KR20220158637A - Non-alcoholic fatty liver artificial tissue model - Google Patents

Abstract

The present invention relates to an artificial tissue model of nonalcoholic fatty liver disease which includes a device comprising a decellularized liver tissue-derived extracellular matrix and a plurality of microchannels, and thus actual nonalcoholic fatty liver disease can be more properly simulated compared to the prior art where the culture is only performed in Matrigel and the decellularized liver tissue-derived extracellular matrix, by including Kupffer cells and hepatic stellate cells. Also, in the artificial tissue model of the present invention, the growth of liver organoids is improved, fat accumulation and inflammation in the liver organoids occur easily through free fatty acid treatment, and nonalcoholic fatty liver phenotype can be expressed more properly.

Description

Non-alcoholic fatty liver artificial tissue model {NON-ALCOHOLIC FATTY LIVER ARTIFICIAL TISSUE MODEL}

The present invention relates to an artificial tissue model of non-alcoholic fatty liver.

Non-alcoholic Fatty Liver (NAFL) disease has fatty liver as the basic lesion, and despite the lack of drinking history, tissue inflammation, necrosis, and fibrosis in the liver parenchyma similar to alcoholic liver damage It is a condition that indicates change. NAFL is basically asymptomatic, and transitions from fatty liver to fatty hepatitis and liver cirrhosis to liver cancer as the condition progresses. Steatohepatitis in NAFL is called Non-Alcoholic SteatoHepatitis (NASH). Particularly recently, metabolic syndrome caused by obesity or diabetes has become a social problem, and NASH is considered to be one of the metabolic syndromes. Lifestyle-related diseases such as obesity, diabetes, hyperlipidemia and hypertension are recognized as complications of NAFL and NASH, and the main characteristic of the clinical condition is an increase in the concentration of alanine aminotransferase (ALT) or hyaluronic acid in the blood, and lowering of total cholesterol or albumin concentration; and the like. However, there are still many unclear points about the onset of NAFL and NASH, and the reality is that effective treatment and therapeutic drugs have not been established. One of the reasons for this lies in the fact that NAFL and NASH are based on human lifestyle-related diseases, and therefore, suitable non-human models for the study of NAFL and NASH have not yet been established.

Elucidation of the pathological conditions of NAFL and NASH, which have the potential to progress to lethal diseases such as liver cirrhosis and liver cancer, is essential for the development of effective treatments and therapeutic drugs, and for this purpose, appropriate models of NAFL and NASH are needed.

Although animal models of NASH have been reported so far (for example, Patent Document 1), non-human animal models of NAFL have hardly been reported. Moreover, due to recent problems related to animal ethics, the need for developing an in vitro model as an alternative to animal models has increased.

Thus, the inventors of the present invention completed the present invention as a result of studying the non-alcoholic fatty liver model.

International Publication No. 2011/013247

One aspect of the present invention is a hydrogel comprising a decellularized liver tissue-derived extracellular matrix (Liver Extracellular Matrix; LEM); liver organoids; a device including a well in which the hydrogel is located and a plurality of microchannels through which free fatty acid flows; And to provide a non-alcoholic fatty liver artificial tissue model comprising a culture medium containing free fatty acids.

Another aspect of the present invention comprises the steps of producing the non-alcoholic fatty liver artificial tissue model; And it is an object to provide a method for producing a non-alcoholic fatty liver artificial tissue model comprising the step of perfusing a culture solution containing free fatty acids into the non-alcoholic fatty liver artificial tissue model.

Another aspect of the present invention comprises the step of processing a candidate substance to the non-alcoholic fatty liver artificial tissue model; And it aims to provide a screening method for non-alcoholic fatty liver treatment drugs comprising the step of comparing a group treated with the candidate substance and a control group.

Hereinafter, the present invention will be described with reference to the accompanying drawings. However, the present invention may be embodied in many different forms and, therefore, is not limited to the embodiments described herein. When a certain component is said to "include", this means that it may further include other components without excluding other components unless otherwise stated.

Unless otherwise defined, it can be performed by conventional techniques commonly used in the fields of molecular biology, microbiology, protein purification, protein engineering, and DNA sequencing and recombinant DNA within the capabilities of those skilled in the art. These techniques are known to those skilled in the art and are described in many standardized texts and reference books.

Unless defined otherwise herein, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art.

A variety of scientific dictionaries are well known and available in the art that contain terms included herein. Although any methods and materials similar or equivalent to those described herein will find use in the practice or testing of the present application, several methods and materials are described. Depending on the context of use by those skilled in the art, since it can be used in various ways, the present invention is not limited to specific methodologies, protocols and reagents. Hereinafter, the present invention will be described in more detail.

One aspect of the present invention is a hydrogel comprising a decellularized liver tissue-derived extracellular matrix (Liver Extracellular Matrix; LEM); liver organoids; Provided is an artificial non-alcoholic fatty liver tissue model including a device including a well in which the hydrogel is located and a plurality of microchannels through which free fatty acid flows, and a culture medium containing free fatty acid.

In one embodiment of the present invention, the decellularized liver tissue-derived extracellular matrix (Liver Extracellular Matrix; LEM) is one in which 95 to 99.9%, more specifically, 96 to 98%, and most specifically, 97.18% of the liver tissue cells are removed. can When decellularization is performed at a level of liver tissue cell removal outside the above range, there are problems in that the quality of the prepared scaffold composition is deteriorated or the economic efficiency of the process is reduced.

The "extracellular matrix" refers to a protein component found in mammals and multicellular organisms, and refers to a natural support for cell culture prepared through tissue decellularization. The extracellular matrix may be further processed through dialysis or crosslinking.

The extracellular matrix includes collagens, elastins, laminins, glycosaminoglycans, proteoglycans, antimicrobials, chemoattractants, and cytokines. ), and mixtures of structural and nonstructural biomolecules, including but not limited to growth factors.

The extracellular matrix may include about 90% of collagen in various forms in mammals. Extracellular matrices derived from various biological tissues may have different overall structures and compositions due to the unique role required for each tissue.

The above "derived", "derived" means a component obtained from the source mentioned by a useful method.

In one embodiment of the present invention, the liver organoid may be derived from human induced pluripotent stem cells (hiPSC) or human liver tissue.

The "organoid" refers to a microscopic biological organ produced in the form of an artificial organ by culturing cells derived from tissues or pluripotent stem cells in a 3D form.

The organoid is a three-dimensional tissue analog including organ-specific cells that arise from stem cells and self-organize (or self-pattern) in a manner similar to the in vivo state. can develop into

The organoids may have the original physiological characteristics of cells and may have an anatomical structure that mimics the original state of a cell mixture (including not only limited cell types but also residual stem cells and adjacent physiological niches). . The organoids can have cells and cell functions more well arranged through a 3-dimensional culture method, and have organ-like morphology and tissue-specific functions having functional properties.

Specifically, when the liver organoid is derived from human induced pluripotent stem cells (hiPSC), the liver organoid may be cultured on the hydrogel. In this method, a hydrogel is first injected into a well to form a hydrogel bed and organoids are cultured there. When cells are injected on the hydrogel bed, an organoid having a three-dimensional structure is formed within 24 hours. . In this case, the organoids can be cultured while being supported by the matrix and receiving the microfluidic flow of the culture medium. In particular, since the organoids are directly exposed to the culture medium, the transfer efficiency of the culture medium to the inside of the organoid is very excellent. Organoids having a three-dimensional structure can be formed through the culture structure of such liver organoids and hydrogels.

In addition, when the liver organoid is derived from human tissue, the liver organoid may be cultured in the hydrogel. When cultured in this structure, the hydrogel condenses over time to form a hard matrix, and since the hydrogel containing organoids remains attached to the bottom of the well during the culture process, the organoids continue to flow microfluidically in the culture medium. There is an effect that can be cultivated while receiving it.

The device includes a well in which a hydrogel is located and a plurality of microchannels through which free fatty acid can flow, and the device can be manufactured using a known material, specifically PDMS polymer. It can be. In addition, the device may be composed of a plurality of chambers, specifically, there are 8 chip units to which media are connected, each unit is composed of 4 culture chambers of D = 7.10 mm standard, and each chamber has D = It may consist of 5 mm wells and 8.66 mm wide media chambers at either end of each unit. When a device is manufactured with the above specifications, it is manufactured with the same specifications as the 96-well plate and can be used as a high-throughput multi-well device.

In addition, when the liver organoid is derived from human induced pluripotent stem cells (hiPSC), the well in which the hydrogel is located may have a depth of 2 to 4 mm, specifically 3 mm, and the liver organoid is derived from human tissue. In this case, the well in which the hydrogel is located may have a depth of 0.5 to 1.5 mm, specifically 1 mm. If the depth of the well is fabricated outside the above range, the flow of the culture solution through the channel may not be properly delivered to the organoid inside the hydrogel.

In one embodiment of the present invention, the concentration of the free fatty acid may be 100 to 900 μM, specifically 200 to 800 μM, and most specifically 800 μM. The free fatty acid flows through the plurality of microchannels provided in the above-described device to the well where the hydrogel is located, and affects liver organoids on or inside the hydrogel. When free fatty acids are used outside the above concentration range, the characteristics of non-alcoholic fatty liver may not appear or the cells in the tissue model may die.

