JP2019033732A - Three-dimensional liver tissue structure and method of producing the same - Google Patents

Abstract

To provide a three-dimensional liver tissue structure with sufficient thickness and high albumin production capability and a method of producing the same, and also to provide a method of evaluating hepatotoxicity of a test compound using the three-dimensional liver tissue structure.SOLUTION: Provided is a method of producing a three-dimensional liver tissue structure, which comprises at least liver cells and has two or more cell layers stacked in a thickness direction, the method being characterized in that the method produces a three-dimensional liver tissue structure, 40% or more of all the cells constituting the three-dimensional liver tissue structure is liver cells and thickness of the three-dimensional liver tissue structure is 150 μm or more. The three-dimensional liver tissue structure is characterized in that the structure comprises at least liver cells and has two or more cell layers stacked in the thickness direction, and the thickness of the structure is 150-500 μm.SELECTED DRAWING: None

Description

  The present invention relates to an artificially constructed liver tissue structure capable of maintaining albumin production ability, that is, liver function for a long time, a method for producing the same, and liver toxicity of a test compound using the obtained liver tissue structure. On how to evaluate

  Evaluation of hepatotoxicity of compounds is important for new drug development, and there is a need for an evaluation system that can provide highly reliable evaluation in an in vitro assay system. In general, in vitro hepatotoxicity evaluation is performed using human hepatocellular carcinoma cell line HepG2 or human primary hepatocytes. In particular, primary cultured hepatocytes are considered to be close to living liver tissue, and thus are useful systems for evaluating liver toxicity. However, when considering the industrialization of toxicity evaluation, there is a problem of individual differences in liver function due to differences in SNP (single nucleotide polymorphism) related to securing the supply source and metabolic activity. On the other hand, HepG2 is a cultured strain, and although there is no problem with supply, it is inactivated hepatoma cells, and it is presumed that biological characteristics are also different from the liver in vivo.

  PXB cell provided by Phoenix Bio Inc. is a cell that is easier to supply and close to human hepatocytes. PXB cells are primary human hepatocytes isolated from mice in which human fetal liver tissue was transplanted into immunodeficient mice and the mouse liver was replaced with human liver tissue at a rate of 70-80%. By subculturing this mouse, it is theoretically possible to supply primary human hepatocytes indefinitely. This PXB cell can be used for evaluation of hepatotoxicity in monolayer culture, but since it is derived from a fetus, it is presumed that the liver function is different from that of an adult.

  In addition, primary cultured hepatocytes cultured by a conventional monolayer culture method have been difficult to maintain for a long time each function of the liver including metabolic functions. In addition, primary cultured hepatocytes cultured in monolayers may require exposure to high concentrations of compounds to detect toxicity, and conversely, toxicity detection by metabolic activation may not be sufficient. There was also a problem that it was not reflected.

  Non-Patent Document 1 reports that, when primary human hepatocytes are monolayer cultured, the amount of albumin produced by primary human hepatocytes is increased by culturing the mixture with mesenchymal stem cells. Albumin production is an index of liver function, and it is expected that liver function is improved by co-culturing primary human hepatocytes with mesenchymal stem cells.

  Attempts have been made to develop a three-dimensional liver tissue having a function closer to the liver in vivo than a hepatocyte cultured in a monolayer. For example, Patent Document 1 discloses a three-dimensional liver tissue in which hepatocytes, endothelial cells, and mesenchymal stem cells are seeded on a biodegradable scaffold material and co-cultured in the scaffold material in a pseudo-microgravity environment. A manufacturing method is disclosed. However, in the method of creating a three-dimensional tissue by using a scaffold material, since the scaffold material itself generally occupies a spatial region in the culture system, a tissue having sufficient density and high morphological strength is formed. It was difficult to do.

  As a method for constructing a three-dimensional tissue that does not require a scaffold, there is a spheroid culture method. Spheroids formed from primary cultured hepatocytes, HepG2 and the like maintain liver function more than monolayer cultures, and hepatotoxicity evaluation using such spheroids is widely performed. However, it is difficult to ensure the same size of spheroids, it is difficult to ensure the reproducibility of liver function, and the lack of interaction with the stroma to culture only hepatocytes. There is also a problem.

