Liver-like or liver tissue-like structure

Abstract

The purpose of the present invention is to provide a liver-like or liver tissue-like structure as a novel in vitro evaluation model, which is useful for the development of a therapeutic agent for MASH. The present invention pertains to a liver-like or liver tissue-like structure comprising: human hepatocytes in which inflammation is induced; and human hepatic stellate cells, wherein said structure is present in a culture medium containing ascorbic acid, a salt thereof, a derivative thereof, or the like, or is cultured in the culture medium.

Description

Liver-like or liver tissue-like structure 【0001】 The present invention relates to a liver-like or liver tissue-like structure or the like. Specifically, it relates to a liver-like or liver tissue-like structure or the like composed of human hepatocytes in which inflammation has been induced and human hepatic stellate cells. 【0002】 Metabolic dysfunction-associated steatohepatitis (MASLD), a liver disease, is a condition closely related to the metabolic syndrome associated with visceral obesity. Furthermore, MASLD includes metabolic dysfunction-associated steatohepatitis (MASH), which can progress to liver cirrhosis and increase the risk of liver cancer. Currently, the number of MASH patients in Japan is estimated to be approximately 2 million, and the number of patients with a series of liver diseases (NAFLD) including non-alcoholic fatty liver disease without inflammation to MASH is estimated to be over 10 million, making it a disease of extremely high social concern. However, the current treatment for MASLD / MASH mainly focuses on correcting visceral obesity through diet therapy and weight loss through exercise, and there is a lack of drug therapies with a high level of evidence-based evaluation. Only recently (March 14, 2024), the US Food and Drug Administration (FDA) quickly approved "Rezdiffra" (generic name: resmetirom), a MASH treatment drug developed by Madrigal Pharmaceuticals in the US, which is the first such case. 【0003】Under these circumstances, pharmaceutical companies worldwide are focusing on developing new MASH treatments, but development is taking time. There are several reasons for this, one of which is that it takes several months to create appropriate animal disease models to evaluate drug efficacy. For this reason, the development of evaluation systems made of human cells as an alternative to animal models, such as in vitro evaluation models using fatty liver or fibrotic liver cells, is attracting attention (see Patent Documents 1 and 2). The characteristics that these evaluation models should possess include, for example, (1) being composed of human liver cells, (2) undergoing fibrosis under appropriate culture conditions, (3) increasing the degree of fibrosis with continued culture, thereby simulating disease progression, (4) being of a size that is easy for testers to handle and easily confirmed by the eye, and (5) having as few types of cells as possible to make up the model, making product manufacturing easy. 【0004】 To date, several in vitro evaluation models have been commercially available. For example, Insphero offers a service using a product with a spheroid approximately 200 μm in diameter, composed of four types of human-derived hepatocytes (Hepatocytes, Liver Endothelial Cells, Kupffer Cells, and Stellarate Cells) (see Patent Document 1 and Non-Patent Document 1). However, none of the commercially available evaluation models satisfy all of the characteristics described in (1) to (5) above. In particular, since MASH is a disease that is highly likely to progress to cirrhosis or liver cancer, therapeutic drugs are required to suppress and improve the progression of liver fibrosis, and therefore there is a high demand for evaluation models that can reproduce the disease progression described in (3) above. Furthermore, all commercially available models are smaller than 200 μm in diameter, making them difficult to see and handle. Prior Art Documents 【0005】 U.S. Patent Application Publication No. 2019 / 0316093 U.S. Patent Application Publication No. 2020 / 0115682 【0006】Radina Kostadinova et al. ,Digital pathology with artificial intelligence analysis provides insight to the efficiency of anti-fibrotic compounds in human 3D MASH model. , Scientific Report, 14, 5885 (2024) (DOI: https: / / doi.org / 10.1038 / s41598-024-55438-2) 【0007】 Under these circumstances, there was a need for the development of new in vitro evaluation models, such as liver-like or liver tissue-like structures, that would be useful in the development of MASH treatments. 【0008】 The present invention has been made in consideration of the above circumstances and provides the following liver-like or liver tissue-like structures, etc. 【0009】 [1] A liver-like or liver tissue-like structure comprising inflamed human hepatic parenchymal cells and human hepatic stellate cells, wherein the structure is present in or cultured in a culture medium containing ascorbic acid, a pharmacoacceptable salt thereof, or a hydrate or solvate thereof, or a derivative thereof. [2] The structure according to [1], wherein the cells constituting the structure exhibit enhanced expression of at least one oxidative stress-related gene selected from the group consisting of SOD2, NQO1, GPX2, GPX7, GPX8, CAPN2, CTNNB1, CRYAB, FBLN5, GSTP1, PRDX4, AOX1, MT1A, MT2A, and PDLIM5. [3] The structure according to [2], wherein the expression level of the enhanced oxidative stress-related genes is at least twice as high as that of the cells constituting the liver-like or liver tissue-like structure cultured in a medium that does not contain ascorbic acid, a pharmacologically acceptable salt thereof, or their hydrates and solvates. 【0010】[4] The structure according to [1], wherein the culture medium further comprises one or more saturated or unsaturated fatty acids having three or more alkyl chains, pharmacologically acceptable salts thereof, hydrates or solvates thereof, or derivatives thereof. [5] The structure according to [4], wherein the saturated or unsaturated fatty acid having three or more alkyl chains is palmitic acid or oleic acid. [6] The structure according to [1], wherein the cells constituting the structure show enhanced expression of at least one inflammation-related gene or cytokine-related gene selected from the group consisting of CRP, SAA1, SAA2, CCL2, CCL20, CXCL1, CXCL2, CXCL6, CXCL8, LGALS1, and GSTP1. 【0011】 [7] The structure according to [6], wherein the expression level of the enhanced inflammation-related gene or cytokine-related gene is at least twice as high as that of the cells constituting the liver-like or liver tissue-like structure cultured in a medium that does not contain ascorbic acid, a pharmacologically acceptable salt thereof, or hydrates and solvates thereof. [8] The structure according to [1], wherein the medium further comprises TGFβ. [9] The structure according to [1], wherein the human hepatic stellate cells are activated human hepatic stellate cells. 【0012】

