Method for culturing hepatic epithelial-like tissue having a junction structure between hepatocytes and bile duct epithelial cells

Abstract

Provided is a novel method for culturing hepatic epithelioid tissue. A method is used for forming hepatic epithelioid tissue having a structure of connections between hepatocytes and cholangiocytes, the method including a step of culturing cholangiocytes on a collagen gel, and a step of inoculating and culturing hepatocytes on the cultured cholangiocytes.

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This patent application claims priority to Japanese Patent Application No. 2019-52282, filed on March 20, 2019, the entire disclosure of which is incorporated herein by reference. Technical Field

[0002] The present invention relates to a method for forming hepatic epithelial-like tissue, and more specifically, to a method for forming hepatic epithelial tissue having a junction structure between hepatocytes and bile duct epithelial cells, the method including a step of culturing bile duct epithelial cells on a collagen gel, and a step of seeding hepatocytes on the cultured bile duct epithelial cells and culturing them. [Background technology]

[0003] Many therapeutic drugs for diseases are metabolized in hepatocytes in the liver and show physiological activity or toxicity. Therefore, in preclinical experiments for the development of new drugs, it is desirable to conduct activity and toxicity tests using hepatocytes that show high metabolic activity equivalent to that in vivo. It is also necessary to develop an assay system that reflects pharmacokinetics in liver tissue.

[0004] It has been reported that liver tissue spheroids with functions similar to those in vivo have been produced by using a mixture of hepatocytes, endothelial cells, and stem cells (for example, Patent Document 1). However, although the spheroid culture method has a certain effect in enhancing the functions of liver cells, the liver tissue structure, particularly the liver epithelial tissue structure, is not reconstructed, and therefore pharmacokinetics at the tissue / organ level is not fully reproduced. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2016-105700 A Summary of the Invention

[0006] The present inventors have found that a hepatic epithelial-like tissue structure having a junction structure between hepatocytes and bile duct epithelial cells similar to that in vivo can be reconstructed ex vivo by using a specific method for forming hepatic epithelial tissue. The present invention is based on these findings.

[0007] Therefore, the present invention provides a method for forming hepatic epithelial-like tissue, which can reconstruct ex vivo (outside the body) a hepatic epithelial-like tissue structure having a junction structure between hepatocytes and bile duct epithelial cells similar to that found in vivo.

[0008] According to the present invention, the following inventions are provided. (1) A method for forming hepatic epithelial-like tissue having a junction structure between hepatocytes and bile duct epithelial cells, comprising the steps of: Culturing bile duct epithelial cells on a collagen gel; and A step of seeding hepatocytes on the cultured bile duct epithelial cells and culturing them. (2) The method for forming a hepatic epithelial-like tissue according to (1), wherein the collagen gel contains 2 to 6 mg / mL of collagen. (3) A method for forming a hepatic epithelial-like tissue according to (1) or (2), in which hepatocytes are seeded and cultured on bile duct epithelial cells that have been cultured on a collagen gel to a density of 20 to 40% of the culture area. (4) A method for forming a hepatic epithelial-like tissue according to any one of (1) to (3), further comprising a step of overlaying a gel containing EHS-gel and culturing the hepatocytes after the culturing step of seeding and culturing the hepatocytes. (5) The method for forming hepatic epithelial-like tissue according to (4), wherein the gel contains EHS-gel at 5% (v / v) or more. (6) The method for forming a hepatic epithelial-like tissue according to any one of (1) to (5), wherein the hepatocytes are small hepatocytes and / or mature hepatocytes. (7) The method for forming a hepatic epithelial-like tissue according to any one of (1) to (5), wherein the hepatocytes are mouse small hepatocytes and / or mouse mature hepatocytes. (8) The method for forming a hepatic epithelial-like tissue according to any one of (1) to (5), wherein the hepatocytes are human hepatic progenitor cells. (9) A hepatic epithelial-like tissue formed by the method for forming hepatic epithelial-like tissue according to any one of (1) to (8). (10) A liver tissue comprising the hepatic epithelial-like tissue described in (9). (11) Ex vivo reconstituted liver tissue with junction structures between hepatocytes and bile duct epithelial cells. (12) The liver tissue according to (10) or (11), in which one or more markers selected from the group consisting of the following (a) to (d) are expressed: (a) one or more hepatocyte metabolic function markers selected from the group consisting of Cps, Tdo2, Cyp, and Ugt1a1; (b) one or more bile duct epithelial cell transporter markers selected from the group consisting of Ae2 and Cftr; (c) one or more cytoskeletal markers of bile duct epithelial cells selected from the group consisting of Ck7 and Ck19, and (d) Cldn4, a molecule that forms tight junctions in bile ducts. (13) The liver tissue according to any one of (10) to (12), in which one or more hepatocyte metabolic function markers selected from the group consisting of Cps, Tdo2, Cyp, and Ugt1a1 are expressed. (14) The liver tissue according to any one of (10) to (13), in which one or more bile duct epithelial cell transporter markers selected from the group consisting of Ae2 and Cftr are expressed. (15) The liver tissue according to any one of (10) to (14), in which one or more cytoskeleton markers for bile duct epithelial cells selected from the group consisting of Ck7 and Ck19 are expressed. (16) A liver tissue according to any one of (10) to (15), in which Cldn4, a molecule that forms tight junctions, is expressed. (17) The liver tissue according to any one of (10) to (16), which is used as a disease model. (18) The liver tissue according to (17), wherein the disease model is a fatty liver model.