In addition, the culture medium containing the free fatty acid induces non-alcoholic fatty liver by continuously exposing the liver organoid to the free fatty acid contained in the culture medium while culturing the liver organoid. Components of the culture may be a mixture of known substances used for liver organoid culture in addition to free fatty acids.

In one embodiment of the present invention, the free fatty acid may be any one selected from oleic acid, palmitic acid, and linoleic acid, specifically oleic acid.

In one embodiment of the present invention, the liver organoid is any one or more of vascular cells, mesenchymal stem cells, Kupffer cells (KC) and hepatic stellate cells (HSC), specifically Kupffer cells ( It may be co-cultured with Kupffer cells (KC) and hepatic stellate cells (HSC). The Kupffer cells are immune cells present in the liver, and secrete cytokines and reactive oxygen species (ROS) when inflammatory Kupffer cells are activated through non-alcoholic fatty liver induction. In addition, the hepatic stellate cells play a role in secreting fibrotic factors in non-alcoholic fatty liver disease. The non-alcoholic fatty liver artificial tissue model of the present invention further specifically reflects the microenvironment of the liver by further including any one or more of the above-described Kupffer cells and hepatic stellate cells as well as liver organoids, and through this, non-alcoholic fatty liver with a high degree of mimicry can be produced

Another aspect of the present invention comprises the steps of producing a non-alcoholic fatty liver artificial tissue model; and perfusing the non-alcoholic fatty liver artificial tissue model with a culture solution containing free fatty acids.

The step of manufacturing the non-alcoholic fatty liver artificial tissue model is a step of manufacturing the non-alcoholic fatty liver artificial tissue model, specifically, by using a PDMS polymer, a well in which a hydrogel is located and free fatty acid can flow. manufacturing a device (microfluidic chip) including a plurality of microchannels; and placing the hydrogel and the liver organoid in the well. Details of the hydrogel, liver organoid, and device are the same as those of the non-alcoholic fatty liver artificial tissue model described above.

The perfusion step is a step of perfusing a culture medium containing free fatty acid into the non-alcoholic fatty liver artificial tissue model. As described above, the culture medium containing free fatty acids flows through the microchannel of the device to the wells where the hydrogel and liver organoids are located, and the liver organoids are continuously exposed to the free fatty acids, thereby exhibiting a non-alcoholic fatty liver phenotype.

In one embodiment of the present invention, the perfusion may be performed by stirring the non-alcoholic fatty liver artificial tissue model on a stirrer. The perfusion of the culture solution containing the free fatty acid may be performed through a known device, but a continuous flow of the culture solution can be made to the liver organoids by using a stirrer with high accessibility and operation convenience. Specifically, when the non-alcoholic fatty liver artificial tissue model is placed on a stirrer, the culture medium is filled, and the stirrer is operated, the culture medium flows and perfusion occurs as it tilts in the left and right directions.

Another aspect of the present invention comprises the step of processing a candidate substance to the non-alcoholic fatty liver artificial tissue model; And it provides a screening method for a non-alcoholic fatty liver treatment drug comprising the step of comparing a group treated with the candidate substance and a control group.

The processing of the candidate material is a step of processing the candidate material on the non-alcoholic fatty liver artificial tissue model, and the treatment method of the candidate material may vary depending on the desired administration route and dosage of the candidate material.

In addition, the step of comparing the group treated with the candidate substance and the control group may be a step of comparing the non-alcoholic fatty liver artificial tissue model treated with the candidate substance and the control group. The control group is non-alcoholic fatty liver treated with or not treated with a conventionally known non-alcoholic fatty liver treatment drug or a known material within the range that does not inhibit or increase the physiological activity of liver organoids in the non-alcoholic fatty liver model. It may be an artificial tissue model.

Comparison between the group treated with the candidate substance and the control group confirmed the level of fat accumulation in liver organoids, survival rate of liver organoids, differentiation of liver organoids, functional analysis, and/or various indicators secreted in liver organoids or culture medium. It can be done by

In addition, the screening method may further include selecting a non-alcoholic fatty liver treatment drug. The selection step is the reduction of fat accumulation in liver organoids, the increase in viability of liver organoids, the differentiation of liver organoids, the recovery of functionality, and/or the increase of secreted improvement indicators in liver organoids or culture medium through the above-described comparison step. If the etc. are confirmed, it may be selected as a non-alcoholic fatty liver treatment drug. When using a conventionally known non-alcoholic fatty liver treatment drug as a control group, if it shows an improved effect compared to the control group, it can be determined and selected as having an improved effect than a conventionally known non-alcoholic fatty liver treatment drug.

The non-alcoholic fatty liver artificial tissue model of the present invention includes a device including a decellularized liver tissue-derived extracellular matrix and a plurality of microchannels, compared to the prior art cultured only in Matrigel and decellularized liver tissue-derived extracellular matrix, Kupffer cells. and hepatic stellate cells can be included to better simulate actual non-alcoholic fatty liver disease, and the growth of liver organoids is improved, as well as fat accumulation and inflammation in liver organoids occur easily through free fatty acid treatment There is an effect that can better simulate the .

In addition, since the non-alcoholic fatty liver artificial tissue model of the present invention simulates non-alcoholic fatty liver, it can be used to screen non-alcoholic fatty liver treatment drugs.

1 is a result of analyzing the extracellular matrix derived from decellularized liver tissue.
2 and 3 are diagrams showing the configuration and mode of action of a device for producing a non-alcoholic fatty liver artificial tissue model.
4 is a result of culturing liver organoids in the device of the present invention.
Figure 5 shows the experimental results for optimizing the free fatty acid treatment concentration.
6 to 10 show the results of selection of optimal culture conditions for the construction of non-alcoholic steatohepatitis (NASH) organoid models, and FIGS. 6 to 8 show optimized results for the construction of non-alcoholic steatohepatitis (NASH) organoid models. Experimental results for selecting a culture platform, and FIG. 9 shows results of simulation of fluid movement in a chip-based culture platform optimized for non-alcoholic steatohepatitis (NASH) induction, and FIG. 10 is non-alcoholic steatohepatitis (NASH) induction. It shows the results of simulation of fatty acid absorption in the chip-based culture platform optimized for .
11 to 14 show the result of confirming the effect of enhancing organoid differentiation through co-culture of liver tissue-specific microenvironmental cells. Kupffer cell differentiation results, FIG. 12 shows hepatic stellate cell (HSC) differentiation for a non-alcoholic steatohepatitis organoid model integrated with the liver tissue microenvironment and FIG. 13 shows the microenvironment of human tissue-derived liver organoids. It shows liver differentiation and functional comparison results according to the presence or absence of cell co-culture. 14 shows the results of comparison of liver differentiation and functionality of human iPSC-derived liver organoids with and without microenvironmental cell co-culture.
15 to 17 show the results of drug efficacy evaluation using a non-alcoholic steatohepatitis (NASH) organoid model, FIG. 15 is the result of verification with Obeticholic acid (OCA), and FIG. 16 is the result of verification with Ezetimibe (Eze). And Figure 17 is the result of verification with Dapagliflozin (DAPA).
18 to 22 show the results of verifying the mechanisms and effects of selected effective drugs based on non-alcoholic steatohepatitis (NASH) organoids. 18 is a result of verifying drug efficacy in a human tissue-derived liver organoid-based non-alcoholic steatohepatitis model, and FIGS. 19 and 20 verify drug efficacy in a human iPSC-derived liver organoid-based non-alcoholic steatohepatitis model. 21 is a result of comparing efficacy of effective drugs and identifying treatment mechanisms using human tissue-derived acute and chronic nonalcoholic steatohepatitis organoid models, and FIG. 22 is human tissue-derived acute and chronic nonalcoholic steatohepatitis organoid model. This is the result of comparing the efficacy of effective drugs using the hepatitis organoid model and identifying the treatment mechanism.
23 to 28 show the results of evaluating the efficacy of new therapeutic drugs discovered in non-alcoholic steatohepatitis (NASH) organoid models and verifying related mechanisms (evaluation of drug efficacy and mechanism study in NASH animal models). 23 to 26 show the results of DAPA drug validation in non-alcoholic steatohepatitis animal models. Figure 27 is a result of comparing the efficacy of effective drugs in acute and chronic nonalcoholic steatohepatitis animal models and identifying the treatment mechanism, Figure 28 is a comparison of the efficacy of effective drugs in acute and chronic nonalcoholic steatohepatitis animal models and treatment It shows the result of identifying the mechanism.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

Example 1: Fabrication of non-alcoholic fatty liver artificial tissue model

Example 1-1. Preparation of decellularized liver tissue-derived extracellular matrix

Porcine liver tissue was isolated and prepared by cutting, and decellularized liver tissue was prepared by removing only the cells of the liver tissue by stirring the liver tissue with 1% Triton X-100 and 0.1% ammonium hydroxide. Thereafter, the decellularized liver tissue was lyophilized and pulverized to prepare a liver extracellular matrix (LEM) derived from the decellularized liver tissue.