[10] The structure according to [1], wherein fibers made of collagen are deposited within the structure.

[11] The structure according to [1], wherein when any cross section thereof is stained with picrosilius red, a region that is stained in a fibrous manner is observed, and the Fibrosis Index (%) calculated from the following formula (I): Fibrosis Index (%) = (area stained with picrosilius red / total area of ​​the sample) × 100 (I) is in the range of 4% to 60%.

[12] The structure according to [1], wherein the culture medium in which the structure is present or the culture medium in which the structure is cultured contains 20 ng or more of procollagen type I C-terminal peptide (PIP) per structure in its supernatant. 【0013】

[13] The structure according to [1], wherein the ratio of the number of human hepatic stellate cells to the total number of cells in the structure is 1 to 10%.

[14] The structure according to [1], wherein the human hepatic parenchymal cells are at least one selected from the group consisting of human primary cultured hepatocytes, iPS cell-derived hepatocytes, hepatocytes induced by direct reprogramming, and hepatocytes taken from the liver of a human hepatocyte chimeric mouse.

[15] The structure according to [1], wherein the equivalent diameter of the circle in the two-dimensional projection is 400 to 1,500 μm.

[16] The structure according to [1], wherein the structure is made up of multiple spheroids produced by a bio-3D printer stacked on top of each other. 【0014】 The present invention provides a liver-like or liver tissue-like structure, etc., as a novel in vitro evaluation model useful for the development of MASH therapeutic drugs. The liver-like or liver tissue-like structure according to the present invention can be made, for example, of a size that is easily visible (preferably 400 to 1,500 μm), making it easy to handle as the above in vitro evaluation model. Furthermore, the degree of fibrosis within the structure can increase with continued culture, making it possible to simulate disease progression, thus possessing usefulness and practicality. 【0015】This figure shows the expression levels of oxidative stress-related genes in the embodiment of the present invention. Left figure: SOD2 expression level, Right figure: AOX1 expression level. This figure shows the expression levels of inflammation-related genes in the embodiment of the present invention. Left figure: CRP expression level, Right figure: SAA2 expression level. This figure shows the expression levels of cytokine-related genes in the embodiment of the present invention. Left figure: CCL2 expression level, Right figure: CXCL6 expression level. This figure shows the expression level of the ACTA2 gene, which indicates activation of hepatic stellate cells, in the embodiment of the present invention. This figure shows the change over time of the amount of procollagen (PIP) secreted into the culture supernatant of the three-dimensional liver-like structure in the embodiment of the present invention. This figure shows the histological analysis results (picrosilius red staining) of the three-dimensional liver-like structure cultured using medium A and medium B in the embodiment of the present invention. This figure shows the change over time of the amount of deposited fibers in the three-dimensional liver-like structure in the embodiment of the present invention. This figure shows the change in fibrosis-related genes due to the addition of Seloncertib in the embodiment of the present invention. This figure shows a schematic diagram of the method (fabrication scheme) for fabricating a fibrotic model of a large three-dimensional liver-like structure in the embodiment of the present invention. This figure shows the histological analysis results (picrosirius red staining image) of the fabricated fibrotic large three-dimensional liver-like structure (see Figure 9) in the embodiment of the present invention. 