Method for producing hepatocytes derived from transparent embryoid bodies by using technique of co-culture with mesenchymal stem cell patch and drug toxicity evaluation method using 3D liver organoids produced thereby

Abstract

The present invention relates to: a method for producing hepatocytes derived from transparent embryoid bodies by using a technique of co-culture with a mesenchymal stem cell patch; and a drug toxicity evaluation method using 3D liver organoids produced thereby. Hepatocytes produced using a technique of co-culture with a mesenchymal stem cell patch, and 3D liver organoids produced using the hepatocytes with a microfluidic chip for three-dimensional cell culture, of the present invention, are differentiated from transparent embryoid bodies exhibiting the characteristics of mature hepatocytes, and thus can reproduce the liver of an actual human body in the same way. Also, the production method enables purification of hepatocytes to high purity. In addition, the 3D liver organoids produced by the production method express the same markers as mature hepatocytes, and thus can be effectively used for drug toxicity evaluation.

Description

Method for producing hepatocytes derived from transparent embryoid bodies using mesenchymal stem cell patch co-culture technique and method for evaluating drug toxicity using 3D liver organoids produced thereby

[0001] The present invention relates to a method for producing hepatocytes derived from transparent embryoid bodies using a mesenchymal stem cell patch co-culture technique and a method for evaluating drug toxicity using 3D liver organoids produced thereby.

[0002] The liver is an important organ for maintaining life and regulating the body's homeostasis. It is one of the body's major organs that performs various essential functions, such as protein synthesis, metabolism, and decomposition of toxic substances, including cholesterol and bile acids.

[0003] The liver is a representative organ with in vivo regenerative potential. Therefore, animal models or tumor-derived hepatocytes have been utilized in research on the mechanisms of liver disease and the development of treatments. However, due to genetic and physiological differences with actual human hepatocytes, primary human hepatocytes are currently considered the most suitable model for in vivo liver models. However, primary human hepatocytes are limited in supply, have issues with cryopreservation, have limited in vitro culture, and present inconsistent cell characteristics.

[0004] To overcome these limitations, studies have been conducted using hepatocytes derived from pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs).

[0005] In particular, liver spheroids or organoids derived from PSCs are emerging as an alternative to in vitro 3D liver models, but they are still limited in maintaining the proliferation capacity and liver functionality of hepatocytes.

[0006] Accordingly, the present inventors developed a method for producing hepatocytes and liver organoids by isolating transparent embryoid bodies from embryoid bodies (EBs) formed from PSCs, and co-culturing the transparent embryoid bodies with mesenchymal stem cell (MSC) patches, and completed the present invention by confirming the reproducibility of a human liver model using hepatocytes and liver organoids produced through the above production method.

[0007] The purpose of the present invention is to confirm that the expression level of the hepatocyte marker AFP in transparent embryoid bodies formed from human pluripotent stem cells is expressed at least 50 times higher than that in opaque embryoid bodies, and to provide a method for producing differentiated hepatocytes from transparent embryoid bodies by co-culturing the transparent embryoid bodies with mesenchymal stem cells through a mesenchymal stem cell patch co-culture technique.

[0008] Another object of the present invention is to provide a method for producing a 3D liver organoid by differentiating the hepatocytes using a 3D cell culture microfluidic chip.

[0009] Another object of the present invention is to provide a 3D liver organoid manufactured by the above method.

[0010] Another object of the present invention is to provide a method for evaluating drug toxicity using a 3D liver organoid manufactured by the above method.

[0011] In order to achieve the above-mentioned purpose, the present invention comprises the steps of: i) isolating a transparent embryoid body (EB) from an embryoid body (EB) formed by culturing human pluripotent stem cells;

[0012] ii) A step of co-culturing the above transparent embryoid body with a mesenchymal stem cell patch to differentiate it into definitive endoderm (DE);

[0013] iii) a step of differentiating the true endoderm into hepatocyte-like cells, which are epithelial cells; and

[0014] iv) A method for producing hepatocytes is provided, including a step of differentiating the hepatocyte-like cells into hepatocytes.

[0015] The present invention also provides a method for producing a 3D liver organoid, including a step of culturing the hepatocytes using a 3D cell culture microfluidic chip.