10 mg of the decellularized liver tissue-derived extracellular matrix was dissolved in a 4 mg/ml pepsin solution (4 mg of pepsin powder dissolved in 1 ml of 0.02 M HCl) for 48 hours. 10X PBS and 1 M NaOH were uniformly mixed in an electrolyte state of neutral pH and 1X PBS buffer, followed by gelation at a temperature of 37 ° C. for 30 minutes to prepare a hydrogel type support composition.

The prepared decellularized liver tissue-derived extracellular matrix was subjected to H&E staining before and after the decellularization process to confirm that the structure of the prepared decellularized matrix was well maintained and all cellular components were removed, and the internal structure was confirmed through scanning electron microscopy images. It was confirmed that the fiber bundles formed a stable polymer network. Through DNA quantitative comparison and quantitative analysis of Glycosaminoglycan (GAG), one of the representative extracellular matrix components, it was confirmed that most of the cellular components were removed and GAG was well preserved (FIG. 1A).

In addition, proteomics was performed to identify the components of the decellularized liver tissue-derived scaffold, and it was confirmed that various liver tissue-specific extracellular matrix (Collagens, Glycoproteins, Proteoglycans) and growth factor proteins were included in LEM. did In addition, Matrigel (MAT), an existing commercialized scaffold, is mostly ECM Glycoproteins, whereas decellularized liver tissue matrix (LEM) is composed of various components in the order of Proteoglycans and ECM regulators. . Among the 10 extracellular matrix (ECM) proteins with the highest amount detected, Biglycan (BGN), Lumican (LUM), and Asporin (ASPN), which are specifically present in LEM, are major proteins involved in ECM remodeling during liver tissue development. and PRELP is known as a protein that plays an important role in maintaining normal hepatocyte structure. Compared to Matrigel, the prepared decellularized liver tissue-derived extracellular matrix (LEM) scaffold contains various extracellular matrix proteins present in actual liver tissue that play an important role in liver structure, development, and function. confirmed (FIG. 1B).

Example 1-2. Fabrication of device for non-alcoholic fatty liver artificial tissue model

In the device manufactured by the microfluidic chip manufacturing method using PDMS polymer, there are 8 chip units to which media are connected, and each unit consists of 4 culture chambers of D=7.10 mm size, each chamber The device was fabricated so that a D = 5 mm standard well (manufactured in two types, H = 3 mm and 1 mm in depth), and media chambers with a horizontal length of 8.66 mm were located at both ends of each unit (FIG. 2A).

Specifically, when manufactured in the form of a developed 32-multiwell device, steatohepatitis can be more efficiently induced and high-throughput drug screening of disease organoids is possible. The high-throughput multi-well device has the same specifications as the existing 96-well plate, so it was manufactured to be compatible with general-purpose 96-well based analysis equipment including plate readers (Fig. 2B).

When the liver organoids are derived from human induced pluripotent stem cells (hiPSC), the liver organoids are cultured on the above extracellular matrix located in wells with a depth of 3 mm, and when the liver organoids are derived from human tissue, the liver organoids are cultured on the wells having a depth of 3 mm. Cultured in the extracellular matrix placed in 1 mm wells (Fig. 3A).

In addition to this, the device was fabricated to enable effective microfluidic flow formation using a commercially available rocking shaker (FIG. 3B).

Experimental Example 1: Confirmation of liver organoid culture in the device of the present invention and optimization of non-alcoholic fatty liver model

Experimental Example 1-1. Confirmation of liver organoid culture in the device of the present invention

In the device described above, liver organoids were cultured based on decellularized liver tissue-derived extracellular matrix (LEM) and compared with conventional well plates in a static culture environment. Specifically, liver organoids prepared from human liver tissue were used, and analysis was performed on the 7th day of culture.

As a result, as confirmed in FIG. 4, it was confirmed that the size of the liver organoid (LEM-chip) cultured in the device of the present invention significantly increased compared to the liver organoid (LEM-plate) cultured on the existing well plate. . When the organoid size was quantitatively analyzed, a significant increase in size was confirmed in the liver organoid group cultured while receiving the medium flow from the device (FIG. 4A).

In addition, when immunostaining of the proliferation related marker KI67 was performed to verify the above results, the control matrigel group cultured on the plate and the scaffold group derived from decellularized liver tissue showed similar proliferation ability, but decellularized liver tissue It was confirmed that KI67 expression significantly increased in the group in which the derived scaffold and the microfluidic device were combined (FIG. 4B). Through this, oxygen and nutrients were supplied smoothly, and it is assumed that the growth capacity of liver organoids was greatly increased and the size of organoids increased.

In addition, when gene expression was compared by quantitative PCR, compared to liver organoids prepared in a static plate culture environment, liver organoids cultured in the device of the present invention showed increased expression of LGR5, KRT19, HNF4A, SOX9 and FOXA2. It was confirmed (Fig. 4C).

Experimental Example 1-2. Optimization of free fatty acid treatment concentration

A disease model by culturing human liver organoids in a decellularized liver tissue-derived extracellular matrix-multiwell microfluidic device platform (LEM-Chip) with the highest steatohepatitis organoid-inducing efficiency and treating them with free fatty acid (Oleic acid) at different concentrations Optimal concentrations for fabrication were tested.

As a result, when human tissue-derived liver organoids were cultured on a platform combining decellularized LEM hydrogel and multi-well device and fatty acids were treated at different concentrations, fat accumulated inside the liver organoids at different concentrations, causing inflammation and It was confirmed that the noid-specific morphology and structure were damaged and deformed (FIG. 5A).

When the amount of fat accumulation inside the liver organoid was confirmed through immunostaining, the higher the concentration of the treated fatty acid, the higher the amount of fat accumulated inside the organoid. It was confirmed that the organoid model could be implemented (Fig. 5B).

As a result of analyzing the gene expression level for each concentration of fatty acid treatment through quantitative PCR analysis, it was confirmed that the characteristics of steatohepatitis organoids were best expressed when treated at a high concentration of 800 μM. Additionally, when comparing urea synthesis ability, which is a liver functional assay, it was confirmed that urea synthesis ability also decreased as the concentration of fatty acid treatment increased (FIG. 5C).

* PLIN2: Lipid droplet marker, HES1: NASH/Fibrosis-related notch signaling marker, CASP3: Cell death marker, LGR5: Stemness marker, HNF4A: Mature hepatocyte marker

Experimental Example 2: Selection of optimal culture conditions for non-alcoholic steatohepatitis (NASH) organoid model construction

Experimental Example 2-1. Selection of optimized culture platform for non-alcoholic steatohepatitis (NASH) organoid model construction (1)

In order to select the optimal culture platform for the construction of non-alcoholic steatohepatitis organoid model, (1) matrigel (MAT), which is a commercially available culture support, (2) decellularized liver tissue support (LEM), (3) decellularization Liver tissue support and multi-well device combined system (LEM-Chip) Human liver organoids were prepared under three culture conditions and treated with 800 µM oleic acid to induce a fatty liver model. Liver organoids were prepared using adult liver stem cells extracted from human liver tissue.

As a result, when liver organoids were cultured in the decellularized LEM hydrogel scaffold, it was confirmed that liver organoids of a shape and size similar to those of the Matrigel group (MAT) were formed. It was confirmed that liver organoids having a larger size were formed due to smooth oxygen supply due to the micro flow of the culture medium. Even when the fatty liver model was induced through fatty acid treatment, it was confirmed that the LEM-chip group induced the best induction of fat accumulation and inflammation inside organoids (FIG. 6A).

In addition, when confirmed by quantitative PCR after fatty acid treatment of liver organoids for 3 days to evaluate NASH disease induction efficiency according to each culture condition, the control matrigel group (MAT) and decellularized liver tissue support group (LEM) In comparison, it was confirmed that NASH disease induction was better in the decellularized liver tissue scaffold-multiwell device group (LEM-Chip) (FIG. 6B). (TNF-α: inflammatory marker, SMA: fatty liver/fibrosis marker, PLIN2: fat accumulation marker, ALB: mature liver differentiation marker)

Through this, a liver organoid culture platform in which a decellularized liver tissue-derived scaffold and a multi-well device were combined was selected as an optimal platform and used for later NASH-induced experiments.

Experimental Example 2-2. Selection of optimized culture platform for non-alcoholic steatohepatitis (NASH) organoid model construction (2)

In order to select the optimal culture platform for the construction of non-alcoholic steatohepatitis organoid model, (1) matrigel (MAT), which is a commercially available culture support, (2) decellularized liver tissue support (LEM), (3) decellularization Liver tissue support and multi-well device combined system (LEM-Chip) Human liver organoids were prepared under three culture conditions and treated with 800 µM oleic acid to induce a fatty liver model. Liver organoids were prepared using adult liver stem cells extracted from human liver tissue.

As a result, when steatohepatitis was induced for 3 days under 3 culture conditions and steatohepatitis induction efficiency was compared through immunostaining, liver organoids cultured under MAT and LEM conditions in a static culture environment (well plate) were more In the organoids cultured in the culture environment coupled with the multi-well device, the expression of SMA and VIM, which are liver fibrosis markers, was observed at much higher levels (FIG. 7A).