[0009] The method for forming hepatic epithelial-like tissue of the present invention is advantageous in that it makes it possible to reconstruct ex vivo a hepatic epithelial-like tissue structure having a junction structure between hepatocytes and bile duct epithelial cells similar to that in the body. [Brief description of the drawings]

[0010] [Figure 1] Figure 1A shows an image taken 1 day after seeding of small hepatocytes. Figure 1B shows an image taken 2 weeks after overlaying collagen gel containing Matrigel (registered trademark). The right side shows an enlarged view of each boxed area, and the arrowheads indicate the connection structure between hepatocytes and bile duct epithelial cells. [Diagram 2] Figure 2 shows that fluorescein diacetate taken up by hepatocytes is decomposed within the hepatocytes to release the fluorescent substance fluorescein, which then flows into the bile duct structure formed by bile duct epithelial cells. The left image shows an image observed with a fluorescence microscope (indicated as "Fluorescein" in the figure), the center image shows an image observed with a phase contrast microscope (indicated as "bright field" in the figure), and the right image shows a superposition of the image observed with a fluorescence microscope and the image observed with a normal microscope (indicated as "Merge" in the figure) (the arrowhead in the figure indicates the connection between the hepatocytes and the bile duct epithelial cells). [Diagram 3] Figure 3 shows the unenlarged image in the upper right corner, and an enlarged image of each boxed area in the image in the upper right corner, showing that bilirubin flows into the bile duct structure formed by bile duct epithelial cells. [Figure 4] Figure 4A shows the amount of albumin secreted (μg / ml / h) over time after overlaying type I collagen gel containing 20% ​​(v / v) Matrigel (after overlaying Matrigel). Figure 4B shows Cyp3A4-like activity (AU) in small hepatocytes (Hep) (small hepatocytes cultured alone) or hepatic epithelial-like tissue (Hep+BEC) (co-culture of small hepatocytes and bile duct epithelial cells) 2 weeks after overlaying Matrigel. [Diagram 5]5 is a diagram showing the relationship between the size of bile duct epithelial cell colonies and the formation of hepatic epithelial-like tissue, in which the vertical axis represents the number of junction structures formed between hepatocytes and bile duct epithelial cells per well, and the horizontal axis represents the density (%) of bile duct epithelial cells in the culture area. [Figure 6] Figure 6A shows images of hepatic epithelial-like tissue formed using collagen gel alone (left), collagen gel containing 20% ​​(v / v) Matrigel (20%MG) (center), and 100% (v / v) Matrigel (100%MG) (right) as the top layer. The arrowheads indicate the connection structures between hepatocytes and bile duct epithelial cells. The graph in Figure 6A shows the number of connection structures between hepatocytes and bile duct epithelial cells versus the Matrigel content (%) in the top layer gel. Figure 6B shows images of hepatic epithelial-like tissue formed using 20W-1 (hepatic progenitor cells) (left), SH (small hepatocytes) (center), and MH (mature hepatocytes) (right). Collagen gel containing 20% ​​(v / v) Matrigel (20%MG) was used as the top layer. Moreover, the graph in FIG. 6B shows the number of junction structures formed between hepatocytes and bile duct epithelial cells per well (vertical axis) in 20W-1, SH, and MH (horizontal axis). [Figure 7] The left image in Fig. 7A shows that Cholyl Lysine Fluorescein (CLF), a bile acid analog, was taken up by hepatocytes and excreted into the bile canaliculi (the right image in Fig. 7A shows a bright-field image superimposed on the fluorescent image in the left image). The left image in Fig. 7B shows that Cholyl Lysine Fluorescein excreted into the bile canaliculi was transported to the bile duct (the right image in Fig. 7B shows a bright-field image superimposed on the fluorescent image in the left image). [Figure 8] Figure 8A is a graph comparing the expression levels of hepatocyte markers on day 1 of mature hepatocyte culture (MH) and one month after co-culture (Hep+Cho). Figure 8B is a graph comparing the expression levels of bile duct markers on day 7 of bile duct epithelial cell culture (Cho) and one month after co-culture (Hep+Cho). [Figure 9]The left image in Figure 9A shows the state in which bile acid analogs (CLFs) were taken up into liver epithelium-like tissue (the right image in Figure 9A shows the fluorescent image in the left image overlaid with a bright field image). Figure 9B shows the fluorescence intensity of CLFs leaked into the medium and detected by adding no methyl-beta-cyclodextrin (MβCD) (control), 25 mM MβCD, or 75 mM MβCD. [Figure 10] FIG. 10A is an image showing the formation of a contact surface between mouse BEC (area shown in red) and human CLIP (hCLiP) at the early stage of co-culture (before and after overlaying collagen gel containing Matrigel). The left image of FIG. 10A shows an image observed by a phase contrast microscope (indicated as "bright field" in the figure), the center image of FIG. 10A shows an image observed by a fluorescent microscope (indicated as "Tomato" in the figure), and the right image of FIG. 10A shows a superposition of an image observed by a fluorescent microscope and an image observed by a normal microscope (indicated as "Merge" in the figure). FIG. 10B shows the flow of CLF into the bile duct (area shown in green) near the contact surface between mouse BEC (area shown in red) and human CLiP (see the left image of FIG. 10B), and shows that CLF taken up by human CLiP is transported to mouse bile duct tissue (see the right image of FIG. 10B). The upper right panel of Fig. 10B shows images observed by a fluorescence microscope using CLF and Tomato, respectively. The lower left panel of Fig. 10B shows a superimposition of the CLF image and the Tomato image, and the lower right panel of Fig. 10B shows a superimposition of the lower left panel of Fig. 10B with an image observed by a phase contrast microscope (indicated as "Merge" in the figure). CLF stands for bile acid analogue, and Tomato is a fluorescent protein that represents bile duct epithelial cells expressing it. [Figure 11]Figure 11 shows that when fatty acids were added to liver epithelial tissue, the activity of glutamate oxaloacetate transaminase (GOT), a marker of liver cell damage detected in the medium, increased over time with incubation. The dotted line represents the control where no fatty acids (oleic acid and palmitic acid) were added, and the solid line represents the case where fatty acids were added. [Figure 12] FIG. 12 shows fluorescence microscopy images stained with Nile Red with and without fatty acid addition. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] [Method for forming hepatic epithelial tissue] The method for forming hepatic epithelial-like tissue of the present invention is a method for forming hepatic epithelial-like tissue having a junction structure between hepatocytes and bile duct epithelial cells, which comprises the following steps: Culturing bile duct epithelial cells on a collagen gel; and A step of seeding hepatocytes on the cultured bile duct epithelial cells and culturing them.