[0016] The present invention also provides a 3D liver organoid manufactured according to the above manufacturing method,

[0017] The above liver organoid provides a liver organoid that expresses at least one selected from the group consisting of albumin (ALB), AFP, α1-antitrypsin (AAT), CK8, CK18, CK19, CYP2C9, CYP2C19, CYP3A4, glucose-6-phosphatase, and UDP-glucuronosyl transferase.

[0018] The present invention also provides a method for evaluating drug toxicity using a 3D liver organoid manufactured according to the above manufacturing method, the method comprising the following steps:

[0019] i) a step of treating the liver organoid with a drug; and

[0020] ii) A step of measuring liver disease markers, cell viability or oxygen consumption rate (OCR) in the liver organoid.

[0021] Hepatocytes produced using the mesenchymal stem cell patch co-culture technique of the present invention and 3D liver organoids produced using a 3D cell culture microfluidic chip can be differentiated from transparent embryoid bodies exhibiting the characteristics of mature hepatocytes, thereby reproducing the liver of an actual human body. Furthermore, the production method enables purification of high-purity hepatocytes. Furthermore, the 3D liver organoids produced by the production method express the same markers as mature hepatocytes, and thus can be usefully utilized in drug toxicity assessment.

[0022] Figure 1 is a schematic diagram showing the process of producing transparent embryoid-derived hepatocytes from human pluripotent stem cells.

[0023] Figure 2 is an image showing a transparent and opaque spheroid.

[0024] Figure 3 is a graph comparing AFP expression according to the shape of the embryo (*p< 0.05).

[0025] Figure 4 is an image showing the process of culturing embryonic stem cells using mesenchymal stem cell patches.

[0026] Figure 5 shows the expression patterns of SOX17, AFP, and HNF4a genes when transparent embryos were cultured by attaching them to mesenchymal stem cell patches.

[0027] Figure 6 shows the expression patterns of the albumin (ALB), AFP, and HNF4a genes in purified and cultured hepatocyte-like cells, which are epithelial cells derived from transparent embryoid bodies.

[0028] Figure 7 is an image showing the process of manufacturing a 3D liver organoid using a microfluidic chip for 3D cell formation.

[0029] Figure 8 shows the spheroid morphology of 3D liver organoids manufactured through 3D cell culture and the expression patterns of albumin (ALB), CK18, and AFP genes.

[0030] Figure 9 is an image showing the drug toxicity evaluation process in which a manufactured 3D liver organoid was treated with azathioprine, a liver toxic drug.

[0031] Figure 10 is a Seahorse XF analyzer image for OCR measurement (A) and a graph (B) showing the OCR results measured after treating 3D liver organoids with different concentrations of azathioprine (1 μM, 10 μM, and 100 μM).

[0032] Hereinafter, the present invention will be described in detail.

[0033] Method for producing liver cells

[0034] In one aspect, the present invention comprises: i) a step of separating a transparent embryoid body (EB) from an embryoid body (EB) formed by culturing human pluripotent stem cells;

[0035] ii) A step of co-culturing the above transparent embryoid body with a mesenchymal stem cell patch to differentiate it into definitive endoderm (DE);

[0036] iii) a step of differentiating the true endoderm into hepatocyte-like cells, which are epithelial cells; and

[0037] iv) It relates to a method for producing hepatocytes, including a step of differentiating the hepatocyte-like cells into hepatocytes.

[0038] In this specification, the term “human Pluripotent Stem Cell (hPSC)” may refer to a cell that has the ability to differentiate into all cells that constitute the human body.

[0039] In one embodiment of the present invention, the human pluripotent stem cell may be a human embryonic stem cell (ESC) or a human induced pluripotent stem cell (iPSC), preferably a human induced pluripotent stem cell (iPSC).

[0040] The term "embryonic stem cell" as used herein refers to a cell derived from the inner cell mass of a blastocyst prior to implantation, and the derived cell is a cell capable of unlimited culture and pluripotent differentiation.

[0041] The term "induced pluripotent stem cell" as used herein refers to a pluripotent differentiated cell created by dedifferentiation from a somatic cell of the body, and is a cell that is formed by making the somatic cell into a state similar to an embryonic stem cell through a reprogramming process such as cell fusion, nuclear transfer, and overexpression of a pluripotent regulatory factor.