  In recent years, spheroid culture using iPS cell-derived hepatoblasts and the like has also been actively studied. By co-culturing iPS-derived hepatoblasts, vascular endothelial cells, mesenchymal hepatocytes, etc., it is possible to create a liver tissue structure that maintains liver function. However, there are problems that the efficiency of differentiation induction from iPS cells to liver tissue is low and that canceration occurs due to gene mutation introduction.

  Non-Patent Document 2 discloses that a cell structure obtained by alternately coating HepG2 surface with fibronectin and gelatin by the so-called LbL (Layer by Layer) method and expressing the cells in a layer is expressed in the liver. Among the characteristic proteins, it has been reported that the expression levels of albumin and CYP (cytochrome P450) are increased as compared with the conventional monolayer-cultured HepG2. Furthermore, in this document, it is also reported that the HepG2 structure obtained by the LbL method has maintained albumin producing ability for almost one month.

JP 2017-085945 A

Fitzpartrick et al., Cell Transplantation, 2015, Vol.24, p.73-83. Matsuzawa et al., JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A, 2015, Vol.103A (4), p.1554-1564. Nishiguchi et al., Macromol Bioscience, 2015, vol.15 (3), p.312-317.

  The present invention provides a three-dimensional liver tissue structure having a sufficient thickness and high albumin-producing ability, a method for producing the same, and a method for evaluating liver toxicity of a test compound using the three-dimensional liver tissue structure. The purpose is to provide.

  The inventors of the present invention have intensively studied to solve the above-mentioned problems, and previously mixed a cell containing at least hepatocytes, a strong electrolyte polymer, and an extracellular matrix component in a cationic buffer solution. It was found that a thicker three-dimensional liver tissue structure can be produced quickly and easily by laminating cells in this mixture in layers in the direction of its own weight to form a three-dimensional structure. Furthermore, the constructed three-dimensional liver tissue structure was found to have a higher albumin production ability than monolayer culture, and the present invention was completed.

The method for producing a three-dimensional liver tissue structure, the three-dimensional liver tissue structure, and the compound hepatic toxicity evaluation method according to the present invention are the following [1] to [20].
[1] A method for producing a three-dimensional liver tissue structure comprising at least hepatocytes and having two or more cell layers laminated in the thickness direction,
A method for producing a three-dimensional liver tissue structure, comprising producing a three-dimensional liver tissue structure in which 40% or more of all cells constituting the structure are hepatocytes and the thickness is 150 μm or more.
[2] (a) A step of mixing a cell containing at least hepatocytes, a strong electrolyte polymer, and an extracellular matrix component in a cationic buffer solution to obtain a mixture;
(B) seeding the mixture obtained by the step (a) in a cell culture vessel;
(C) After the step (b), the liquid component is removed from the cell mixture in the cell culture container, and a cell structure in which cells containing at least hepatocytes are laminated in a multilayer structure is three-dimensionally formed in the cell culture container. Obtaining a typical liver tissue structure;
The method for producing a three-dimensional liver tissue structure according to [1] above, comprising:
[3] The strong electrolyte polymer is selected from the group consisting of heparin, glycosaminoglycan, dextran sulfate, rhamnan sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, polyacrylamide-2-methylpropane sulfonic acid, and polyacrylic acid. The method for producing a three-dimensional liver tissue structure according to [2], which is one or more of the above.
[4] The above-mentioned [2] or [3], wherein the extracellular matrix component is one or more selected from the group consisting of collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, fibrillin, and proteoglycan. A method for producing a three-dimensional liver tissue structure.
[5] The three-dimensional liver tissue according to any one of [2] to [4], wherein the cationic buffer is Tris-HCl buffer, Tris-maleic acid buffer, bis-Tris-buffer, or HEPES. Manufacturing method of structure.
[6] The method for producing a three-dimensional liver tissue structure according to any one of [1] to [5], wherein the hepatocytes are non-cancerous cells.
[7] The method for producing a three-dimensional liver tissue structure according to any one of [1] to [6], wherein the thickness of the obtained three-dimensional liver tissue structure is 500 μm or less.
[8] The method for producing a three-dimensional liver tissue structure according to any one of [1] to [7], wherein the cells in the step (a) further include mesenchymal stem cells.
[9] The method for producing a three-dimensional liver tissue structure according to any one of [1] to [8], wherein the cells in the step (a) further include cells constituting stroma.
[10] The above [9], wherein the cells in the step (a) further include one or more selected from the group consisting of vascular endothelial cells and lymphatic endothelial cells as the cells constituting the stroma. A method for producing a three-dimensional liver tissue structure.
[11] A three-dimensional liver tissue structure characterized by being a cell structure including at least hepatocytes, having two or more cell layers laminated in the thickness direction and having a thickness of 150 to 500 μm.
[12] The three-dimensional liver tissue structure according to the above [11], wherein 40% or more of all cells constituting the three-dimensional liver tissue structure are hepatocytes.
[13] The three-dimensional liver tissue structure according to [11] or [12], further including a mesenchymal stem cell as a cell constituting the three-dimensional liver tissue structure.
[14] The three-dimensional liver tissue structure according to any one of the above [11] to [13], which further includes a cell constituting the stroma as a cell constituting the cell structure.
[15] The three-dimensional body according to [14], wherein the three-dimensional liver tissue structure further includes at least one selected from the group consisting of vascular endothelial cells and lymphatic endothelial cells as cells constituting the stroma. Liver tissue structure.
[16] The three-dimensional liver tissue structure according to any one of [11] to [15], wherein the total number of cells constituting the three-dimensional liver tissue structure is 1.2 × 10 ⁵ or more.
[17] The three-dimensional liver tissue structure according to any one of [11] to [16], wherein the amount of albumin produced by the three-dimensional liver tissue structure is 500 ng / mL or more as of the ninth day of culture.
[18] The three-dimensional liver tissue according to any one of [11] to [17], wherein the amount of albumin produced by the three-dimensional liver tissue structure is 500 ng / mL or more at the time of culture on the 11th day after the structure is constructed. Structure.
[19] A culturing step of culturing the three-dimensional liver tissue structure according to any one of [11] to [18] in a state in which the test compound is contacted;
Evaluation of the hepatotoxicity of the compound, wherein the presence or absence of hepatic toxicity of the test compound is evaluated using the survival rate or albumin producing ability of the hepatocytes in the three-dimensional liver tissue structure after the culturing step as an index. Method.
[20] The method for evaluating the hepatotoxicity of the compound according to [19], wherein all hepatocytes contained in the three-dimensional liver tissue structure have a common genotype of at least one drug metabolizing enzyme.

