the drug to be assessed with a liver organoid obtained according to the methods of claims 1-7, or a liver organoid derived from iPSC cells as described in claim 8 or 9; Optionally, the method further includes: After exposing the drug to be evaluated to liver organoids derived from iPSC cells provided by the present invention for a period of time, biochemical indicators are detected and / or liver toxicity is assessed.

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