[0042] The term "mesenchymal stem cell" in this specification refers to a stromal cell with multipotency, which is a stem cell with limited but diverse differentiation potential.

[0043] In one embodiment of the present invention, step i) may be performed in a medium to which a serum replacement has been added.

[0044] The term "medium" in this specification refers to a medium capable of supporting proliferation, survival and differentiation of transparent embryoid bodies and hepatocytes in vitro, and includes all conventional media suitable for culturing and differentiating transparent embryoid bodies and hepatocytes used in the relevant field, and specifically, the media include, but are not limited to, DMEM (Dulbeco's Modified Eagle's Media), DMEM / F12, Advanced DMEM / F12, α-MEM (Minimum Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), BME (Basal Medium Eagle), RPMI1640 and Cellartis-HEP medium.

[0045] In another embodiment of the present invention, the medium to which the serum substitute is added may be a DMEM medium.

[0046] In another embodiment of the present invention, the transparent fibroblast may express at least one selected from the group consisting of SOX17, GATA4, FOXA2, CXCR4, and EOMES, and preferably may express SOX17.

[0047] In another embodiment of the present invention, the AFP expression level of the transparent embryo may be at least 50 times that of the opaque embryo.

[0048] In another embodiment of the present invention, step i) may be performed for 1 to 4 days, preferably 2 days.

[0049] In another embodiment of the present invention, the mesenchymal stem cell patch may be composed of mesenchymal stem cell-conditioned medium (MSC-CM).

[0050] In another embodiment of the present invention, step ii) may be performed in a medium supplemented with 10% FBS.

[0051] In another embodiment of the present invention, step ii) may be performed in DMEM medium.

[0052] In another embodiment of the present invention, step ii) may be performed through 3D floating culture.

[0053] In another embodiment of the present invention, step ii) may be performed in a culture dish coated with any one selected from the group consisting of Pluronic F-68, Pluronic F-127, Pluronic F-188, Pluronic F-237, Pluronic F-338 and Pluronic F-407.

[0054] In another embodiment of the present invention, step ii) may be preferably performed in a culture dish coated with Pluronic F-127 for culturing the transparent embryo.

[0055] In another embodiment of the present invention, step ii) may be performed for 6 to 10 days, preferably 8 days.

[0056] In another embodiment of the present invention, step iii) may be performed through 2D attachment culture.

[0057] In another embodiment of the present invention, step iii) may be performed in a culture dish coated with any one selected from the group consisting of Matrigel, laminin, fibronectin, gelatin, and collagen.

[0058] In another embodiment of the present invention, step iii) may be preferably performed in a culture dish coated with collagen for hepatocyte differentiation.

[0059] In another embodiment of the present invention, the collagen may be collagen type Ⅰ.

[0060] In another embodiment of the present invention, step iii) may be performed in DMEM medium.

[0061] In another embodiment of the present invention, step iii) may be performed for 3 to 7 days, preferably 5 days.

[0062] In another embodiment of the present invention, the hepatocyte-like cells may be enzyme-treated. Specifically, the enzyme may be at least one selected from the group consisting of trypsin, collagenase, hyaluronidase, heparinase, elastase, pronase, DNase, dispase, papain, and chymotrypsin, but is not limited thereto.

[0063] In another embodiment of the present invention, preferably, the enzyme may be dispase.

[0064] In another embodiment of the present invention, step iv) may be performed in Cellartis-HEP medium.

[0065] In another embodiment of the present invention, the differentiated hepatocytes of step iv) may be subcultured in the Cellartis-HEP medium.

[0066] In another embodiment of the present invention, step iv) may be performed for 3 to 7 days, preferably 5 days.

[0067] In another embodiment of the present invention, the differentiated hepatocytes of step iv) may express at least one selected from the group consisting of albumin (ALB), AFP, FOXA1, FOXA2, HNF3B, HNF4a, CYP1A2, CYP2C9, CYP2C19 and CYP3A4, and preferably, may express albumin (ALB), AFP and HNF4a.

[0068] In another embodiment of the present invention, the differentiated hepatocytes of step iv) may exhibit a high survival rate while maintaining their shape even after freezing and thawing.