  According to the present invention, a three-dimensional liver tissue structure having a sufficient thickness and having a high albumin producing ability can be provided. Moreover, the obtained three-dimensional liver tissue structure can be used for evaluation of the hepatotoxicity of the compound.

FIG. 1 is an HE-stained image of a paraffin-embedded section on the 15th day of culture of the cell structure constructed in Example 1. FIG. 2 is a transmitted light image of the cell structure constructed in Example 1 on the 11th day of culture. FIG. 3 shows the amount of albumin (ng / ng) in each culture supernatant collected on days 1, 3, 5, 7, 9, 11, 13, and 15 of the cell structure constructed in Example 1 from the start of culture. It is the figure which showed the result of having measured mL) with time.

<Three-dimensional liver tissue structure>
The three-dimensional liver tissue structure according to the present invention includes at least hepatocytes, and two or more cell layers are laminated in the thickness direction. In the present invention and the present specification, the “three-dimensional liver tissue structure” means a three-dimensional cell aggregate including at least hepatocytes, and the “thickness of the three-dimensional liver tissue structure” It means the length of the structure in its own weight direction. The self-weight direction is a direction in which gravity is applied and is also referred to as a thickness direction.

  The hepatocytes constituting the three-dimensional liver tissue structure according to the present invention may be primary hepatocytes collected from the liver of an animal, cells cultured from primary hepatocytes, or primary hepatocytes. May be a cultured cell line that is established as a stock cell, or hepatoblasts artificially differentiated from stem cells. Primary hepatocytes include primary human hepatocytes such as PXB Cell. Examples of the cultured cell line include inactivated hepatoma cell-derived cell lines such as HepG2. Examples of stem cells that are differentiated into hepatoblasts include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells, and the like. The hepatocytes contained in the three-dimensional liver tissue structure according to the present invention are preferably non-cancerous cells, such as primary hepatocytes and hepatoblasts, and PXB Cell is more preferable in terms of ease of handling. . The three-dimensional liver tissue structure according to the present invention has a sufficient thickness. Therefore, the amount of hepatocytes contained per structure is large, and therefore it can be expected to have a sufficient albumin producing ability. . In addition, a non-cancerous cell means the cell which is not cancerous, ie, the cell which has not acquired infinite proliferation ability.