【0016】 The present invention will now be described in detail. The scope of the present invention is not limited to this description, and modifications may be made as appropriate, without preserving the spirit of the invention, in addition to the examples given below. This specification encompasses the entirety of Japanese Patent Application No. 2023-045232 (filed March 22, 2023), which forms the basis of this application's priority claim. Furthermore, all publications cited herein, such as prior art documents, and published applications, patent gazettes, and other patent documents, regardless of their purpose, are incorporated herein by reference in their entirety. 【0017】The present invention will now be described in detail. The scope of the present invention is not limited to this description, and modifications can be made as appropriate, insofar as they do not impair the spirit of the invention, in addition to the examples given below. This specification encompasses the entirety of Japanese Patent Application No. 2024-214663 (filed December 9, 2024), which forms the basis of this application's priority claim. Furthermore, all publications cited herein, such as prior art documents, and published gazettes, patent gazettes, and other patent documents, are incorporated herein by reference. 【0018】 As described above, the present invention relates to a liver-like or liver tissue-like structure (hereinafter sometimes simply referred to as "the liver-like structure of the present invention"). The structure is a three-dimensional, three-dimensional structure, for example, a structure with a roughly spherical shape. The liver-like structure of the present invention is composed of human hepatic parenchymal cells and human hepatic stellate cells. 【0019】 Examples of human hepatocytes include at least one selected from the group consisting of, for example, primary human hepatocytes, iPS cell-derived hepatocytes, hepatocytes induced by direct reprogramming, and hepatocytes collected from the liver of human hepatocyte chimeric mice. 【0020】 In the liver-like structure of the present invention, the ratio of the number of human hepatic stellate cells to the total number of cells in the structure is preferably, for example, 1 to 10%, and may be 2 to 6%. 【0021】 The liver-like structure of the present invention may be formed by stacking multiple spheroids (for example, five or more, eight or more, or ten or more, but not limited to these) that have been fabricated using a bio-3D printer (for example, the Regenova® bio-3D printer manufactured by Cyfuse Co., Ltd.). The spheroid is composed of at least human hepatic parenchymal cells and human hepatic stellate cells. 【0022】 The liver-like structure of the present invention is present in or cultured in ascorbic acid, a pharmaceutically acceptable salt thereof, or a hydrate or solvate thereof, or a derivative thereof (hereinafter sometimes simply referred to as "ascorbic acid, etc."). 【0023】 The culture medium used can be any medium commonly used for culturing hepatocytes. Examples of such media include DMEM, RPMI-1640, DMEM / F12, and Williams' Medium E. Alternatively, commercially available hepatocyte culture media (such as Primary Hepatocyte Maintenance Supplements (CM series, Life Technologies)) can also be used. 【0024】 When cultured in a medium containing ascorbic acid, etc., and / or present in said medium, the liver-like structure of the present invention undergoes fibrosis (deposition of fibers) within the structure. The fibrosis within the liver-like structure of the present invention may be enhanced (fibrosis may increase) with increasing culture time in a medium containing ascorbic acid, etc., and / or elapsed time in the medium. The liver-like structure of the present invention includes structures cultured in a medium, and such cultured structures may also include structures isolated from the medium (structures removed from the medium). 【0025】 The aforementioned ascorbic acid is preferably water-soluble ascorbic acid. Water-soluble ascorbic acid can be commercially available ascorbic acid sold as a pharmaceutical or quasi-drug, and these usually refer to the L-isomer. 