[0069] In one embodiment of the present invention, transparent EBs were isolated from embryoid bodies (EBs) formed from iPSCs, and as a result of culturing them in a medium containing MSC-CM patches, it was confirmed that SOX17 antibody, an endoderm cell marker involved in hepatocyte differentiation, and AFP antibody, a hepatocyte marker, were expressed in more than about 80% of transparent EBs (Fig. 6, A and B). In addition, as a result of 2D adherent culture after 3D floating culture of transparent EBs, cell morphology and expression of AFP, a hepatocyte marker, were confirmed, and a high increase in the expression of HNF4a and AFP was confirmed in hepatocyte-like cells, which are epithelial cells separated through dispase enzyme treatment, compared to transparent EBs (Fig. 6, C and D).

[0070] That is, hepatocytes produced by the hepatocyte production method according to the present invention exhibit the characteristics of mature hepatocytes, and high-purity hepatocytes can be produced through separation of transparent embryoid bodies.

[0071] Method for producing liver organoids

[0072] Another aspect of the present invention relates to a method for producing a 3D liver organoid, comprising a step of culturing the liver cells produced by the above production method using a 3D cell culture microfluidic chip.

[0073] As used herein, the term "organoid," also called an organoid, refers to a three-dimensional cell aggregate formed through self-renewal and self-organization from adult stem cells (ASCs), embryonic stem cells, and induced pluripotent stem cells (iPSCs). Organoids are miniature, simplified in vitro three-dimensional (3D) organs that mimic the anatomical structure of actual tissues. By constructing organoids from patient tissues, disease modeling and drug toxicity assessment based on the patient's genetic information are possible.

[0074] In one embodiment of the present invention, the method for producing a 3D liver organoid by differentiating the hepatocytes into organoids may specifically use the method disclosed in Korean Patent No. 10-2155868 (Patent Document 1) to form organoids from hepatocytes, and may start culturing by seeding hepatocytes onto the microfluidic chip (Fig. 7).

[0075] In one embodiment of the present invention, the method for producing a 3D liver organoid may additionally include a step of aggregating liver cells after performing the culturing step.

[0076] 3D liver organoids manufactured by the manufacturing method

[0077] In another aspect, the present invention provides a 3D liver organoid manufactured according to the liver organoid manufacturing method,

[0078] The above liver organoid relates to a liver organoid, wherein the liver organoid expresses at least one selected from the group consisting of albumin (ALB), AFP, α1-antitrypsin (AAT), CK8, CK18, CK19, CYP2C9, CYP2C19, CYP3A4, glucose-6-phosphatase, and UDP-glucuronosyl transferase.

[0079] In one embodiment of the present invention, the liver organoid may express albumin (ALB), AFP, and CK18.

[0080] In one embodiment of the present invention, the 3D liver organoids are produced by 3D culturing hepatocytes differentiated from transparent embryoid bodies using a 3D cell culture microfluidic chip, thereby stably producing organoids without separate cell transfer, maintaining normal karyotypes, and maintaining the characteristics and functions of mature hepatocytes. In particular, the 3D liver organoids were confirmed to exhibit the characteristics of mature hepatocytes by performing specific morphological analysis and confirming the expression of hepatocyte-specific factors AFP, CK18, and ALB (Fig. 8).

[0081] Drug toxicity evaluation method using liver organoids

[0082] In another aspect, the present invention relates to a method for evaluating drug toxicity using a liver organoid manufactured according to the above 3D liver organoid manufacturing method, the method comprising the following steps:

[0083] i) a step of treating the liver organoid with a drug; and

[0084] ii) A step of measuring liver disease diagnostic markers, cell viability or oxygen consumption rate (OCR) in the liver organoid.

[0085] The term "drug" in this specification may refer to an individual nucleic acid, protein, other extract or natural product that is presumed or randomly selected to have the potential to prevent or treat liver disease according to conventional selection methods; and a drug that is presumed or confirmed to have toxic side effects that is currently on the market.

[0086] In one embodiment of the present invention, the toxicity assessment of a drug may be performed by treating the liver organoid with the drug, measuring liver disease diagnostic markers, cell viability, or oxygen consumption rate, and comparing it with a control group that was not treated with the drug.