  The hepatocytes contained in the three-dimensional liver tissue structure according to the present invention may be one type or two or more types. For example, the three-dimensional liver tissue structure according to the present invention may contain a plurality of hepatocytes having different genotypes of proteins involved in liver function. Conversely, all hepatocytes contained in the three-dimensional liver tissue structure according to the present invention may have the same genotype of a protein involved in liver function. Examples of proteins involved in liver function include drug metabolizing enzymes.

  The hepatocytes constituting the three-dimensional liver tissue structure according to the present invention may be cells derived from the liver of any animal. For example, hepatocytes derived from animals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice and rats can be used.

  The three-dimensional liver tissue structure according to the present invention may be constructed only from hepatocytes or may contain cells other than hepatocytes. When cells other than hepatocytes are included, the abundance ratio of the hepatocytes to the total cells constituting the three-dimensional liver tissue structure (cell number ratio, ratio (%)) is not particularly limited, but stable tissue And the formed three-dimensional liver tissue structure exerts liver function, at least 40% of all cells constituting the three-dimensional liver tissue structure are preferably hepatocytes, and more than 50% Are more preferably hepatocytes, more preferably 75% or more are hepatocytes, and even more preferably 80% or more are hepatocytes.

  Cells other than the hepatocytes contained in the three-dimensional liver tissue structure according to the present invention are not particularly limited. The cells other than hepatocytes may be mature somatic cells or undifferentiated cells such as stem cells. Examples of somatic cells include nerve cells, dendritic cells, immune cells, vascular endothelial cells, lymphatic endothelial cells, fibroblasts, epithelial cells, cardiomyocytes, islet cells, smooth muscle cells, bone cells, alveolar epithelial cells And spleen cells. Stem cells include ES cells, iPS cells, mesenchymal stem cells, and the like. The cells other than the hepatocytes contained in the three-dimensional liver tissue structure may be normal cells, or may be cells in which any cell function is enhanced or suppressed, such as cancer cells. . A cancer cell is a cell derived from a somatic cell and having acquired unlimited proliferation ability.

  The cells (including hepatocytes) contained in the three-dimensional liver tissue structure according to the present invention may be cells collected from animals, or may be cells obtained by culturing cells collected from animals. A cell obtained by subjecting cells collected from an animal to various treatments or a cultured cell line may be used. In the case of cells collected from animals, the collection site is not particularly limited, and may be somatic cells derived from bone, muscle, viscera, nerves, brain, bone, skin, blood, etc., or germ cells. It may be an embryonic stem cell (ES cell). The biological species from which the cells constituting the three-dimensional liver tissue structure according to the present invention are not particularly limited, and examples thereof include humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, rats, and the like. Cells derived from these animals can be used. A cell obtained by culturing a cell collected from an animal may be a primary cultured cell or a subcultured cell. Examples of cells that have been subjected to various treatments include iPS cells and cells after differentiation induction. Moreover, the three-dimensional liver tissue structure according to the present invention may be composed only of cells derived from the same species, or may be composed of cells derived from a plurality of species.

  The cells other than the hepatocytes contained in the three-dimensional liver tissue structure according to the present invention may be one type or two or more types. The three-dimensional liver tissue structure according to the present invention preferably includes hepatocytes and mesenchymal stem cells from the viewpoint that the albumin producing ability of the formed three-dimensional liver tissue structure can be stably obtained for a longer period of time. When a three-dimensional liver tissue structure contains mesenchymal stem cells, a sufficient effect can be obtained by the coexistence of mesenchymal stem cells, so the abundance ratio of mesenchymal stem cells in all cells constituting the three-dimensional liver tissue structure Is preferably 10 to 60%, more preferably 10 to 40%, and still more preferably 15 to 30%.

  The three-dimensional liver tissue structure according to the present invention may include cells constituting the stroma (stromal cells). A more homogeneous structure can be obtained by constructing a tissue structure from hepatocytes in the presence of stromal cells. In addition, a three-dimensional liver tissue structure constructed in the presence of stromal cells can maintain albumin production ability for a longer period. The reason why albumin production ability can be maintained for a longer period is unclear, but by coexisting with stromal cells, hepatocytes in the three-dimensional liver tissue structure exist in a state close to the environment of the liver tissue in the animal body It is assumed that it is possible.