【0026】Derivatives of ascorbic acid include, for example, those that undergo metabolism in the body such as oxidation, reduction, hydrolysis, or conjugation. Specifically, examples include ester derivatives or ether derivatives of ascorbic acid. Examples of ester derivatives include phosphate ester derivatives of L-ascorbic acid such as L-ascorbic acid monophosphate, L-ascorbic acid diphosphate, or L-ascorbic acid triphosphate; and L-ascorbic acid-2-sulfate. Examples of ether derivatives include L-ascorbic acid-2-glucoside. Among these, L-ascorbic acid, phosphate ester derivatives of L-ascorbic acid, L-ascorbic acid-2-sulfate, and L-ascorbic acid-2-glucoside are preferred, and due to their high efficacy, L-ascorbic acid, L-ascorbic acid monophosphate, and L-ascorbic acid-2-glucoside are particularly preferred. 【0027】Pharmacologically acceptable salts of ascorbic acid and its derivatives are not limited to, but preferably include, for example, hydrohalides (e.g., hydrochlorides, hydrobroms, and hydroiodides), inorganic salts (e.g., sulfates, nitrates, perchlorates, phosphates, carbonates, and bicarbonates), organic carboxylates (e.g., acetates, trifluoroacetates, maleates, tartrates, fumarates, and citrates), organic sulfonates (e.g., methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, benzenesulfonates, toluenesulfonates, and camphorsulfonates), amino acid salts (e.g., aspartates and glutamates), quaternary amine salts, alkali metal salts (e.g., sodium salts and potassium salts), and alkaline earth metal salts (e.g., magnesium salts and calcium salts). More specifically, examples include salts with organic bases (e.g., salts with tertiary amines such as trimethylamine salt, triethylamine salt, monoethanolamine salt, triethanolamine salt, pyridine salt, and basic ammonium salts such as arginine), and salts with inorganic bases (e.g., alkali metal salts such as ammonium salt, sodium salt, and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, and aluminum salt). Particularly preferred salts are magnesium salt, sodium salt, and potassium salt, specifically including sodium ascorbate, sodium ascorbate monophosphate, sodium ascorbate diphosphate, sodium ascorbate triphosphate, magnesium ascorbate phosphate, and sodium ascorbic acid-2-sulfate. 【0028】 In the present invention, the concentration of ascorbic acid, etc., in the culture medium is preferably 50 to 600 μmol / l, and may be 100 to 400 μmol / l, or 150 to 300 μmol / l. 【0029】The human hepatocytes used in the liver-like structure of the present invention are inflamed. This inflammation has the same characteristics as the inflammation observed in metabolic dysfunction-associated steatohepatitis (MASH), or is a similar state of inflammation. Prior to the definition of MASH, it was understood as non-alcoholic steatohepatitis (NASH). 【0030】 More specifically, inflammation of human hepatocytes is a state characterized by, for example, (1) increased expression of at least one oxidative stress-related gene selected from the group consisting of SOD2, NQO1, GPX2, GPX7, GPX8, CAPN2, CTNNB1, CRYAB, FBLN5, GSTP1, PRDX4, AOX1, MT1A, MT2A, and PDLIM5 in the cells constituting the liver-like structure of the present invention, and / or (2) increased expression of at least one inflammation-related gene or cytokine-related gene selected from the group consisting of CRP, SAA1, SAA2, CCL2, CCL20, CXCL1, CXCL2, CXCL6, CXCL8, LGALS1, and GSTP1 in the cells constituting the liver-like structure of the present invention. 【0031】 Here, the expression level of the enhanced oxidative stress-related gene, and / or the expression level of the enhanced oxidative stress-related gene, is preferably at least twice the expression level of cells constituting liver-like or liver tissue-like structures when cultured in a medium that does not contain ascorbic acid, its pharmacologically acceptable salts, and their hydrates and solvates (i.e., a medium that does not contain ascorbic acid, etc.), and may be at least three times, four times, or five times higher. 