[0087] In another embodiment of the present invention, when treating the liver organoid with a drug, if a liver disease diagnostic marker increases / decreases, or cell viability or oxygen consumption rate decreases, the drug may be determined to be a hepatotoxic substance. The oxygen consumption rate is used to determine mitochondrial functionality, and a decrease in the oxygen consumption rate can confirm a decrease in mitochondrial respiration.

[0088] In another embodiment of the present invention, the liver disease may be any one selected from the group consisting of hepatitis virus, non-alcoholic fatty liver disease, liver inflammation, non-alcoholic steatohepatitis, cholestatic liver disease, liver fibrosis, cirrhosis, liver failure, and liver cancer.

[0089] In one embodiment of the present invention, when the liver organoids were treated with the hepatotoxic drug azathioprine, a decrease in OCR was observed, confirming metabolic toxicity through impaired mitochondrial respiration (Fig. 10). This confirms that the liver organoids exhibit the inherent sensitivity and accuracy of liver tissue to drug toxicity, and can be used as a human liver model for assessing drug toxicity.

[0090] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and the scope of the present invention is not limited by these examples.

[0091] Example 1. Production of hepatocytes from stem cells

[0092] 1.1. Formation of the reproductive body

[0093] First, to form embryoid bodies (EBs) for hepatocyte differentiation, undifferentiated iPSCs (produced in-house at Chung-Ang University) were cultured in TeSR-E8 medium at 37°C for 4 days, and then iPSCs were detached from the bottom of the culture dish using dispase (Gibco) enzyme. EB formation was induced by culturing in DMEM medium supplemented with serum replacement (Gibco) for 2 days.

[0094] 1.2. Comparison of AFP expression according to the type of reproductive body

[0095] The formed embryoid bodies were cultured for 8 days in DMEM medium supplemented with 10% FBS (hyclone). Afterwards, transparent embryoid bodies and opaque embryoid bodies were separated by a floating method (Fig. 2).

[0096] After separation, cDNA synthesis was performed using each embryoid body, followed by qPCR. First, RNA was isolated from each embryoid body using TRIzol (Invitrogen), and cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Applied Biosystems). qRT-PCR was performed using FastStart Essential DNA Green Master (Roche) with a Light Cycler 96 system (Roche). GAPDH and AFP primers were designed using the Primer 3 program, and relative gene expression was analyzed by calculating the average Ct value for the technology, and the results are shown in Figure 3 of the present invention.

[0097] Genetic analysis results showed that the expression of the AFP gene in transparent embryos was approximately 50 times higher than in opaque embryos (Fig. 3).

[0098] 1.3. Exploration of processing factor conditions to improve the differentiation efficiency of transparent spheroids

[0099] The optimal conditions for differentiation of transparent spheroids to maximize the formation of transparent spheroids were analyzed through various factors and concentrations.

[0100]

[0101] First, as shown in Table 1 below, embryoid bodies were collected through 8-day culture under suspension culture conditions in DMEM medium with different concentrations of FBS (hyclone), and the ratio of transparent embryoid body formation was analyzed using an inverted microscope (ECLIPSE Ts2, Nikon) through a manual separation method. The results are shown in Table 1 below.

[0102] Processing FactorFBS (%)Processing Conditions (Concentration)51020Formation Rate (%)52620

[0103] The analysis results showed that 26% of transparent embryoid bodies were formed in 10% FBS, confirming that the concentration of FBS with the best differentiation efficiency was 10%.

[0104] Next, the hepatocyte stimulating factors Activin A (R&D system), BMP4 (R&D system), HGF (R&D system) and MSC-CM (MSC-Conditioned media) (self-produced) were added to the 10% FBS - DMEM medium under the conditions shown in Table 2 below, and the transparent embryoid body formation rate was analyzed under suspension culture conditions, and the results are shown in Table 2 below.