In addition, when the NASH disease induction efficiency according to each culture condition was confirmed by Oil red O staining (intracellular fat staining), the control matrigel group (MAT) and the decellularized liver tissue support group (LEM) in a static culture environment In comparison, it was confirmed that fatty acid accumulation inside the liver organoid was better induced in the decellularized liver tissue scaffold-multiwell device group (LEM-Chip) (FIG. 7B).

Also, when confirming collagen accumulated inside organoids through Masson's Trichrome (MT) staining, it was confirmed that the amount of accumulated collagen was higher in the LEM-Chip group than in the MAT and LEM groups. Through this, compared to static culture conditions, fatty acids treated under culture conditions using a microfluidic chip are best induced to accumulate into liver organoids by microflow through microchannels and fibrosis is best induced. It was confirmed (Fig. 7C).

Based on these results, a liver organoid culture platform in which a decellularized liver tissue-derived scaffold and a multi-well device were combined was selected as the optimal platform and used for NASH-induced experiments in the future.

Experimental example 2-3. Selection of optimized culture platform for non-alcoholic steatohepatitis (NASH) organoid model construction (3)

When non-alcoholic steatohepatitis (NASH) was induced in liver organoids, in the LEM-Chip condition selected as the optimal group, compared to other culture conditions, the amount and variety of inflammatory cytokines secreted increased. An immuno-antibody array was performed. Analysis was performed on the normal group and the NASH-induced group in which human tissue-derived liver organoids were cultured on the MAT scaffold as a control group, and the NASH-induced group while culturing under LEM culture conditions and LEM-Chip conditions.

When immuno-antibody array was performed on 40 inflammatory cytokines, quantitative analysis was performed on 7 major inflammatory cytokines with high secretion. As a result, seven cytokines (MIP-1δ, sTNF RI, RANTES, TIMP-2, IL-11, MCP-1, IL-8) tended to increase in the NASH-induced group compared to the Normal group, especially It was confirmed that the highest amount of inflammatory cytokine secretion was detected in the NASH organoid group cultured in the LEM-Chip (FIG. 8). Through this, it was confirmed that the culture platform combining the LEM scaffold and microfluidic chip developed in the present invention is the most optimized platform for inducing NASH model, rather than inducing steatohepatitis in the existing culture platform.

Experimental Example 2-4. Simulation of fluid movement in a chip-based culture platform optimized for induction of non-alcoholic steatohepatitis (NASH)

When non-alcoholic steatohepatitis (NASH) is induced by treating free fatty acids in the culture medium, fatty acid absorption into the organoid under the chip condition with the flow of the culture medium is higher than when NASH is induced under the static plate condition without the existing culture medium flow. A prediction simulation was performed for fluid movement within the chip for acceleration.

The flow of fluid over time was predicted by performing fluid simulation for the chip condition manufactured to induce non-alcoholic steatohepatitis. Since the flow formed by the stirrer changes the speed and direction of the fluid according to the movement of the stirrer, it was confirmed that adjusting the speed and angle of the stirrer can provide a more diverse fluid flow than the existing static plate culture environment ( Fig. 9). Through this, it was predicted that free fatty acids contained in the culture medium could be more efficiently transferred into the organoids in a dynamic chip environment with culture medium flow compared to a plate environment without fluid flow.

Experimental Example 2-5. Simulation of fatty acid absorption in a chip-based culture platform optimized for non-alcoholic steatohepatitis (NASH) induction

When non-alcoholic steatohepatitis (NASH) is induced by treating free fatty acids in the culture medium, fatty acids into the organoids are more concentrated under the chip condition with the flow of the culture medium than when NASH is induced under the static plate condition without the flow of the culture medium. In order to confirm whether absorption can occur more efficiently, prediction simulations of fatty acid absorption under each condition were performed.

Fatty acid uptake of organoids was predicted for 60 minutes through fluid flow and mass transfer simulations for static well plate conditions and dynamic chip conditions. As a result, it was confirmed that in the dynamic chip culture system, the movement and exchange of free fatty acids contained in the culture medium is promoted through the flow of fluid, and therefore, the absorption of fatty acids into the organoids increases rapidly compared to the well plate condition, which is a static culture system. (FIG. 10).

Experimental Example 3: Confirmation of organoid differentiation enhancement effect through co-culture of liver tissue-specific microenvironmental cells

Experimental Example 3-1. Differentiation of Kupffer cells for non-alcoholic steatohepatitis organoid model integrated with liver tissue microenvironment

Kupffer cells are immune cells present in the liver and are known to play an important role in disease phenotypes, such as secreting cytokines and reactive oxygen species (ROS) when inflammatory Kupffer cells are activated in the induction of non-alcoholic fatty liver. Therefore, Kupffer cells that can be co-cultured with liver organoids were differentiated from human induced pluripotent stem cells to prepare a NASH model equipped with such a liver tissue microenvironment.

As a result, it was confirmed that embryoid bodies (EBs) were prepared from human induced pluripotent stem cells and then differentiated into Kupffer cells through a macrophage precursor stage (FIG. 11A).

And when protein marker expression of the prepared Kupffer cells was analyzed through immunostaining, it was confirmed that monocyte and M2 immune cell markers F4/80 and CD163 and Kupffer cell-related marker CD34 were well expressed ( Figure 11B).

Experimental Example 3-2. Differentiation of hepatic stellate cells (HSCs) for a non-alcoholic steatohepatitis organoid model integrated with the liver tissue microenvironment

In the expression of non-alcoholic fatty liver inflammation, it is known that hepatic stellate cells play a role in secreting important fibrotic factors as they are activated. Therefore, in order to prepare a NASH model equipped with such a liver tissue microenvironment, hepatic stellate cells that can be co-cultured with liver organoids were differentiated from human induced pluripotent stem cells.

As a result, it was confirmed that human induced pluripotent stem cells were differentiated into hepatic stellate cells through mesoderm and mesothelial cells, and subculture was possible (FIG. 12A).

In addition, when marker expression was confirmed through immunostaining 14 days after hepatic stellate cell differentiation, COL1 and FN, which are extracellular matrix (ECM) markers secreted by hepatic stellate cells, and Vimentin and a-SMA, which are astrocyte differentiation markers, It was confirmed that this expression was well expressed (FIG. 12B).

Experimental Example 3-3. Comparison of liver differentiation and functionality of human tissue-derived liver organoids with and without microenvironmental cell co-culture

When vascular endothelial cells (EC), Kupffer cells (KC), and hepatic stellate cells (HSC) present in real liver are co-cultured with human tissue-derived normal liver organoid (LO), liver differentiation markers of liver organoids And, in order to confirm the functional improvement, these cells were included in liver organoids cultured on Matrigel (MAT) and liver organoids cultured on decellularized liver tissue-derived support (LEM) and cultured together. A group in which microenvironmental cells were not co-cultured was used as a control group. Human umbilical vein endothelial cells (HUVECs) were used as vascular endothelial cells, and Kupffer cells and hepatic stellate cells were previously differentiated from human induced pluripotent stem cells (iPSCs). 25,000 cells of vascular endothelial cells, 25,000 cells of Kupffer cells, and 20,000 cells of hepatic stellate cells were added to a 30 mL MAT or LEM hydrogel in which liver organoids were cultured and co-cultured, and quantitative PCR and urea synthesis performance analysis were performed 3 days later.

When vascular cells, hepatic stellate cells, and Kupffer cells were co-cultured on human liver organoids cultured on control Matrigel and decellularized LEM hydrogel scaffolds, microenvironmental cells were distributed around the liver organoids in both groups and co-cultured It was confirmed that this is possible (Fig. 13A).

In addition, when comparing the expression of liver differentiation markers through quantitative PCR analysis, the expression of liver differentiation markers in liver organoids cultured on LEM hydrogel was superior to that of the Matrigel group, and the co-culture group containing microenvironmental cells It was confirmed that the differentiation potential was further improved compared to the other groups (FIG. 13B).

In addition, when liver functionality was compared through analysis of urea synthesis ability, which is a representative liver function indicator, similarly, liver organoids cultured in LEM hydrogel had superior urea synthesis ability compared to the control matrigel group, and microenvironmental cells were included. It was confirmed that the urea synthesis ability of the co-culture group was further improved compared to the group without it (FIG. 13C). Through this, it can be seen that the liver organoid model in which the liver tissue-specific microenvironment is integrated has the best liver differentiation and functionality in the LEM hydrogel condition.

Experimental Example 3-4. Comparison of liver differentiation and functionality of human iPSC-derived liver organoids with and without microenvironmental cell co-culture

In addition to iPSC-derived hepatocytes (H), vascular endothelial cells (E), and mesenchymal stem cells (M) included in the production of existing iPSC-derived liver organoids, Kupffer cells (K) and hepatic stellate cells (S) additionally present in the liver When additionally co-cultured, these microenvironmental cells were additionally included in human iPSC-derived liver organoids (HEM) cultured on a decellularized liver tissue-derived scaffold (LEM) to confirm liver differentiation markers and functional enhancement. cultured (HEMKS). Specifically, in the case of co-cultured liver organoids (H:E:M:K:S), organoids were prepared by mixing a total of 400,000 cells at a ratio of 10:7:2:2:1. A group (HEM) in which these microenvironmental cells were not co-cultured was applied as a control group. Kupffer cells and hepatic stellate cells were previously differentiated from human iPSCs. After 5 days of co-culture, comparison between the HEM group and the HEMKS group was performed through immunostaining and quantitative PCR.