[0012] The hepatic epithelial-like tissue formed by the method of the present invention is not particularly limited as long as it is a tissue having at least a junction structure between hepatocytes and bile duct epithelial cells.

[0013] In the hepatic epithelial-like tissue forming method of the present invention, the collagen in the collagen gel used for culturing bile duct epithelial cells may be, for example, type I collagen, type III collagen, or type IV collagen, but type I collagen is preferred. Type I collagen may be a product of Nitta Gelatin Co., Ltd., Fujifilm, Nippi, etc., but it is preferred to use collagen manufactured by Corning. In the hepatic epithelial tissue forming method of the present invention, the collagen gel used for culturing bile duct epithelial cells can be prepared, for example, by adding the collagen as a solution to a well of a culture plate that has been cooled in advance, and then leaving it to gel for a certain period of time.

[0014] In the method for forming hepatic epithelial-like tissue of the present invention, the collagen contained in the collagen gel for use in culturing bile duct epithelial cells is not limited as long as it is capable of reconstructing ex vivo a hepatic epithelial-like tissue structure having a junction structure between hepatocytes and bile duct epithelial cells similar to that in vivo, but is preferably contained at 2 to 6 mg / mL, more preferably 3 to 5 mg / mL, and even more preferably 4 mg / mL.

[0015] Bile duct epithelial cells (also known as BECs) are cells that are positive for the BEC markers cytokeratin 19 (CK19) and epithelial cell adhesion molecule (EpCAM) and express transcription factors such as hepatocyte nuclear factor 1 beta (HNF1b) and grainyhead-like 2 (GRHL2).

[0016] The conditions for culturing bile duct epithelial cells on a collagen gel can be any conditions for culturing normal cells, but for example, the cells are preferably cultured at 36 to 38° C., preferably 37° C. As for the culture period, as described below, the cells are preferably cultured until the density of the bile duct epithelial cells reaches 20 to 40% of the culture area, and preferably cultured for about 3 to 7 days.

[0017] According to a preferred embodiment of the method for forming hepatic epithelial-like tissue of the present invention, there is provided a method for forming hepatic epithelial tissue, comprising the step of seeding and culturing hepatic cells on bile duct epithelial cells whose density has reached 20-40% (preferably 25-35%) of the culture area (for example, the area of ​​the well of a culture plate) by culturing on a collagen gel. The method for measuring the density of the culture area of ​​bile duct epithelial cells is not particularly limited, and for example, it can be measured using image analysis software (manufactured by Olympus Corporation) using a photograph taken with a microscope. By seeding and culturing hepatic cells on bile duct epithelial cells whose density has reached 20-40% of the culture area, the number of junction structures between hepatic cells and bile duct epithelial cells in the formed hepatic tissue can be increased, which is advantageous in that a hepatic epithelial-like tissue structure having a junction structure between hepatic cells and bile duct epithelial cells similar to that in vivo can be further reconstructed ex vivo.

[0018] The hepatocytes used in the method for forming hepatic epithelial-like tissue of the present invention are not particularly limited as long as they are cells derived from the liver, but are preferably small hepatocytes and / or mature hepatocytes, more preferably small hepatocytes or mature hepatocytes, and even more preferably small hepatocytes. Here, the small hepatocytes and mature hepatocytes are preferably mouse small hepatocytes and mouse mature hepatocytes, respectively.

[0019] According to another embodiment of the hepatocytes used in the method for forming hepatic epithelium-like tissue of the present invention, human hepatic progenitor cells are preferred, and human reprogrammed hepatocytes (Human chemically induced hepatocyte progenotors: hCLiPs) are more preferred.