[0105] Treatment Factor 10% FBS Activin A (ng / ml) BMP4 (ng / ml) HGF (ng / ml) MSC-CM (%) Treatment Condition (Concentration) 10 50 100 10 20 50 10 20 50 10 20 50 Formation Rate (%) 2 2 3 8 3 1 1 8 2 6 2 4 2 0 3 5 2 8 4 2 4 6 2 7

[0106] As a result of the analysis, it was confirmed that the highest rate of transparent embryoid body formation was 46% under the culture condition in which 20% MSC-CM was added to 10% FBS - DMEM culture medium.

[0107] Meanwhile, in order to explore the optimization of additional factor combinations, the mixing conditions of hepatocyte stimulating factors (50 ng / ml Activin A, 20 ng / ml BMP4, and 20 ng / ml HGF) at each concentration that showed a high rate of formation of transparent spheroids in Table 2 above and 20% MSC-CM were explored, and the results are as shown in Table 3 below.

[0108] Treatment factorMSC-CMMSC-CM + ActivinAMSC-CM + BMP4MSC-CM + HGFFormation rate(%)46423936

[0109] Through the above results, it was confirmed that the culture condition optimized for the formation of transparent spheroids was the condition in which 20% MSC-CM was treated alone in a 10% FBS-DMEM culture medium.

[0110] 1.4. Exploration of co-culture conditions with mesenchymal stem cells to improve differentiation efficiency of transparent embryoid bodies.

[0111] In order to confirm the differentiation rate of transparent embryoid bodies through co-culture of mesenchymal stem cells according to the addition conditions of mesenchymal stem cell conditioned medium (MSC-CM), collagen extracellular matrix (Baobab Healthcare) was mixed with MSC, and an MSC-CM patch was made using a pipette tip. Then, iPSCs were cultured on the MSC-CM patch for 8 days under co-culture conditions, and the differentiation rate of transparent embryoid bodies was compared with that of the MSC-CM direct treatment group, and the results are shown in Table 4 below.

[0112] Treatment factor MSC-CM (20%) MSC-CM (patch) formation rate (%) 4665

[0113] As a result of the analysis, when MSC-CM was directly treated in the medium, transparent embryoid bodies were formed in 46% of cases, and when MSC-CM patches were produced and treated in the medium, transparent embryoid bodies were formed in 65% of cases, confirming that transparent embryoid bodies were effectively cultured under co-culture conditions using MSC-CM patches.

[0114] 1.5. Differentiation of transparent spheroids into hepatocytes using MSC-CM patches

[0115] After embryoid body formation, hepatocyte differentiation was induced by suspension culture on a culture dish coated with Pluronic F-127 in DMEM medium containing 10% FBS for approximately 8 days, and then adherent culture was performed using a culture dish coated with collagen type I in DMEM medium containing 10% FBS for maintenance culture for 5 days.

[0116] After cell purification, the cells were subcultured for 5 days with hepatocyte culture medium (Cellatis-HEP, Lonza), and then frozen and thawed. Hepatocyte differentiation was performed in DMEM containing 10% FBS serum as a basic method. However, depending on the conditions, Activin A was added at 10 ng / ml to 100 ng / ml, BMP4 at 10 to 50 ng / ml, and HGF at 10 to 50 ng / ml, and MSC-CM patches were added at a ratio of 10 to 50% to induce differentiation.

[0117] Example 2. Preparation of liver organoids

[0118] In order to produce liver organoids using the hepatocytes obtained after the differentiation of transparent embryoid bodies through the mesenchymal stem cell patch co-culture technique and the hepatocyte differentiation induction and purification process through this, the hepatocytes produced through Example 1 were seeded and aggregated using a 3D cell culture microfluidic chip disclosed in Korean Patent No. 10-2155868 to produce liver organoids (Fig. 7). The produced liver organoids were observed through an inverted microscope (ECLIPSE Ts2, Nikon) to confirm the spheroid shape (Fig. 8A).

[0119] Example 3. Characterization of hepatocytes and liver organoids

[0120]

[0121] 3.1. Analysis of expression factors of transparent spheroids

[0122] Co-cultured hepatocytes were isolated and single cells were separated using 0.25% trypsin-EDTA (gibco) enzyme, and the expression patterns were confirmed using flow cytometry (FACS, Sony) equipment using hepatocyte-specific proteins SOX17, AFP, and HNF4a. In addition, the isolated hepatocytes were fixed with 4% paraformaldehyde (Sigma), stained with hepatocyte-specific proteins SOX17 and AFP, and then three-dimensional structures were photographed using a confocal microscope and immunofluorescence staining analysis was performed.