As a result, when immunostaining was performed for the Kupffer cell marker CD68 and hepatic stellate cell markers PDGFRB, GFAP, FN, and SMA, markers of each microenvironmental cell were not expressed in HEM organoids, whereas in HEMKS organoids It was confirmed that glycolytic cell-specific markers were expressed (FIG. 14A). In particular, when comparing the expression of SOX17, AFP, and ALB to confirm that the expression of hepatic differentiation markers is further enhanced by Kupffer cells and hepatic stellate cells, in the HEMKS group co-cultured with Kupffer cells and hepatic stellate cells, the expression of liver differentiation markers It was confirmed that expression was also increased.

In addition, when comparing the expression of each marker gene for the HEMKS organoid group in which a liver-specific microenvironment was established through quantitative PCR analysis and the HEM organoid group in which there was no microenvironmental cells, the expression of liver differentiation markers (HNF4A, ALB) It was confirmed that expression significantly increased in HEMKS organoids (FIG. 14B). In addition, in the case of HEMKS organoids, since hepatic stellate cells were added compared to the existing HEM organoids, it was confirmed that the expression of PDGFRB and COL1A1, which are mainly expressed in mature hepatic stellate cells, increased in the HEMKS group. Even in the case of vascular cells present in the existing HEM organoids, it was confirmed that the expression increased more than 10-fold in the HEMKS organoids additionally co-cultured with microenvironmental cells.

Through these results, it can be seen that the liver organoid model in which the liver tissue-specific microenvironment is integrated has higher liver differentiation and functionality than the existing liver organoid model, and therefore, it is different from the organoid in which the liver microenvironment is integrated. It is expected that more accurate disease phenotypes can be derived by proceeding with alcoholic steatohepatitis modeling.

Experimental Example 4: Evaluation of drug efficacy using non-alcoholic steatohepatitis (NASH) organoid model

Experimental Example 4-1. Evaluation of drug efficacy using non-alcoholic steatohepatitis organoids derived from human tissue (1)

Liver organoids in which a liver tissue microenvironment [vascular (E) + Kupffer cells (K) + hepatic stellate cells (S)] were established under culture conditions in which a decellularized liver tissue-derived LEM scaffold and a multi-well microfluidic device were combined Hepatitis (NASH) was induced and treated with obeticholic acid (OCA) at different concentrations. OCA (Obeticholic acid) is a semi-synthetic bile acid analogue that has been previously used to treat cholangitis and is a drug candidate whose efficacy has recently been verified in non-alcoholic steatohepatitis clinical trials. The NASH group was treated with 800 μM fatty acid for 3 days to induce disease, and the NASH + OCA drug group was treated with 800 μM fatty acid and the corresponding drug for 3 days.

As a result, compared to the organoids of the normal group (Normal), fatty liver organoids (NASH) accumulate fat inside and the organoid-specific shape and structure are damaged, whereas the concentration increases in the group treated with Obeticholic acid drug. It was confirmed that the shape and structure of organoids were restored and the amount of accumulated fat was reduced (FIG. 15A).

In addition, when the viability analysis of normal liver organoids treated with the drug was performed to evaluate the cytotoxicity according to the drug concentration, it was 80% or more at 1 μM, 70% at 10 μM, and 60% at 100 μM concentration conditions. It was confirmed that the survival rate was about % (FIG. 15B). That is, it was confirmed that liver toxicity increased as the concentration of OCA treatment increased.

In addition, when disease marker gene expression was compared by treatment concentration of effective drug through quantitative PCR analysis, the expression of inflammation-related markers (TNFα) and fibrosis-related markers (SMA) increased in the fatty liver organoid group (NASH), and OCA drugs It was confirmed that it decreased when treated with . However, at a drug concentration of 100 μM, it was confirmed that the expression of inflammatory markers and fibrosis-related markers was further increased due to drug toxicity (FIG. 15C).

Through these results, the NASH liver organoid model co-cultured with EC, HSC, and KC cells under LEM-Chip culture conditions was verified as an in vitro non-alcoholic steatohepatitis model for drug efficacy and toxicity evaluation.

Experimental Example 4-2. Evaluation of drug efficacy using human tissue-derived non-alcoholic steatohepatitis organoids (2)

Liver organoids in which a liver tissue microenvironment [vascular (E) + Kupffer cells (K) + hepatic stellate cells (S)] were established under culture conditions in which a decellularized liver tissue-derived LEM scaffold and a multi-well microfluidic device were combined Hepatitis (NASH) was induced and treated with the drug Ezetimibe (Eze) at different concentrations. Eze (Ezetimibe) is a cholesterol absorption inhibitor and is a drug candidate previously used to treat hyperglycemic cholesterol and lipid abnormalities. The NASH group was treated with 800 μM fatty acid for 3 days to induce disease, and the NASH + Eze drug group was treated with 800 μM fatty acid and the corresponding drug for 3 days.

As a result, compared to the organoids of the normal group (Normal), fatty liver organoids (NASH) have internal fat accumulation and the organoid-specific shape and structure are damaged. It was confirmed that the shape and structure of organoids were restored and the amount of accumulated fat was reduced (FIG. 16A).

In addition, when the viability analysis of normal liver organoids treated with the drug was performed to evaluate the cytotoxicity according to the drug concentration, the viability was more than 70% in the 1-10 μM condition and about 50% in the 100 μM concentration condition. It was confirmed that the survival rate was shown (FIG. 16B).

In addition, when disease marker gene expression was compared for each treatment concentration of the effective drug through quantitative PCR analysis, the expression of inflammation-related markers (TNFα) and fibrosis-related markers (SMA) increased in the fatty liver organoid group (NASH), and Ezetimibe drug It was confirmed that it decreased when treated with . When treated at a concentration of 10 μM or more, it was confirmed that the expression of inflammatory and fibrotic markers was reduced to a level similar to that of normal organoids (FIG. 16C).

Through these results, the NASH liver organoid model co-cultured with EC, HSC, and KC cells under LEM-Chip culture conditions was verified as an in vitro non-alcoholic steatohepatitis model for drug efficacy and toxicity evaluation.

Experimental Example 4-3. Evaluation of drug efficacy using non-alcoholic steatohepatitis organoids derived from human tissue (3)

Liver organoids in which a liver tissue microenvironment [vascular (E) + Kupffer cells (K) + hepatic stellate cells (S)] were established under culture conditions in which a decellularized liver tissue-derived LEM scaffold and a multi-well microfluidic device were combined Hepatitis (NASH) was induced and treated with the drug Dapagliflozin (DAPA) at different concentrations. DAPA (Dapagliflozin) is a sodium-glucose transporter inhibitor and is a drug candidate previously used to treat diabetes. The NASH group was treated with 800 μM fatty acid for 3 days to induce disease, and the NASH + DAPA drug group was treated with 800 μM fatty acid and the corresponding drug for 3 days.

As a result, compared to the organoids of the normal group (Normal), fatty liver organoids (NASH) accumulate fat inside and damage the organoid-specific shape and structure. It was confirmed that the shape and structure of organoids were restored and the amount of accumulated fat was reduced (FIG. 17A).

In addition, when the viability analysis of normal liver organoids treated with the drug was performed to evaluate the cytotoxicity according to the drug concentration, the viability was not affected up to 1 μM concentration, and the viability was not affected by 80% or more under the 10 μM condition. In the μM concentration condition, it was confirmed that the survival rate was about 60% (FIG. 17B).

In addition, when disease marker gene expression was compared by treatment concentration of effective drug through quantitative PCR analysis, inflammation-related marker (TNFα), fibrosis-related marker (SMA), fat accumulation marker (PLIN2), and hepatic stellate cell activation marker (PDGFRB) ) were all increased in the fatty liver organoid group (NASH) and decreased according to the DAPA treatment concentration. In the case of ALB expression, which is a marker for liver differentiation, it was confirmed that it decreased in the fatty liver organoid group (NASH) and then recovered again when treated with DAPA drug (FIG. 17C).

Through these results, the NASH liver organoid model co-cultured with EC, HSC, and KC cells under LEM-Chip culture conditions was verified as an in vitro non-alcoholic steatohepatitis model for drug efficacy and toxicity evaluation.

Experimental Example 5: Identification of mechanisms and effects of selected effective drugs based on non-alcoholic steatohepatitis (NASH) organoids

Experimental Example 5-1. Drug validation in human tissue-derived liver organoid-based non-alcoholic steatohepatitis model

After NASH was induced in a human tissue-derived liver organoid model integrated with the liver microenvironment on the LEM-Chip culture platform, an analysis was conducted to determine whether the previously selected dapagliflozin (DAPA) drug had a therapeutic effect on steatohepatitis. did In order to additionally verify the need for vascular cells (E), Kupffer cells (K), and hepatic stellate cells (S) when creating the NASH disease model, an organoid model in which microenvironmental cells were not co-cultured was used as a control.