【0032】Conditions for enhancing the expression of oxidative stress-related genes include, for example, further containing in the culture medium one or more saturated or unsaturated fatty acids with three or more alkyl chains, pharmacologically acceptable salts thereof, hydrates or solvates thereof, or derivatives thereof. Examples of saturated or unsaturated fatty acids with three or more alkyl chains include well-known compounds and are not particularly limited, but examples include palmitic acid and oleic acid. Here, the explanations for ascorbic acid, etc., mentioned above can be appropriately applied to the pharmacologically acceptable salts, hydrates or solvates, or derivatives. The concentration of saturated or unsaturated fatty acids with three or more alkyl chains in the culture medium is preferably in the range of 50 μmol / l to 800 μmol / l, and may be 100 to 500 μmol / l. 【0033】 Furthermore, conditions that enhance the expression of inflammation-related genes or cytokine-related genes include, for example, further containing TGFβ in the culture medium. The concentration of TGFβ in the culture medium is preferably in the range of 0.1 to 20 ng / ml, and may be 2 to 12 ng / ml. 【0034】 The human hepatic stellate cells used in the liver-like structure of the present invention are preferably activated human hepatic stellate cells. Hepatic stellate cells are activated during liver injury and exhibit a myofibroblast-like morphology. Activated hepatic stellate cells secrete cytokines such as HGF, which are necessary for hepatocyte regeneration, thereby promoting liver regeneration, while also producing collagen and TGFβ, and thus contributing to liver fibrosis. In the liver-like structure of the present invention, as described above, inflammation is induced in the human hepatic parenchymal cells, so the human hepatic stellate cells contained in the structure can be in an activated state. Fibrosis (deposition of fibers, specifically deposition of collagen fibers) in the liver-like structure of the present invention can be directly brought about by the activation of these human hepatic stellate cells. 【0035】In the liver-like structure of the present invention, intracellular fibrosis can be confirmed, for example, by picrosilius red staining, and more specifically, regions that are stained in a fibrous manner can be observed. Preferably, when any cross-section of the structure is stained with picrosilius red, regions that are stained in a fibrous manner are observed, and the Fibrosis Index (%) calculated from the following formula (I) is in the range of 4% to 60%, or in the range of 8% to 50%. 【0036】 Fibrosis Index (FI) (%) = (Picrosilius Red stained area / Total sample area) × 100 (I) 【0037】 Furthermore, intracellular fibrosis in the liver-like structure of the present invention can also be confirmed by the content of procollagen type I C-terminal peptide (PIP) in the supernatant of the culture medium. Specifically, it is preferable that the culture medium in which the structure is present or the culture medium in which the structure is cultured contains 20 ng or more of PIP per structure in its supernatant, or it may contain 15 ng or more. 【0038】 In this case, it is preferable that the liver-like structure of the present invention has an FI% and PIP content that increase with the progression of the culture period of the structure. This allows the degree of fibrosis within the structure to increase with continued culture, and consequently, the progression of the disease can be simulated. 【0039】 For ease of use as an in vitro evaluation model, the liver-like structure of the present invention is preferably of a size that is easily visible. Specifically, the diameter of the circle equivalent in the two-dimensional projection of the structure is preferably 400 to 1,500 μm, or it may be 450 to 1,200 μm. As mentioned above, when creating a liver-like structure by stacking multiple spheroids produced by a bio-3D printer, it is preferable to produce spheroids with a diameter of approximately 400 to 600 μm in the two-dimensional projection and stack them to create the liver-like structure of the present invention. 【0040】Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto. In the following examples, the "liver-like or liver tissue-like structure" according to the present invention may be referred to as a "three-dimensional liver-like structure". 