[0020] Here, small hepatocytes do not simply mean small cells derived from the liver, but refer to a special type of small liver-derived cells that can be isolated from the liver and are reported in Mitaka et al., Hepatology, 16, 440-447 (1992), Mitaka et al., Hepatology, 29, 111-135 (1999), WO 01 / 092481, WO 02 / 088332, and WO 2006 / 001473, and that show a phenotype similar to that of mature hepatocytes with respect to markers such as albumin, transferrin, CK8, and CK18, and that have ultrastructural characteristics of hepatocytes, but also have high proliferation ability.

[0021] Small hepatocytes have a lower specific gravity than mature hepatocytes (liver parenchymal cells), and when a suspension containing a liver-derived cell population obtained by collagenase treatment of liver tissue or the like is centrifuged at low gravitational acceleration, the mature hepatocytes mainly migrate to the precipitate, whereas the small hepatocytes mainly migrate to the supernatant. In order to efficiently separate the small hepatocytes from the mature hepatocytes, the centrifugation is preferably performed at 50×g for 1 minute or less.

[0022] In addition, small hepatocytes express CD44, whereas mature hepatocytes and nonparenchymal liver cells do not. Based on these characteristics, small hepatocytes are understood to be distinct from mature hepatocytes and nonparenchymal liver cells.

[0023] According to a preferred embodiment of the method for forming hepatic epithelial-like tissue of the present invention, there is provided a method for forming hepatic epithelial-like tissue having a junction structure between hepatocytes and bile duct epithelial cells, which comprises a step of overlaying a gel containing EHS-gel and culturing the gel after a step of seeding and culturing hepatocytes. By performing such an additional step, a hepatic epithelial-like tissue structure having a junction structure between hepatocytes and bile duct epithelial cells similar to that in vivo can be further reconstructed ex vivo.

[0024] The period from seeding of hepatocytes until overlaying the gel containing EHS-gel is not particularly limited, but it is preferable to overlay after culturing for several days, and more preferably after 2 to 3 days.

[0025] The culture period for layering and culturing a gel containing EHS-gel is not particularly limited as long as it is a period during which liver epithelial-like tissue is formed. For example, it is preferable to culture at 37°C for at least 1 week, preferably at least 1 to 2 weeks, and more preferably at least 2 weeks.

[0026] EHS-gel is a gel containing basement membrane components purified from mouse sarcoma (Engelbreth-Holm-Swarm: EHS), and can be prepared as described in, for example, Kleinman et al. "Basement membrane complexes with biological activity" Biochemistry. 1986 Jan 28; 25(2): 312-8. As commercially available EHS-gel, for example, EHS gel basement membrane matrix (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), ECM Gel from Engelbreth-Holm-Swarm murine sarcoma (manufactured by Sigma), or Matrigel (manufactured by Corning) can be used. In addition, the EHS-gel of the present invention is preferably a gel containing laminin protein, and the gel containing EHS-gel preferably contains 40 to 80% laminin protein, more preferably 50 to 65% laminin protein. As the EHS-gel containing laminin protein, Matrigel (manufactured by Corning) can be suitably used. Here, laminin is a protein in which α chains, β chains, and γ chains are associated in a 1:1:1 ratio, and it is known that there are five types of α chains, two types of β chains, and two types of γ chains in humans.

[0027] The EHS-gel contained in the overlaying gel is not limited as long as it is capable of reconstructing ex vivo a hepatic epithelium-like tissue structure having a junction structure between hepatocytes and bile duct epithelial cells similar to that in vivo, but is preferably contained at 5% (v / v) or more, more preferably 10% (v / v) or more. The overlaying "gel containing EHS-gel" may contain only EHS-gel, but when the "gel containing EHS-gel" contains gels other than EHS-gel, it is preferable to contain collagen gel, and more preferably type I collagen gel (preferably collagen gel manufactured by Koken Co., Ltd.).

[0028] According to another aspect of the present invention, there is provided a hepatic epithelial-like tissue formed by the method of the present invention for forming hepatic epithelial-like tissue. The hepatic epithelial-like tissue formed by the method of the present invention for forming hepatic epithelial-like tissue is a hepatic epithelial-like tissue structure reconstructed ex vivo having a junction structure between hepatocytes and bile duct epithelial cells similar to that in vivo.

[0029] According to a preferred embodiment of the present invention, Culturing bile duct epithelial cells on a collagen gel containing 2 to 6 mg / mL of collagen; A step of seeding and culturing hepatocytes (preferably small hepatocytes or mature hepatocytes) on bile duct epithelial cells cultured on a collagen gel to a density of 20 to 40% in the culture area; and A process of layering gel containing EHS-gel and culturing The present invention provides a method for forming a hepatic epithelium-like tissue having a junction structure between hepatocytes and bile duct epithelial cells, comprising:

[0030] According to another aspect of the present invention, there is provided a hepatic epithelial-like tissue formed by the method of the present invention for forming hepatic epithelial-like tissue.

[0031] [Liver tissue] According to one aspect of the present invention, liver tissue is formed that contains hepatic epithelial-like tissue formed by the above-mentioned method for forming hepatic epithelial-like tissue of the present invention. Moreover, since the hepatic epithelial-like tissue formed by the method for forming hepatic epithelial-like tissue of the present invention has a junction structure between hepatocytes and bile duct epithelial cells, according to another aspect of the present invention, ex vivo reconstructed liver tissue having a junction structure between hepatocytes and bile duct epithelial cells is provided.