[0123] Flow cytometry analysis results confirmed 79.80% expression of SOX17 antibody, an endoderm cell marker involved in hepatocyte differentiation (Fig. 5A), and immunofluorescence staining results showed that SOX17 antibody and hepatocyte marker AFP antibody were expressed in more than about 80% of cells in the clear embryoid body (Fig. 5B).

[0124] Next, after culturing the 3D floating cultured transparent embryoid bodies for 5 days under 2D attachment culture conditions, the cell morphology (epithelial and mesenchymal) and the expression of hepatocyte markers were analyzed. As a result, AFP expression was confirmed in epithelial morphology cells, but AFP expression was not confirmed in mesenchymal morphology cells (C in Figure 5). When only epithelial morphology cells were separated using dispase (Gibco) enzyme and flow cytometric analysis was performed, it was confirmed that HNF4a and AFP expression increased compared to the transparent embryoid bodies (D in Figure 5).

[0125]

[0126] 3.2. Analysis of expression factors of hepatocyte-like cells, which are epithelial cells

[0127] Hepatocyte-like cells, which are epithelial cells derived from passaged and frozen / thawed clear embryoid bodies, were dissociated into single cells using 0.25% trypsin-EDTA (gibco) enzyme, and immunofluorescence staining was performed using hepatocyte-specific proteins AFP, HNF4a, and albumin (ALB). First, hepatocytes isolated as single cells were fixed with 4% (w / v) paraformaldehyde for 20 minutes, stained with AFP (1:200), HNF4a (1:200), and albumin (ALB) (1:200) antibodies diluted in 0.03% Triton X-100 for 24 hours at 4°C, and then stained with Alexa Fluor-488 and Alexa Fluor-594 IgG antibodies (ThermoFisher Scientific) for fluorescence expression for 1 hour at room temperature, followed by analysis using FACS. Immunostaining was also performed in the same manner on attached hepatocytes, and the immunofluorescence staining expression pattern was analyzed using a flow cytometer (FACS / Sony). The cell morphology was observed using a fluorescence microscope (ECLIPSE Ti2, Nikon).

[0128] As a result of the analysis, it was confirmed that the epithelial-shaped cells derived from the transparent embryonic body were maintained for up to 2 passages, and that the morphology and characteristics of the cells were maintained even when the cells were frozen and then thawed (Fig. 6A). In the epithelial-shaped cells, the expression of hepatocyte markers HNF4a, AFP, and albumin (ALB) was confirmed (Fig. 6B). As a result of immunofluorescence staining, the expression of hepatocyte markers albumin (ALB) and HNF4a was confirmed (Fig. 6C).

[0129] 3.3 Analysis of expression factors in liver organoids

[0130] To analyze the expression factors of liver organoids manufactured using a microfluidic chip, immunofluorescence staining analysis was performed, and CK18, AFP, and albumin (ALB) antibodies were used as expression factors.

[0131] First, liver organoids were fixed with 4% (w / v) paraformaldehyde for 20 min at 4°C, stained with antibodies to CK18 (Abcam) and AFP (Abcam) diluted in 0.03% Triton X-100 for 24 h at 4°C, and then cell nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Abcam), and the images were analyzed using a fluorescence microscope Ti2 (Nikon, Japan) (Fig. 8B).

[0132] Additionally, liver organoids were fixed with 4% (w / v) paraformaldehyde for 20 minutes, stained with albumin (ALB) (1:200) antibody diluted in 0.03% Triton X-100 for 24 hours at 4°C, and then stained with Alexa Fluor-594 IgG antibody (ThermoFisher Scientific) for 1 hour at room temperature for fluorescence expression, and then analyzed using a flow cytometer (FACS, Sony) (Fig. 8C).

[0133] Example 4. Drug toxicity analysis through metabolic measurements in liver organoids.

[0134] Using the 3D liver organoids of the present invention, we conducted a study on drug hepatotoxicity by measuring oxygen consumption rates (OCR). To this end, we analyzed the toxicity response of azathioprine (Korea United Pharmaceuticals), an autoimmune disease treatment that exhibits hepatotoxicity.