【0041】 <Preparation of three-dimensional liver-like structure> 1×10 ４ cells of primary cultured human liver cells and 5×10 ２ cells of human liver stellate cells were seeded into each well of a U-bottom 96-well plate (Nunc lon ＴＭ Sphere ＴＭ Microplate 96 well (Thermo Fisher Scientific Fisher Scientific, 174925)) with a non-adhesive surface treatment, and cultured in an incubator at 37°C for 3 days to prepare spherical three-dimensional liver-like structures. The average equivalent diameter of the circle in the two-dimensional projection diagram of the three-dimensional liver-like structure was 500 μm, and the standard deviation was 20 μm. In addition, the three-dimensional liver-like structure could be continuously cultured for about 4 weeks using a maintenance medium for hepatocytes. Here, the maintenance medium may contain compound A83-01 at a concentration of 1 to 10 nmol / l. 【0042】 <Induction of oxidative stress and inflammation> The three-dimensional liver-like structure prepared in Example 1 was cultured in a maintenance medium for hepatocytes, for example, Williams Medium E (product number A12176-01) / Hepatocyte Maintenance Supplement Pack (product number CM4000) manufactured by Gibco, in medium A supplemented with the compounds shown in Table 1 below for 3 days (Day 0 to Day 3), and then switched to medium B and cultured for 2 weeks (Day 3 to Day 17). As shown in Table 1 below, the compounds added to medium A and medium B are different for each sample. Among Samples -1 to -6 in Table 1, Samples -1 and -2 correspond to the examples of the present invention, and Samples -3 to -6 correspond to the comparative examples. 【0043】 【0044】(1) The results of examining the survival rates determined from the ATP amounts after culturing for 3 days in medium A followed by 4 days (total 7 days) and 11 days (total 14 days) in medium B are shown in Table 2 below. In medium A and medium B, when culturing for 7 days or more under culture conditions containing 500 μmol / l of PA or TGFβ (Sample - 4, Sample - 5), the survival rate significantly decreased. In medium B, only when the PA concentration was 100 μmol / l or less or in the culture conditions without TGFβ (Sample - 1, Sample - 2, Sample - 3), a survival rate of 85% or more was maintained. 【0045】 【0046】 (2) Gene expression analysis Samples - 1 to Samples - 3 and Sample - 6 were each sampled on Day 3, Day 10, and Day 17, and mRNA was extracted using ReliPrep™ RNA Tissue Miniprep System (Promega, Z6112). Gene expression profiles were obtained by RNAseq analysis using a next-generation sequencer and by quantitative PCR method. 【0047】 The expression levels of the oxidative stress-related genes SOD2 and AOX1 were higher in Samples - 1, Samples - 2, and Samples - 3 compared to Sample - 6 without additives, indicating that the liver cells in the samples were under oxidative stress conditions (Figure 1). On the other hand, the expression levels of the inflammation-related genes CRP and SAA2 increased in Samples - 1, Samples - 2, and Samples - 3 when cultured in medium A (on the 3rd day of culture), and a significant increase in the expression level was observed after the 10th day of culture when the medium was changed to medium B containing an ascorbic acid derivative (Samples - 1, Samples - 2; Figure 2). Also, for the genes of cytokines CCL2, CCL20, CXCL6, and CXCL8, a significant increase in the expression level was observed when the medium was changed to medium B containing an ascorbic acid derivative (Figure 3). Furthermore, the expression level of the ACTA2 gene was higher in Samples - 1 and Samples - 2 compared to Sample - 3 without an ascorbic acid derivative in the medium, indicating that the activation of hepatic stellate cells was progressing (Figure 4). 【0048】From these results, we found that under the conditions of Sample-1 and Sample-2, oxidative stress, inflammation, cytokines, and stellate cell activation can be induced while maintaining a high survival rate of hepatocytes. 