As a result, immunostaining was performed on the normal organoid group (Normal - EKS) and the normal group (Normal + EKS) where liver-specific microenvironmental cells were not co-cultured, and microenvironmental cells were compared. It was confirmed that cells expressing CD68, a Kupffer cell marker, and α-SMA, a hepatic stellate cell marker, were distributed around the organoids in the co-cultured organoid group (FIG. 18A). When NASH was induced in the organoid group in which these cells were co-cultured, it was confirmed that some fatty acids were accumulated inside the organoid and also in the surrounding cells of the microenvironment, and it was confirmed that fatty acid accumulation was reduced in the group treated with DAPA drug. In addition, in the case of α-SMA, a hepatic stellate cell marker and at the same time a liver fibrosis marker, in the NASH + EKS group, it was also expressed in the organoids, surrounding matrix, and immune cells, whereas α-SMA expression decreased in the DAPA-treated group. confirmed that

In addition, through quantitative PCR analysis, NASH was induced for the group in which the microenvironment was established and the group in which only organoids were cultured, respectively, resulting in inflammation (IL-6, IL-1β, TNF-α) depending on the presence or absence of co-cultured cells (EKS). , fibrosis (SMA, COL1A1, TGF-β) and fatty acid biosynthesis (SREBPC1, FAS, ACC)-related genes, when NASH was induced in organoids that did not contain co-cultured cells, inflammation, fibrosis and fatty acid Although the expression of genes related to biosynthesis increased, when NASH was induced in organoids containing co-cultured cells, the expression of each gene related to inflammation, fibrosis, and fatty acid biosynthesis increased significantly (FIG. 18B). In addition, compared to normal organoids without co-cultured cells (Normal - EKS), normal organoids with co-cultured cells (Normal + EKS) contain stromal cells and immune cells, thereby increasing the number of genes for inflammation, fibrosis and fatty acid biosynthesis. Because of differences in the initial expression itself, inducing NASH models in organoids without co-cultured cells may provide inaccurate information regarding inflammation, fibrosis, and fatty acid biosynthesis. In addition, when the NASH organoid model integrated with the microenvironment was treated with the DAPA drug, it was confirmed that the expression of genes related to inflammation, fibrosis, and fatty acid biosynthesis was reduced and steatohepatitis was improved.

Through these results, the human tissue-derived NASH organoid model integrated with the LEM-Chip-based liver microenvironment developed in the present invention is a model that can present a more accurate non-alcoholic steatohepatitis disease phenotype than existing organoid models. At the same time, it was demonstrated that the newly discovered DAPA drug based on the model showed an effective treatment effect for steatohepatitis.

Experimental Example 5-2. Drug validation in human iPSC-derived liver organoid-based non-alcoholic steatohepatitis model (1)

To verify the drug efficacy of Dapagliflozin (DAPA) in the human iPSC-derived liver organoid (HEMKS) steatohepatitis model cultured on the LEM-Chip platform, the normal group, the group treated with 800 μM oleic acid for 5 days to induce NASH, and NASH + DAPA groups treated with 800 μM oleic acid and 50 μM DAPA were compared and analyzed. The NASH organoid model was subjected to immunostaining and western blot analysis for markers related to inflammatory response, fibrosis, and mTORC1 mechanism.

As a result, when comparing the expression of fibrosis-related marker SMA, fatty acid biosynthesis-related marker ACC, mTORC1 mechanism-related marker p-S6, and p-4EBP1 through immunostaining, these markers were significantly increased in the NASH group compared to normal organoids. and it was confirmed that the group treated with the DAPA drug showed a decreasing pattern (FIG. 19A). Through CD68 staining, a Kupffer cell marker, it was confirmed that Kupffer cells were mainly distributed in areas where the p-S6 marker, which caused a lot of inflammatory response, was overexpressed in the NASH group. Through ALB, a liver functional marker, and BODIPY staining, which marks accumulated fatty acids, it was confirmed that accumulated fatty acids increased and ALB decreased in the NASH group, while fatty acid accumulation decreased and ALB expression increased in the group treated with the DAPA drug. It was confirmed that the functionality was improved.

In addition, when the expression of each marker protein was confirmed through western blot analysis, fibrosis-related markers (SMA, Col1A1), inflammation-related markers (IL1-β, p-IkB/IkB), and mTORC1-related markers (p-4EBP1/4EBP1, p -p70S6K1/p70S6K1, p-S6/S6) expression was increased in NASH organoids and decreased in the group treated with DAPA drug (FIG. 19B).

Through these results, in the human iPSC-derived NASH organoid model prepared under LEM-Chip culture conditions, inflammation and fibrosis responses increased similar to actual steatohepatitis, whereas inflammatory responses, fibrosis and mTORC1-related protein expression by the newly discovered DAPA drug It was demonstrated that the recovery was similar to this normal.

Experimental Example 5-3. Drug validation in human iPSC-derived liver organoid-based non-alcoholic steatohepatitis model (2)

To verify the drug efficacy of Dapagliflozin (DAPA) in the human iPSC-derived liver organoid (HEMKS) steatohepatitis model cultured on the LEM-Chip platform, the normal group, the group treated with 800 μM oleic acid for 5 days to induce NASH, and NASH + DAPA groups treated with 800 μM oleic acid and 50 μM DAPA were compared and analyzed.

As a result, when Masson's Trichrome (MT) staining was performed to confirm the accumulation of collagen due to fibrosis inside the organoid, the amount of collagen accumulation due to accompanying fibrosis was significantly increased in the NASH group compared to the normal organoid group and treated with DAPA. The decrease in the Haejun group was confirmed through histological staining and quantitative analysis (FIG. 20A).

In addition, through Oil red O staining, which stains fatty acids accumulated in cells, it was confirmed that fat accumulation, which is stained red inside organoids, significantly increased, and that fatty acid accumulation was reduced in the group treated with the DAPA drug (FIG. 20B). )

Through these results, it was confirmed that the efficacy of the DAPA drug can be verified in the human iPSC-derived liver organoid (HEMKS) steatohepatitis model cultured on the LEM-Chip platform.

Experimental Example 5-4. Comparison of effective drug efficacy and identification of treatment mechanism using human tissue-derived acute and chronic non-alcoholic steatohepatitis organoid models (1)

In order to identify and validate the treatment mechanism of Dapagliflozin (DAPA) drug in human tissue-derived liver organoid steatohepatitis (NASH) model cultured on the LEM-Chip platform, Normal group, NASH-induced group, and NASH + DAPA group were selected. Comparative analysis was performed. In the case of the acute NASH model, organoids were treated with 800 μM oleic acid for 3 days and 800 μM oleic acid + 50 μM DAPA for 3 days, respectively. In the chronic NASH model, organoids were treated with 800 μM oleic acid for 5 days. + 20 ng/ml TGF-β treatment group and 800 μM Oleic acid + 20 ng/ml TGF-β + 50 μM DAPA treatment group for 5 days. Western blot analysis was performed on markers related to inflammatory response, fibrosis, YAP/TAZ, and mTORC1 mechanisms on human tissue-derived acute and chronic NASH organoid models.

Through Western blot analysis, fibrosis markers α-SMA, COL1A1, inflammatory markers p-IkB/IkB, pro-IL1β, YAP/TAZ mechanism-related markers p-YAP/YAP and mTORC1 mechanism-related markers p-S6/S6, p-p70S6K1 As a result of comparing the expression of /p70S6K1 and p-4EBP1/4EBP1 proteins, the expression of protein markers significantly increased in both the acute and chronic NASH groups compared to normal organoids in the case of proteins related to fibrosis and inflammatory response, which was treated with DAPA drug. It was confirmed that it decreased in one group (FIG. 21A). However, in the case of YAP/TAZ mechanism-related proteins and mTORC1 mechanism-related proteins, (1) the expression increased in acute NASH organoids and decreased in the DAPA drug-treated group, whereas (2) in chronic NASH organoids It was confirmed that the expression of these proteins significantly decreased and recovered to a level similar to normal by the DAPA drug.

And, even when quantification was performed through three repetitions of the experiment, it was confirmed that the expression of markers related to fibrosis and inflammatory response was significantly reduced by DAPA in both acute and chronic NASH models (FIG. 21B). However, the expression of YAP/TAZ mechanism-related proteins and mTORC1 mechanism-related proteins increased in the acute model and significantly decreased in the chronic model, and it was confirmed that the expression was restored similarly to normal organoids by the DAPA drug.

Through these results, when acute and chronic nonalcoholic steatohepatitis were induced in human tissue-derived organoid models, the expression of YAP/TAZ and mTORC1-related proteins increased in the acute model and decreased in the chronic model. It was confirmed that the over- or reduced protein expression was restored to a normal level by DAPA, a newly discovered effective drug. Therefore, it is verified that the human NASH organoid model constructed in the present invention can be used not only for discovering effective drugs for NASH treatment, but also for research to identify the therapeutic mechanism at the molecular level of the drug.