[0032] The above-mentioned liver tissue is not particularly limited, but is preferably a liver tissue in which one or more markers selected from the group consisting of the following (a) to (d) are expressed: (a) one or more hepatocyte metabolic function markers selected from the group consisting of Cps, Tdo2, Cyp, and Ugt1a1; (b) one or more bile duct epithelial cell transporter markers selected from the group consisting of Ae2 and Cftr; (c) one or more cytoskeletal markers of bile duct epithelial cells selected from the group consisting of Ck7 and Ck19, and (d) Cldn4, a molecule that forms tight junctions in bile ducts.

[0033] Here, Cps is carbamylphosphate synthetase, Tdo2 is tryptophan 2,3-dioxygenase, Cyp is cytochrome P450, preferably Cyp1a2 or Cyp7a1, Ugt1a1 is UDP-glucuronosyl-transferase-1 family polypeptide A11a1, Ae2 is anion exchanger 2, Cftr is cystic fibrosis transmembrane conductance regulator, Ck7 is cytokeratin 7, Ck19 is cytokeratin 19, and Cldn4 is claudin 4.

[0034] According to a preferred embodiment of the present invention, there is provided liver tissue in which one or more hepatocyte metabolic function markers selected from the group consisting of Cps, Tdo2, Cyp, and Ugt1a1 are expressed.

[0035] According to a preferred embodiment of the present invention, there is provided liver tissue in which one or more bile duct epithelial cell transporter markers selected from the group consisting of Ae2 and Cftr are expressed.

[0036] According to a preferred embodiment of the present invention, there is provided liver tissue in which one or more hepatocyte metabolic function markers selected from the group consisting of Ck7 and Ck19 are expressed.

[0037] According to a preferred embodiment of the present invention, there is provided liver tissue in which Cldn4, a molecule that forms tight junctions, is expressed.

[0038] According to a more preferred embodiment of the present invention, there is provided liver tissue in which all of the markers Cps, Tdo2, Cyp, Ugt1a1, Ae2, Cftr, Ck7, Ck19, and Cldn4 are expressed.

[0039] The liver tissue of the present invention can be used as a disease model. By using the liver tissue of the present invention, any model of disease originating from liver tissue can be prepared by known methods, for example, fatty liver model, acute liver injury model caused by drugs, liver fibrosis model, etc., can be mentioned, and among these, fatty liver model can be preferably used. The fatty liver model can be prepared by adding oleic acid and / or palmitic acid to the liver tissue of the present invention. In the construction of fatty liver models that have already been reported, spheroid culture is often used (see, for example, Patent Document 1). Therefore, in order to confirm the morphology of hepatocytes and confirm that lipid droplets have accumulated in the cytoplasm, it is necessary to prepare slices or observe using a confocal microscope or the like. In the fatty liver model using the liver tissue of the present invention, the morphology of hepatocytes can be easily observed, and therefore it is possible to simply and clearly show that lipid droplets have accumulated in the cytoplasm.

[0040] According to a more preferred embodiment of the present invention, the disease model of liver tissue of the present invention can be used to screen for therapeutic agents for the disease, preferably for screening for therapeutic agents for fatty liver, acute liver damage caused by drugs or the like, or liver fibrosis, and therefore, a method for screening therapeutic agents is suitably provided. EXAMPLES

[0041] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples.

[0042] 1. Cell Isolation from Liver Collagenase perfusion was performed on the livers of 8-10 week old male mice purchased from Sankyo Labo Services, and the resulting cell suspension was centrifuged at 50xg for 1 minute. The supernatant was centrifuged at 115xg for 3 minutes, and the resulting cells were suspended in HBSS (Hank's Balanced Salt Solution) (Sigma-Aldrich), an equal amount of Percoll (GE Healthcare Japan) was added, and the mixture was centrifuged at 180xg for 15 minutes to remove dead cells, and these were used as small hepatocytes.

[0043] After collagenase perfusion, the undigested tissue was cut into pieces, and the pieces were stirred and digested in a collagenase and hyaluronidase solution. After reacting with an antibody against EpCAM (Biolegend), a surface marker of bile duct epithelial cells, EpCAM(+) cells were isolated using MACS (Miltenyi Biotec). These were used as bile duct epithelial cells in the following.

[0044] 2.Culture A 4 mg / ml type I collagen solution was prepared and added to the wells of a pre-cooled culture plate (Greiner), and allowed to stand at 37°C for 30-60 minutes to gel. Bile duct epithelial cells suspended in DMEM / F12 medium (Sigma-Aldrich) containing 10% serum (fetal bovine serum), 1xITS (insulin-transferrin-selenium), 0.1 μM dexamethasone, 5 ng / ml EGF (epidermal growth factor), and 5 ng / ml HGF (hepatocyte growth factor) were added to 5x10 4 After culturing at 37°C for 4-5 days, when the density of bile duct epithelial cells reached about 30% of the culture area, small hepatocytes were added at 5x10 4 The cells were seeded per well. The density of bile duct epithelial cells in the culture area was measured using a photograph taken under a microscope with image analysis software (Olympus). After 2 days of culture at 37°C, 10ng / ml recombinant mouse oncostatin M (mOSM) (R&D) was added, and after another day, 1.6mg / ml type I collagen gel containing 20% ​​(v / v) Matrigel (registered trademark) (Corning) (EHS-gel) was overlaid. After gelation at 37°C for 3 to 4 hours, DMEM / F12 medium (Sigma-Aldrich) containing serum, 1xITS, dexamethasone, and 1% DMSO was added. Culture was continued while changing the medium once a week.