【0049】 <Induction of Fibrosis> Procollagen type I C-terminal peptide (PIP) secreted into the culture supernatant of three-dimensional liver-like structures prepared under the conditions of Sample-1, Sample-2, Sample-3, and Sample-6 in Table 1 was quantified using the ELIZA kit (Figure 5). Furthermore, after paraffin fixation, 3-6 μm thick section samples were prepared using a microtome and stained with picrosilius red, which selectively stains collagen fibers. The Fibrosis Index (FI) (%) was calculated from the area of ​​the red-stained fibrous portion in the obtained stained image (Figure 6) (Figure 7). FI (%) is calculated using the following formula (I): FI (%) = (Picrosilius red stained area / Total sample area) × 100 (I) 【0050】 It was found that only the samples of the present invention (Sample-1 and Sample-2), cultured using a medium containing an ascorbic acid derivative, resulted in the deposition of fibers in the tissue from the third day of culture onward. Furthermore, the amount of fiber deposition showed the same trend in both indicators: FI (%) determined from stained images and the amount of procollagen type I C-terminal peptide (PIP) secreted in the culture supernatant (i.e., it tended to increase with the number of culture days), indicating that it can be used as a model for disease progression. 【0051】 <Evaluation of the efficacy of the therapeutic agent> ASK1 (Apoptosis Signal-regulating Kinase 1) plays an important role in inducing hepatocyte death and also contributes to the induction of inflammatory cytokines in inflammatory cells, and its inhibition may improve the pathogenesis of MASH. A fibrosis model was prepared by culturing for 10 days under the conditions of sample-1 in Table 1. Next, the ASK1 inhibitor Seloncertib was dissolved in medium B at a concentration of 10 μmol / l and culture was continued. After continuing the culture for 2 weeks, the sample was collected and ReliaPrep ＴＭmRNA was extracted using the RNA Tissue Miniprep System (Promega, Z6112). Gene expression profiles were obtained from RNAseq analysis using a next-generation sequencer (Figure 8). It was found that the addition of Seloncertib reduced the expression level of the ACTA2 gene by approximately 50%, indicating an inhibitory effect on hepatic stellate cell activation. In addition, the expression levels of ECM-producing genes such as COL1A1, COL1A2, COL1A3, ELN, and FN, as well as the TIMP gene which inactivates the collagen-degrading enzyme MMP, were also reduced, indicating that all of these have an inhibitory effect on fibrosis. 【0052】 To improve handling during culture, a fibrotic model of a larger-sized three-dimensional liver-like structure (large liver-like structure) was created. Using the scheme shown in Figure 9, nine spheroids were stacked using the Regenova® bio 3D printer (registered trademark) manufactured by Cyfuse Co., Ltd. (https: / / www.cyfusebio.com / product / regenova), and after maturation culture, a large liver-like structure of approximately 1 mm in size was created. During the enlargement process, samples 7 to 10 were created as fibrotic large liver-like structures by changing the timing of the change to a culture medium with the same composition and concentration as sample 1 of Example 1. Each of the created samples was immobilized, and the excised sections were stained with picrosilius red to obtain pathological images of the fibrotic regions (Figure 10). 【0053】 In samples 7 and 8, the peripheral portion of the three-dimensional liver-like structure was fibrotic. In samples 9 and 10, the fibrosis had spread to the interior of the three-dimensional liver-like structure. Thus, it was demonstrated that the area of ​​fibrosis can be controlled by combining a culture medium that induces fibrosis with the 3D printing process. 【0054】The present invention provides a liver-like or liver tissue-like structure, etc., as a novel in vitro evaluation model useful for the development of MASH therapeutic drugs. The liver-like or liver tissue-like structure according to the present invention can be made, for example, of a size that is easily visible (preferably 400 to 1,500 μm), making it easy to handle as the above in vitro evaluation model. Furthermore, the degree of fibrosis within the structure can increase with continued culture, making it possible to simulate disease progression, thus possessing usefulness and practicality.