  Examples of stromal cells include endothelial cells, fibroblasts, nerve cells, dendritic cells, macrophages, mast cells, epithelial cells, cardiomyocytes, hepatocytes, pancreatic islet cells, tissue stem cells, smooth muscle cells and the like. The stromal cells contained in the three-dimensional liver tissue structure according to the present invention may be one type or two or more types.

  The vascular network structure and the lymphatic network structure are considered to be important for the growth of a three-dimensional liver tissue structure and the expression of a function similar to a living body. For this reason, the three-dimensional liver tissue structure according to the present invention preferably has a vascular network structure. That is, as a three-dimensional liver tissue structure according to the present invention, a vascular network structure such as a lymphatic vessel and / or a blood vessel is three-dimensionally constructed in a laminated body of cells that do not form a vascular vessel, What has constructed | assembled the structure | tissue closer to a living body is preferable. The vascular network structure may be formed only inside the three-dimensional liver tissue structure, or may be formed so that at least a part thereof is exposed on the surface or bottom surface of the three-dimensional liver tissue structure. . In the present invention and the present specification, the “vascular network structure” refers to a network structure such as a vascular network or a lymphatic network in a living tissue.

  The vascular network structure can be formed by including endothelial cells constituting the vasculature as stromal cells. The endothelial cells contained in the three-dimensional liver tissue structure according to the present invention may be vascular endothelial cells or lymphatic endothelial cells. Further, both vascular endothelial cells and lymphatic endothelial cells may be included.

  The number of endothelial cells in the three-dimensional liver tissue structure according to the present invention is not particularly limited as long as it is sufficient to form a vascular network structure. In addition, it can be appropriately determined in consideration of the cell types of endothelial cells and cells other than endothelial cells. For example, a three-dimensional structure in which a vascular network structure is formed by setting the abundance ratio (number ratio) of endothelial cells to the total cells constituting the three-dimensional liver tissue structure according to the present invention to 0.1% or more. Liver tissue structures can be prepared. The abundance ratio of endothelial cells in all cells constituting the three-dimensional liver tissue structure is preferably 10.0% or more, more preferably 10 to 60%, and more preferably 30 to 60%. Further preferred.

  When the three-dimensional liver tissue structure according to the present invention has a vascular network structure, it may contain hepatocytes and other cells than the endothelial cells constituting the vasculature. The other cells are not particularly limited as long as they do not inhibit the function of hepatocytes or the formation of a vascular network by endothelial cells. For example, the other cell is preferably a cell that constitutes a tissue surrounding the vascular in vivo because the endothelial cell easily forms a vascular network that retains its original function and shape. Furthermore, a three-dimensional liver tissue structure including at least fibroblasts as cells other than endothelial cells is more preferable because it can be more closely approximated to liver tissue in vivo, and a three-dimensional liver including vascular endothelial cells and fibroblasts is more preferable. More preferred is a tissue structure, a three-dimensional liver tissue structure containing lymphatic endothelial cells and fibroblasts, or a three-dimensional liver tissue structure containing vascular endothelial cells, lymphatic endothelial cells and fibroblasts. The cells other than the endothelial cells contained in the three-dimensional liver tissue structure may be cells derived from the same species as the endothelial cells or cells derived from different species. Moreover, the number of cells other than the endothelial cells contained in the three-dimensional liver tissue structure may be one, or two or more.

  The three-dimensional liver tissue structure according to the present invention may be a structure in which hepatocytes are scattered throughout the structure, or a structure in which hepatocytes are present only in a specific cell layer. Also good. In the three-dimensional liver tissue structure, the cell layer (hepatocyte layer) containing hepatocytes may be only one layer or two or more layers.

  The size and shape of the three-dimensional liver tissue structure according to the present invention are not particularly limited. Since the vascular network structure in a state closer to the vasculature formed in the tissue in the living body can be formed and more accurate evaluation is possible, the thickness of the three-dimensional liver tissue structure is 5 μm. The above is preferable, 50 μm or more is more preferable, 100 μm or more is further preferable, and 150 μm or more is more preferable. The thickness of the three-dimensional liver tissue structure is preferably 500 μm or less, more preferably 400 μm or less, and even more preferably 300 μm or less. The number of cell layers of the three-dimensional liver tissue structure according to the present invention is preferably about 2 to 60 layers, more preferably about 5 to 60 layers, and further preferably about 10 to 60 layers.