[0045] Test Example 1: Confirmation of the junction structure between hepatocytes and bile duct epithelial cells Images taken 1 day after seeding of small hepatocytes and 2 weeks after overlaying with type I collagen gel containing 20% ​​(v / v) Matrigel (after Matrigel overlay) are shown in Figure 1. In the image taken 2 weeks after overlaying with Matrigel, the junction structure between hepatocytes and bile duct epithelial cells was clearly observed (see Figure 1B).

[0046] Test Example 2: Confirmation of the junction structure between hepatocytes and bile duct epithelial cells Fluorescein diacetate (FDA) (Tokyo Chemical Industry Co., Ltd.) was incorporated into the hepatic epithelial-like tissue two weeks after the above-mentioned Matrigel overlay, and observed under a phase-contrast microscope and a fluorescence microscope. In the above-mentioned hepatic epithelial-like tissue two weeks after the Matrigel overlay, a connection structure (arrowhead) was formed between the hepatocytes and bile duct epithelial cells, indicating that fluorescein had flowed into the bile duct (see Figure 2). FDA is taken up by hepatocytes and hydrolyzed to become fluorescent fluorescein, which is then excreted into the bile canaliculi.

[0047] Test Example 3: Bilirubin uptake by ex vivo reconstituted liver epithelial-like tissue The hepatic epithelial-like tissue two weeks after the above-mentioned Matrigel overlay was cultured for 3 to 5 days in a medium containing 10 μg / ml bilirubin (Fujifilm Wako Pure Chemical Industries, Ltd.), fixed with neutral formalin, and stained with Fouchest's reagent (20% TCA (trichloroacetic acid) (Fujifilm Wako Pure Chemical Industries, Ltd.) / 1% Ferric Chloride (Fujifilm Wako Pure Chemical Industries, Ltd.)), and observed under a microscope. As shown in Figure 3, it was found that the bilirubin taken up by the hepatocytes flows into the bile duct structure formed by the bile duct epithelial cells.

[0048] Test Example 4: Examination of the effect of co-culture with hepatocytes and bile duct epithelial cells on hepatocyte function following the formation of junction structures The amount of albumin secreted from small hepatocytes (Hep) (small hepatocytes cultured alone for 2 weeks at 37°C under the same conditions as the co-culture) and from hepatic epithelial-like tissue (Hep+BEC) 2 weeks after the above-mentioned Matrigel overlay (co-culture of small hepatocytes and bile duct epithelial cells) was quantified using the Sandwich ELISA method using an anti-albumin antibody (Bethyl).It was found that Hep+BEC secreted sufficient albumin from 2 weeks after the Matrigel overlay for approximately 1 month, and that hepatic differentiation function was maintained (see Figure 4A). In addition, the Cyp3A4 activity, an indicator of cytochrome P450 activity, was measured for the above Hep and Hep+BEC using the P450-Glo CYP3A4 Assay System (Promega). As a result, it was found that Hep+BEC had higher Cyp3A4 activity than Hep, and that Hep+BEC induced higher liver function than Hep (see Figure 4B).

[0049] Test Example 5: Examination of the relationship between the size of bile duct epithelial cell colonies and the formation of hepatic epithelial-like tissue In the above "2. Culture", small hepatocytes were seeded when the density of bile duct epithelial cells was about 30% of the culture area, but in order to consider the optimal timing for seeding small hepatocytes, densities other than the above were also examined. Specifically, the seeding timing of small hepatocytes was shifted using the density of the culture area of ​​bile duct epithelial cells as an index, and the number of connection structures between hepatocytes and bile duct epithelial cells per well of liver epithelial-like tissue two weeks after overlaying with Matrigel was measured using image analysis software (Olympus) using photos taken under a microscope. According to the measurement results, it was found that when hepatocytes were seeded when the density of bile duct epithelial cells was about 30% of the culture area, the number of connection structures formed between hepatocytes and bile duct epithelial cells increased, and liver epithelial-like tissue structures having connection structures between hepatocytes and bile duct epithelial cells similar to those in vivo could be better reconstructed ex vivo (see Figure 5).

[0050] Test Example 6: Examination of the relationship between the composition of the overlying gel and the type of hepatocytes used in co-culture and the formation of hepatic epithelial-like tissue We investigated the relationship between the content (%) of EHS-gel (Matrigel) in the overlay gel and the number of junction structures between hepatocytes and bile duct epithelial cells formed in the liver epithelial tissue. Specifically, we investigated the percentage of Matrigel used in the above "2. Culture" at three concentrations: 0% (v / v), 20% (v / v), and 100% (v / v). When the overlay gel contained 20% (v / v) or 100% (v / v), the number of junction structures between hepatocytes and bile duct epithelial cells was high, and it was found that even with EHS-gel (Matrigel) alone, a liver epithelial-like tissue structure with junction structures between hepatocytes and bile duct epithelial cells similar to those in vivo could be reconstructed ex vivo (see Figure 6A). Next, the type of hepatocytes used for co-culture with bile duct epithelial cells was examined. Specifically, instead of the small hepatocytes (SH) used in the above "2. Culture", mature hepatocytes (MH) or a hepatic progenitor cell line (20W-1) established from small hepatocytes of 20-week-old mice were used, and the effect of the number of junction structures formed between hepatocytes and bile duct epithelial cells in the formed hepatic epithelial-like tissue 2 weeks after Matrigel overlay was examined. When mature hepatocytes were used, the number of junction structures formed between hepatocytes and bile duct epithelial cells was slightly lower than when small hepatocytes were used, but a good number of junction structures was shown. On the other hand, when the hepatic progenitor cell line was used, no junction structures were formed between hepatocytes and bile duct epithelial cells at all (see Figure 6B).