Experimental Example 5-5. Comparison of efficacy of effective drugs and identification of treatment mechanisms using human tissue-derived acute and chronic non-alcoholic steatohepatitis organoid models (2)

In order to confirm the reproducibility of the protein expression results confirmed by western blot analysis for acute and chronic NASH organoid models derived from human tissue cultured on the LEM-Chip platform, immunostaining was performed for markers related to liver function, fibrosis, and mTORC1 mechanism. .

In order to efficiently induce acute and chronic NASH in the human tissue-derived organoid model cultured on the LEM-Chip platform, an optimized model was established by applying different concentrations of fatty acid treatment, treatment period, and fibrosis-inducing cytokine treatment. In the case of the acute NASH model, organoids were treated with only 800 μM oleic acid for 3 days as in the previous condition, and in the case of the chronic NASH model, organoids were treated with 800 μM oleic acid and 20 ng/ml TGF-β, a fibrotic cytokine, for 5 days. caused it. In the case of the DAPA drug test, 800 μM Oleic acid + 50 μM DAPA was treated for 3 days for the acute model, and 800 μM Oleic acid + 20 ng/ml TGF-β + 50 μM DAPA was treated for 5 days for the chronic model. (FIG. 22A).

When immunostaining was performed on the acute model induced by treating the human tissue-derived NASH organoid model with fatty acids for 3 days, AFP, a liver function-related marker, decreased in the NASH organoid model, but in the DAPA drug-treated group It was confirmed that the functionality was partially restored, and in the case of the fibrosis-related α-SMA marker, it was confirmed that the expression increased in the NASH-induced group and decreased in the DAPA drug-treated group. Comparing the expression of p-S6 and p-P70S6K1 related to the mTORC1 mechanism, it was confirmed that the expression of p-S6 and p-P70S6K1 was increased in the acute NASH model compared to normal organoids and decreased by the DAPA drug (FIG. 22B).

When immunostaining was performed on a chronic model induced by treating a human tissue-derived NASH organoid model with fatty acids and TGF-β for 5 days, AFP, a liver function-related marker, decreased in the NASH organoid model, then DAPA drug was used. It was confirmed that some of the functionality was recovered in the treated group, and in the case of the fibrosis-related α-SMA marker, it was confirmed that the expression increased in the NASH-induced group and decreased in the DAPA drug-treated group. In particular, it can be confirmed that the α-SMA marker expression greatly increased in the NASH model induced chronically rather than acutely. Comparing the expression of p-S6 and p-P70S6K1 related to the mTORC1 mechanism, it was confirmed that the expression decreased in the chronic NASH model compared to normal organoids and was restored by the DAPA drug (FIG. 22C).

Through these results, when acute and chronic non-alcoholic steatohepatitis were induced in human tissue-derived organoid models, mTORC1-related protein expression increased in the acute model and decreased in the chronic model, identical to the western blot analysis results. It was observed that the over- or reduced protein expression was restored to normal levels by DAPA, a newly discovered effective drug.

Experimental Example 6: Efficacy evaluation and related mechanism verification of novel therapeutic drugs discovered in non-alcoholic steatohepatitis (NASH) organoid model (Evaluation of drug efficacy and mechanism study in NASH animal model)

Experimental Example 6-1. DAPA drug efficacy verification in non-alcoholic steatohepatitis animal model (1)

In order to confirm that Dapagliflozin (DAPA), which was discovered through a steatohepatitis (NASH) organoid model and whose efficacy in NASH treatment was verified, is also effective in an actual NASH animal model, early non-alcoholic steatohepatitis was induced in mice and DAPA drug was administered to 5 It was administered once on day 2 at a concentration of mg/kg.

The Fa/R (Fasting overnight, refed with high-carbohydrate, fat-free diet) model induced in this experiment is a model that can well simulate the fatty acid biosynthesis process (De novo lipogenesis) of patients with actual non-alcoholic steatohepatitis. This is a model that acutely induces steatohepatitis with a high-carbohydrate/fat-free diet for the rest of the day after not feeding during the day. DAPA drug was administered once at a concentration of 5 mg/kg by oral administration at the time of administration of the Fa/R feed (FIG. 23A).

When liver tissues were collected from mice in the normal, Fa/R, and Fa/R + DAPA groups and subjected to H&E staining and MT staining, a lot of fatty acid accumulation was observed in the liver tissues of the Fa/R group, and collagen due to the fibrotic reaction was observed. It was confirmed that it accumulated inside. In contrast, in the group administered with DAPA, fatty acid accumulation was reduced and fibrosis symptoms were improved. Even when the collagen-stained area was quantitatively analyzed, it was confirmed that the collagen area was decreased in the liver tissue of the DAPA drug-administered group (FIG. 23B).

In addition, 3-Nitrotyrosine (3-NT) is accumulated due to an increase in reactive oxygen species in the liver and is highly expressed in steatohepatitis or fibrotic liver tissue. To confirm the effect of reducing oxidative stress by DAPA drug, protein components of liver tissue were extracted and western whole blotting analysis was conducted for 3-NT protein. In the Fa/R group, 3-NT expression increased. However, it was confirmed that a significant decrease was observed in the group treated with the DAPA drug (FIG. 23C).

The liver is responsible for the function of detoxifying lipopolysaccharide (LPS), and when fatty liver is induced, the function of hepatocytes decreases and the amount of serum LPS increases. Since the accumulation of collagen increases as fibrosis occurs, hydroxyproline, which is present in large amounts in collagen increased due to fibrosis, is also used as an indicator of fibrosis caused by steatohepatitis. Serum LPS and albumin were measured by blood analysis through whole blood collection on day 3, and the concentration of hydroxyproline was quantified through ELISA analysis for the corresponding protein. Compared to the normal group, it was confirmed that the amount of LPS and hydroxyproline increased in the Fa/R group and decreased in the group treated with the DAPA drug (FIG. 23D). Serum albumin, as an indicator of liver function, decreased in the Fa/R group and was recovered by the DAPA drug.

Through these results, it was verified that the DAPA drug, whose efficacy was verified in the NASH organoid model, was equally effective in an animal model that induced acute early non-alcoholic steatohepatitis. It was confirmed that it can be applied as an in vitro model for material discovery.

Experimental Example 6-2. DAPA drug efficacy verification in non-alcoholic steatohepatitis animal model (2)

To confirm that Dapagliflozin (DAPA), whose efficacy has been verified in the NASH organoid model, is also effective in the actual NASH animal model, early non-alcoholic steatohepatitis was induced in mice and the drug was orally administered (5 mg/day). kg). Analysis was performed on Day 3 when model induction and drug administration were completed.

When TUNEL staining to detect apoptosis was performed on the liver tissues of mice in the normal, Fa/R, and Fa/R + DAPA groups, TUNEL intensity increased in the Fa/R group and decreased by DAPA drug. By confirming, it was confirmed that necrosis of tissues due to steatohepatitis was inhibited by the DAPA drug (FIG. 24A).

Even when immunohistochemistry (IHC) analysis was performed for 3-Nitrotyrosine (3-NT) protein in liver tissue to confirm the effect of reducing oxidative stress by DAPA, the Fa/R group Compared to the increase in cells expressing 3-NT due to ballooning of hepatocytes, a significant decrease was confirmed in the group treated with the DAPA drug (FIG. 24B).

When quantitative PCR analysis was performed on the liver tissue of each group, the expression of inflammatory response-related IL-6, IL-1β, and TNF-α and the expression of fibrosis-related COL1A1, SMA, and TGF-β were found in the acute NASH model Fa// It was confirmed that it increased in the R group and decreased by the DAPA drug (FIG. 24C).

When ALT, which is an indicator of liver toxicity, was also analyzed through hematology analysis, ALT was increased about 10 times in the Fa/R model compared to the normal mouse group, and it was confirmed that the ALT level was significantly decreased by the DAPA drug (FIG. 24D).

Through these results, it was verified that the DAPA drug, whose efficacy was verified in the NASH organoid model, was effective in an animal model that induced acute non-alcoholic steatohepatitis.

Example 6-3. DAPA drug efficacy verification in non-alcoholic steatohepatitis animal model (3)

In order to confirm that Dapagliflozin (DAPA), whose efficacy has been verified in the NASH organoid model, is also effective in the actual NASH animal model, non-alcoholic steatohepatitis with severe lesions was induced in mice and the drug was administered.

The CDAHFD (Choline-deficient, L-amino acid-defined, high-fat diet) model induced in this experiment is a model that can well simulate NASH-related fibrosis accompanied by fibrosis in patients with severe non-alcoholic steatohepatitis. This is a model of chronic steatohepatitis caused by choline-deficient high-fat diet for 8 weeks. The DAPA drug was injected via oral administration at a concentration of 5 mg/kg in an administration cycle of 3 times a week from 4 weeks after the CDAHFD diet was induced (FIG. 25A).