Claims

1. A liver-like or liver tissue-like structure comprising inflamed human hepatic parenchymal cells and human hepatic stellate cells, wherein the structure is present in or cultured in a culture medium containing ascorbic acid, a pharmacoagulably acceptable salt thereof, or a hydrate or solvate thereof, or a derivative thereof.

2. The structure according to claim 1, wherein the cells constituting the structure have increased expression of at least one oxidative stress-related gene selected from the group consisting of SOD2, NQO1, GPX2, GPX7, GPX8, CAPN2, CTNNB1, CRYAB, FBLN5, GSTP1, PRDX4, AOX1, MT1A, MT2A, and PDLIM5.

3. The structure according to claim 2, wherein the expression level of the enhanced oxidative stress-related genes is at least twice as high as that of the cells constituting the liver-like or liver tissue-like structure cultured in a medium that does not contain ascorbic acid, a pharmacologically acceptable salt thereof, or their hydrates and solvates.

4. The structure according to claim 1, wherein the culture medium further comprises one or more saturated or unsaturated fatty acids having three or more alkyl chains, pharmaceutically acceptable salts thereof, hydrates or solvates thereof, or derivatives thereof.

5. The structure according to claim 4, wherein the saturated or unsaturated fatty acid having three or more alkyl chains is palmitic acid or oleic acid.

6. The structure according to claim 1, wherein the cells constituting the structure have enhanced expression of at least one inflammation-related gene or cytokine-related gene selected from the group consisting of CRP, SAA1, SAA2, CCL2, CCL20, CXCL1, CXCL2, CXCL6, CXCL8, LGALS1, and GSTP1.

7. The structure according to claim 6, wherein the enhanced expression levels of inflammation-related genes or cytokine-related genes are at least twice as high as those of the cells constituting the liver-like or liver tissue-like structure cultured in a medium that does not contain ascorbic acid, a pharmacologically acceptable salt thereof, or their hydrates and solvates.

8. The structure according to claim 1, wherein the culture medium further comprises TGFβ.

9. The structure according to claim 1, wherein the human hepatic stellate cells are activated human hepatic stellate cells.

10. The structure according to claim 1, wherein collagen fibers are deposited within the structure.

11. The structure according to claim 1, wherein when any cross section thereof is stained with picrosilius red, a fibrous stained region is observed, and the Fibrosis Index (%) calculated from the following formula (I): Fibrosis Index (%) = (Picrosilius Red Stained Area / Total Sample Area) × 100 (I) is in the range of 4% to 60%.

12. The structure according to claim 1, wherein the culture medium in which the structure is present or the culture medium in which the structure is cultured contains 20 ng or more of procollagen type I C-terminal peptide (PIP) per structure in the supernatant thereof.

13. The structure according to claim 1, wherein the ratio of the number of human hepatic stellate cells to the total number of cells in the structure is 1 to 10%.

14. The structure according to claim 1, wherein the human hepatocytes are at least one selected from the group consisting of human primary cultured hepatocytes, iPS cell-derived hepatocytes, hepatocytes induced by direct reprogramming, and hepatocytes taken from the liver of a human hepatocyte chimeric mouse.

15. The structure according to claim 1, wherein the structure has a circular equivalent diameter of 400 to 1,500 μm in the two-dimensional projection view.

16. The structure according to claim 1, wherein the structure is made up of multiple spheroids produced by a bio-3D printer stacked on top of each other.

Images