[0051] Test Example 7: Examination of collagen concentration in gel used for lower layer The effect of collagen concentration in the gel used for initial culture of bile duct epithelial cells on the ability to form a junction structure between hepatocytes and bile duct epithelial cells in the liver epithelial-like tissue was examined. Specifically, in the above "2. Culture", the collagen concentration in the gel used for initial culture of bile duct epithelial cells was adjusted to 2, 4, or 6 mg / ml, and the effect on the ability to form a junction structure between hepatocytes and bile duct epithelial cells in the liver epithelial-like tissue formed 2 weeks after Matrigel overlay was examined. As a result, good junction structure formation ability between hepatocytes and bile duct epithelial cells was shown at all concentrations of 2, 4, and 6 mg / ml, but the best junction structure formation ability between hepatocytes and bile duct epithelial cells was shown at 4 mg / ml (data not shown). It is known that bile duct epithelial cells have low proliferation efficiency and dedifferentiate even when cultured on a normal cell culture dish.

[0052] Test Example 8: Examination of metabolite transport from hepatocytes to bile ducts In the above "2. Cultivation", one month after layering 1.6 mg / ml type I collagen gel containing EHS-gel, CLF, a bile acid analog, was added to the medium and cultured. It was found that CLF was taken up by hepatocytes and excreted into the bile canaliculi (30 minutes later), and then transported to the bile duct (6 hours later). Figure 7A is an image showing that CLF was taken up by hepatocytes and excreted into the bile canaliculi, and Figure 7B is an image showing that CLF excreted into the bile canaliculi is transported to the bile duct. This indicates that in addition to FDA and bilirubin, bile acids are also taken up by hepatocytes and then transported to the bile duct.

[0053] Test Example 9: Examination of the expression of hepatocyte and bile duct epithelial cell markers by organoid-constituting cells The gene expression levels of hepatocyte metabolic function markers Cps, Tdo2, Cyp1a2, Cyp7a1, and Ugt1a1 were compared between the first day of culture of mature hepatocytes (MH) similar to those used in Test Example 6 (represented as "MH" in Figure 8A) and liver tissue containing the above-mentioned hepatic epithelial-like tissue one month after co-culture with bile duct epithelial cells and small hepatocytes in "2. Culture" above (represented as "Hep+Cho" in Figure 8A). In addition, the expression levels of Ae2 and Cftr, which are transporter markers of bile duct epithelial cells, Ck7 and Ck19, which are cytoskeleton markers of bile duct epithelial cells, and Cldn4, which is a molecule that forms tight junctions, were compared between the bile duct epithelial cells (Cho) used in "2. Culture" above on the 7th day of culture (represented as "Cho" in Figure 8B) and the liver tissue containing the above-mentioned hepatic epithelial-like tissue one month after co-culture of bile duct epithelial cells and small hepatocytes in "2. Culture" above (represented as "Hep+Cho" in Figure 8B). The results are shown in Figure 8A and Figure 8B.

[0054] The results in Figure 8A show that in this culture system, the hepatocyte metabolic function markers Cps, Tdo2, Cyp, and Ugt1a1 were expressed equally between the hepatic epithelial-like tissue and mature hepatocytes, with no significant difference in their expression levels.

[0055] The results in Figure 8B show that in this culture system, bile duct epithelial cell transporters (Ae2, Cftr), cytoskeleton (Ck7, Ck19), and tight junction-forming molecules (Cldn4) were expressed equally between hepatic epithelial-like tissue and bile duct epithelial cells, with no significant difference in their expression levels.

[0056] Test Example 10: Examination of recovery of metabolites by suppressing the function of cell-cell adhesion Methyl beta cyclodextrin (MβCD) has been reported as a substance that can extract cholesterol from cell membranes (Christoroforides E et al. Org. Chem 2018). Claudins, which form tight junctions, are localized in cholesterol-rich membrane domains, and are therefore extracted from the cell membrane by the addition of MβCD (Shigetomi K et al. J Cell Biol. 2018). In the above "2. Culture", when bile acid analogues (CLF) were added to the cells one month after the addition of EHS-gel, the CLF was taken up into the cells (green fluorescent area) and accumulated in the lumen within the organoid (see Figure 9A), but when MβCD was added to this, fluorescence was detected in the medium (see Figure 9B). This was caused by the inhibition of the barrier function of the epithelial cells within the organoid, which released CLF into the medium, and it was found that this release was dependent on the concentration of MβCD added (see Figure 9B).