[0135] After treating liver organoids with different concentrations of azathioprine (1 μM, 10 μM, and 100 μM), the OCR of the liver organoids was measured after about 30 minutes (20 to 40 minutes) using a real-time cell metabolism analyzer (Seahores XF analyzer, Agilent). After seeding the liver organoids on cell culture mini plates provided by the analyzer, the toxicity of azathioprine was analyzed (Fig. 10A). In order to confirm the mitochondrial spare capacity, a kit (Agilent) containing 2 μM oligomycin, 0.5 μM FCCP, 2 μM rotenone, and 2 μM antimycin A as mitochondrial electron transport inhibitors and cellular respiration inhibitors was used at each step, and the basal culture medium was treated as a control.

[0136] OCR analysis results confirmed metabolic toxicity of liver organoids at 10 μM and 100 μM azathioprine (Fig. 10B).

Claims

1. i) A step of isolating a transparent embryoid body (EB) from an embryoid body (EB) formed by culturing human pluripotent stem cells; ii) a step of co-culturing the above transparent embryoid body with a patch of mesenchymal stem cells to differentiate it into definitive endoderm (DE); iii) a step of differentiating the above true endoderm into hepatocyte-like cells, which are epithelium-type cells; and iv) A method for producing hepatocytes, comprising a step of differentiating the hepatocyte-like cells into hepatocytes.

2. In paragraph 1, A method for producing hepatocytes, wherein the human pluripotent stem cells are human embryonic stem cells (hESCs) or human induced pluripotent stem cells (iPSCs).

3. In paragraph 1, A method for producing hepatocytes, wherein step i) above is performed in a medium containing a serum replacement.

4. In paragraph 1, A method for producing hepatocytes, wherein the transparent stromal cell body expresses at least one selected from the group consisting of SOX17, GATA4, FOXA2, CXCR4, and EOMES.

5. In paragraph 1, A method for producing liver cells, wherein the AFP expression amount of the transparent embryo is at least 50 times that of the opaque embryo.

6. In paragraph 1, A method for producing hepatocytes, wherein the above mesenchymal stem cell patch is composed of a mesenchymal stem cell culture medium (mesenchymal stem cell-conditioned medium, MSC-CM).

7. In paragraph 1, A method for producing hepatocytes, wherein step ii) above is performed in a medium supplemented with 5 to 20% FBS.

8. In paragraph 1, A method for producing hepatocytes, wherein the co-culture in step ii) above is performed through 3D floating culture.

9. In paragraph 1, A method for producing hepatocytes, wherein the step iii) above is performed through a culture process for culturing true endoderm into 2D attachment.

10. In paragraph 1, A method for producing hepatocytes, wherein the hepatocyte-like cells of step iv) above are enzyme-treated.

11. In Article 10, A method for producing hepatocytes, wherein the enzyme is at least one selected from the group consisting of trypsin, collagenase, hyaluronidase, heparinase, elastase, pronase, DNase, dispase, papain, and chymotrypsin.

12. In paragraph 1, A method for producing liver cells, wherein the above liver cells express at least one selected from the group consisting of albumin (ALB), AFP, FOXA1, FOXA2, HNF3B, HNF4a, CYP1A2, CYP2C9, CYP2C19 and CYP3A4.

13. A method for manufacturing a 3D liver organoid, comprising a step of culturing liver cells manufactured according to the manufacturing method of Article 1 using a microfluidic chip for 3D cell culture.

14. A 3D liver organoid manufactured according to the manufacturing method of Article 13, The above liver organoid is a 3D liver organoid expressing at least one selected from the group consisting of albumin (ALB), AFP, α1-antitrypsin (AAT), CK8, CK18, CK19, CYP2C9, CYP2C19, CYP3A4, glucose-6-phosphatase, and UDP-glucuronosyl transferase.

15. A method for evaluating drug toxicity using a 3D liver organoid manufactured according to the manufacturing method of Article 13, comprising the following steps: i) a step of treating the liver organoid with a drug; and ii) A step of measuring liver disease diagnostic markers, cell viability or oxygen consumption rate (OCR) in the liver organoid.

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