When H&E staining and MT staining were performed on liver tissues collected at week 8 from mice in the normal, CDAHFD, and CDAHFD + DAPA groups, fatty acid accumulation occurred severely in the CDAHFD group, and collagen was excessively accumulated inside due to the fibrotic reaction. Confirmed. Through this, it can be confirmed that the CDAHFD model is a model that induces fibrosis more strongly than the previous Fa/R model. In contrast, in the group administered with the DAPA drug, it was confirmed that fat accumulation was reduced and fibrosis symptoms were improved. Even when the collagen-stained area was quantitatively analyzed, it was confirmed that it was significantly decreased in the group administered with the DAPA drug (cv: central vein) (FIG. 25B).

3-Nitrotyrosine (3-NT) accumulates due to increased reactive oxygen species in the liver and is highly expressed in steatohepatitis or fibrotic liver tissue. To confirm the effect of reducing oxidative stress by DAPA drug, protein components of liver tissue were extracted at 8 weeks and western whole blotting analysis was conducted for 3-NT protein. In the CDAHFD group, 3-NT expression was Compared to the increase, it was confirmed that the group treated with the DAPA drug significantly decreased (FIG. 25C).

Similar to the previous Fa/R model, when the amounts of serum LPS and hydroxyproline, which are indicators of liver toxicity, were checked at week 8, the amounts of serum LPS and hydroxyproline were increased in the CDAHFD group compared to the normal group, and the DAPA drug-treated A decrease was observed in the group. Serum albumin, as an indicator of liver function, decreased in the CDAHFD group and was recovered by the DAPA drug (FIG. 25D).

Through these results, it was verified that the DAPA drug, whose efficacy was verified in the NASH organoid model, was equally effective in an animal model that induced chronic non-alcoholic steatohepatitis. It was confirmed that it can be applied as an in vitro model for excavation.

Experimental Example 6-4. Validation of DAPA drug efficacy in non-alcoholic steatohepatitis animal model (4)

In order to confirm that Dapagliflozin (DAPA), whose efficacy has been verified in the NASH organoid model, is also effective in the actual NASH animal model, non-alcoholic steatohepatitis with severe lesions was induced in mice and the drug was administered. The drug was orally administered three times a week at a concentration of 5 mg/kg from the 4th week. Quantitative PCR analysis was performed at the end of the 8th week experiment.

When the size of the liver tissue was checked for each group at the end of the 8th week of the experiment, the CDAHFD chronic non-alcoholic steatohepatitis model showed severe ballooning of the liver tissue and increased in volume. The liver tissue of the DAPA drug-treated group In the case of , it was confirmed that the ballooning phenomenon was reduced (FIG. 26A).

Quantitative PCR analysis was performed on liver tissues collected from mice of the normal, CDAHFD, and CDAHFD + DAPA groups at week 8. Expression of IL-6, IL-1β, and TNF-α related to inflammatory response and COL1A1 and SMA related to fibrosis , It was confirmed that the expression of TGF-β was increased in the CDAHFD group, which is a chronic NASH model, and decreased by the DAPA drug (FIG. 26B).

Through these results, it was verified that the DAPA drug, whose efficacy was verified in the NASH organoid model, had an improvement effect on steatohepatitis in both animal models that induced acute early NASH or chronic NASH.

Experimental Example 6-5. Comparison of efficacy of effective drugs in acute and chronic non-alcoholic steatohepatitis animal models and identification of treatment mechanisms (1)

In order to verify that the DAPA drug, which has been validated in acute and chronic non-alcoholic steatohepatitis animal models, works in animal models according to the same treatment mechanism previously identified in acute and chronic NASH liver organoid models, western blot analysis and based on this Marker quantification analysis was performed. Acute and chronic non-alcoholic steatohepatitis animal models were induced by Fa/R and CDAHFD feed, respectively.

Western blot analysis compared the expression of fibrosis-related α-SMA, COL1A1, inflammation-related p-IkB/IkB, pro-IL1β, mTORC1 mechanism-related p-S6/S6, p-p70S6K1/p70S6K1, and p-4EBP1/4EBP1. Compared to the liver tissue of normal mice, the expression of marker proteins related to fibrosis and inflammatory response was significantly increased in both acute (Fa/R) and chronic (CDAHFD) liver tissues of the NASH group, and in the DAPA drug-treated group, the corresponding markers It was confirmed that their expression decreased. In the case of mTORC1 mechanism-related proteins, their expression increased in the acute NASH animal model (Fa/R) and decreased in the DAPA drug-treated group, whereas in the chronic NASH animal model (CDAHFD), mTORC1-related protein expression significantly decreased and DAPA drug It was confirmed that it was recovered to a level similar to normal by (FIG. 27A).

Even when quantitative analysis was performed through repeated Western blot experiments, the amounts of fibrosis-related proteins and inflammation-related proteins increased in acute and chronic models and were lowered by DAPA drugs, and in the case of mTORC1-related proteins, the increased expression level in acute models was correlated with DAPA drugs. recovered by Conversely, in the chronic model, the expression level of mTORC1-related protein was significantly reduced and improved by DAPA drug. However, in the case of p-4EBP1/4EBP1, unlike the chronic NASH organoid model, no significant decrease was observed by DAPA treatment in the chronic NASH animal model (FIG. 27B).

Through these results, as in human tissue-derived acute and chronic NASH organoid models, in acute and chronic NASH animal models, the treatment mechanism related to mTORC1 of DAPA, a newly discovered effective drug, is mostly consistent, and protein overexpressed or reduced by DAPA drugs It was confirmed that the expression was restored to a normal level.

Experimental Example 6-6. Comparison of efficacy of effective drugs in acute and chronic non-alcoholic steatohepatitis animal models and identification of treatment mechanisms (2)

In acute and chronic non-alcoholic steatohepatitis animal models, immunostaining was performed on the liver tissue to confirm that the protein expression pattern for each marker was consistent with the previous western blot analysis of the DAPA-treated liver tissue. . Acute and chronic non-alcoholic steatohepatitis animal models were induced by Fa/R and CDAHFD feed, respectively.

When fibrosis-related α-SMA was stained through immunostaining, it was confirmed that the expression was increased in Fa/R and CDAHFD models compared to normal liver tissue and decreased by DAPA drug. Through staining for the mTORC1 mechanism-related p-S6 protein, it was confirmed that the corresponding marker increased and decreased by DAPA in the acute model, and the reduced expression was recovered by DAPA in the chronic model, as previously verified. Through immunostaining for another mTORC1 mechanism-related p-4EBP1 protein, it was confirmed that its expression was increased in both acute and chronic NASH models, but its expression was decreased by DAPA drug. This is a result that is generally consistent with the western blot analysis results previously confirmed (FIG. 28).

Therefore, it was confirmed through immunostaining analysis that most of the mTORC1-related therapeutic mechanisms of DAPA, a newly discovered effective drug, were identical in acute and chronic NASH animal models, as in human tissue-derived acute and chronic NASH organoid models.

So far, the present invention has been looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.

Claims (12)

a hydrogel containing a decellularized liver tissue-derived extracellular matrix (Liver Extracellular Matrix; LEM);
liver organoids;
a device including a well in which the hydrogel is located and a plurality of microchannels through which free fatty acid flows; and
A non-alcoholic fatty liver artificial tissue model containing a culture medium containing free fatty acids.
According to claim 1,
The non-alcoholic fatty liver artificial tissue model in which the decellularized liver tissue-derived extracellular matrix is 95 to 99.9% of liver tissue cells removed.
According to claim 1,
The liver organoid is a non-alcoholic fatty liver artificial tissue model derived from human induced pluripotent stem cells (hiPSC) or human liver tissue.
According to claim 3,
When the liver organoids are derived from human induced pluripotent stem cells (hiPSC), the liver organoids are cultured on the hydrogel artificial non-alcoholic fatty liver tissue model.
According to claim 3,
When the liver organoid is derived from human tissue, the non-alcoholic fatty liver artificial tissue model, wherein the liver organoid is cultured in the hydrogel.
According to claim 4,
The well in which the hydrogel is located is a non-alcoholic fatty liver artificial tissue model having a depth of 2 to 4 mm.
According to claim 5,
The well in which the hydrogel is located is a non-alcoholic fatty liver artificial tissue model having a depth of 0.5 to 1.5 mm.
According to claim 1,
The free fatty acid (free fatty acid) is a non-alcoholic fatty liver artificial tissue model having a concentration of 100 to 900 μM.
According to claim 1,
The liver organoid is a non-alcoholic fatty liver artificial tissue model co-cultured with any one or more of vascular cells, mesenchymal stem cells, Kupffer cells (KC) and hepatic stellate cells (HSC).
Producing the non-alcoholic fatty liver artificial tissue model of claim 1; and
A method for producing a non-alcoholic fatty liver artificial tissue model comprising the step of perfusing a culture solution containing free fatty acids into the non-alcoholic fatty liver artificial tissue model.
According to claim 10,
The perfusion step is a method of manufacturing a non-alcoholic fatty liver artificial tissue model made by stirring the non-alcoholic fatty liver artificial tissue model on a stirrer.
Processing a candidate substance to the non-alcoholic fatty liver artificial tissue model of claim 1; and
A method for screening a non-alcoholic fatty liver treatment drug comprising the step of comparing a group treated with the candidate substance and a control group.

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