[0057] Test Example 11: Study of co-culture of human reprogramming hepatocytes and mouse BEC To create organoids that reflect human metabolic functions, we co-cultured human reprogrammed hepatocytes with mouse bile duct epithelial cells. Specifically, mouse bile duct epithelial cells were cultured on collagen gel (collagen content in collagen gel: 4 mg / ml) for 5 days, and then CLiPs were seeded. Two days later, human recombinant Oncostatin M (hOSM) (R&D) was added, and one day later, a collagen gel containing 40% EHSgel (Matrigel) was layered on top to perform co-culture. Human reprogrammed hepatocytes (Human chemically induced hepatocyte progenotors: hCLiPs) are human liver progenitor cells established from frozen human hepatocytes (Katsuda T. et al. Cell Stem Cell 2017; eLife 2019).

[0058] (A) It can be seen that a contact surface is formed between mouse BECs (area indicated in red) and human CLiPs at the early stage of co-culture (see FIG. 10A). (B) CLF was added 1 week after the collagen gel containing EHSgel (Matrigel) was layered on top of the EHSgel. The inflow of CLF into the bile duct was observed near the contact surface between mouse BEC and CLiP, indicating that the CLF taken up by the CLiP was transported to the mouse bile duct tissue (see Figure 10B).

[0059] Test Example 12: Study on the construction of an in vitro fatty liver model As one of the attempts to create an injury model using the liver tissue according to the present invention, the effect of adding fatty acids to the culture medium on hepatocytes was examined. Fatty acids were added to the culture to examine injury to hepatocytes and accumulation of fat. As fatty acids, oleic acid and palmitic acid, which accumulate in hepatocytes in steatohepatitis, were used. Specifically, the bile duct epithelial cells (BEC) used in the above "2. Culture" were cultured on collagen gel, and the small hepatocytes (SH) used in the above "2. Culture" were added to the culture solution, and 1.6 mg / ml type I collagen gel containing 20% ​​(v / v) Matrigel (registered trademark) (manufactured by Corning) (EHS-gel) was overlaid. After further culturing at 37°C for 10 days, 0.5 mM oleic acid (OA) and 0.5 mM palmitic acid (PA) were added and cultured.

[0060] The culture supernatant was collected over time and the activity of GOT, a marker of hepatocyte damage, was measured. GOT activity was confirmed in the culture supernatant by adding fatty acids (oleic acid and palmitic acid) compared to no addition (control), indicating that hepatocytes were sustainedly damaged (see Figure 11).

[0061] In addition, fatty acids were added to the medium and cultured for 3 days, after which accumulation of lipid droplets was examined using Nile red. It was found that lipid droplets stained with Nile red accumulated in the cytoplasm of hepatocytes (increased red area) in the medium containing fatty acids compared to the medium not containing fatty acids (see FIG. 12). This suggests that an in vitro fatty liver model can be constructed using the liver tissue of the present invention.

Claims

1. A method for forming a hepatic epithelial-like tissue culture having a junction structure between hepatocytes and bile duct epithelial cells, comprising the steps of: Culturing bile duct epithelial cells on the collagen gel; seeding and culturing hepatocytes on the cultured bile duct epithelial cells; and After the culture step of seeding and culturing hepatocytes, a gel containing EHS-gel is layered on top of the hepatocytes. The process of nourishing Here, the hepatocytes are small hepatocytes and / or mature hepatocytes, or human hepatic progenitor cells.

2. The method according to claim 1, wherein the collagen gel contains 2 to 6 mg / mL of collagen.

3. 3. The method according to claim 1, wherein hepatocytes are seeded and cultured on bile duct epithelial cells that have been cultured on a collagen gel to a density of 20 to 40% of the culture area.

4. The method according to any one of claims 1 to 3, wherein the gel contains EHS-gel at 5% (v / v) or more.

5. The method according to any one of claims 1 to 4, wherein the hepatocytes are mouse small hepatocytes and / or mouse mature hepatocytes.

6. The method according to any one of claims 1 to 4, wherein the hepatocytes are human hepatic progenitor cells.

7. A hepatic epithelial-like tissue culture formed by the method of any one of claims 1 to 6.

8. A liver tissue culture comprising the liver epithelial-like tissue culture of claim 7.

9. The liver tissue culture of claim 8, which is reconstituted ex vivo.

10. The liver tissue culture according to claim 9, in which one or more markers selected from the group consisting of the following (a) to (d) are expressed: (a) one or more hepatocyte metabolic function markers selected from the group consisting of Cps, Tdo2, Cyp, and Ugt1a1; (b) one or more bile duct epithelial cell transporter markers selected from the group consisting of Ae2 and Cftr; (c) one or more cytoskeletal markers of bile duct epithelial cells selected from the group consisting of Ck7 and Ck19; and (d) Cldn4, a molecule that forms tight junctions in bile ducts.

11. The liver tissue culture according to any one of claims 8 to 10, wherein one or more hepatocyte metabolic function markers selected from the group consisting of Cps, Tdo2, Cyp, and Ugt1a1 are expressed.

12. The liver tissue culture according to any one of claims 8 to 11, wherein one or more bile duct epithelial cell transporter markers selected from the group consisting of Ae2 and Cftr are expressed.

13. The liver tissue culture according to any one of claims 8 to 12, wherein one or more cytoskeleton markers for bile duct epithelial cells selected from the group consisting of Ck7 and Ck19 are expressed.

14. The liver tissue culture according to any one of claims 8 to 13, in which Cldn4, a molecule that forms tight junctions, is expressed.

15. The liver tissue culture according to any one of claims 8 to 14, which is used as a disease model.

16. The liver tissue culture of claim 15, wherein the disease model is a fatty liver model.

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