  The number of cell layers constituting the three-dimensional liver tissue structure is obtained by dividing the total number of cells constituting the three-dimensional structure by the number of cells per layer (the number of cells necessary for constituting one layer). Measured by The number of cells per layer can be examined by culturing the cells in advance in a flat manner so as to be confluent in a cell culture container used when forming a three-dimensional liver tissue structure. Specifically, the number of cell layers of a three-dimensional liver tissue structure formed in a certain cell culture container is determined by measuring the total number of cells constituting the three-dimensional liver tissue structure, and per one layer of the cell culture container. It can be calculated by dividing by the number of cells.

The number of cells constituting the three-dimensional liver tissue structure according to the present invention is not particularly limited, and the thickness and shape of the three-dimensional liver tissue structure to be constructed, the size of the cell culture container used for the construction, etc. It is determined as appropriate in consideration. For example, the total number of cells constituting the three-dimensional liver tissue structure of the present invention may be a 1.0 × 10 ⁵ or more, preferably 1.2 × 10 ⁵ or more, 1.0 × 10 ⁶ or more are more preferable, and 1.0 × 10 ⁷ or more are more preferable. The upper limit of the total number of cells constituting the three-dimensional liver tissue structure according to the present invention is not particularly limited, and can be, for example, 1.0 × 10 ¹² cells.

  Generally, the three-dimensional liver tissue structure according to the present invention is constructed in a cell culture container. The cell culture vessel is not particularly limited as long as it can construct a three-dimensional liver tissue structure and can culture the constructed three-dimensional liver tissue structure. Specific examples of the cell culture container include dishes, cell culture inserts (eg, Transwell (registered trademark) insert, Netwell (registered trademark) insert, Falcon (registered trademark) cell culture insert, Millicell (registered trademark) cell culture). Inserts, etc.), tubes, flasks, bottles, plates and the like. In the construction of the three-dimensional liver tissue structure according to the present invention, dish or various cell culture inserts are preferable because evaluation using the three-dimensional liver tissue structure can be performed more appropriately.

  The three-dimensional liver tissue structure according to the present invention may be a structure composed of multiple cell layers including hepatocytes, and the construction method is not particularly limited. For example, it may be a method of constructing one layer at a time and sequentially laminating it, or a method of constructing two or more cell layers at a time, and combining the two construction methods as appropriate to form a multilayer cell layer. It may be a method of construction. Further, the three-dimensional liver tissue structure according to the present invention may be a multilayer structure in which the cell types constituting each cell layer are different for each layer, and the cell types constituting each cell layer are all of the structure. The layers may be common. For example, a method may be used in which a layer is formed for each cell type and the cell layers are sequentially stacked. A cell mixture solution in which a plurality of types of cells are mixed is prepared in advance, and a multilayer is prepared from the cell mixture solution It may be a method of constructing a three-dimensional liver tissue structure of a structure at a time.

  As a method of constructing one layer at a time and sequentially laminating, for example, a method described in Japanese Patent No. 4919464, that is, a step of forming a cell layer, and the formed cell layer is converted into an ECM (extracellular matrix). And a step of contacting the solution containing the component (1) and the step of alternately laminating the cell layers. For example, when performing the method, a cell mixture in which all cells constituting the three-dimensional liver tissue structure are mixed is prepared in advance, and each cell layer is formed by this cell mixture. A three-dimensional liver tissue structure in which a vascular network structure is formed and hepatocytes are scattered throughout the structure can be constructed. In addition, by forming each cell layer for each cell type, a vascular network structure is formed only in the layer composed of endothelial cells, and a three-dimensional liver tissue in which hepatocytes are present only in a specific cell layer A structure can be constructed.

  In addition, for example, when performing the above method, a cell mixture in which all cells constituting a layer containing stromal cells and mesenchymal stem cells are mixed in advance with all cells constituting a layer containing hepatocytes. Each cell mixture is prepared separately. First, a cell layer composed of a single layer or multiple layers is constructed by sequentially laminating a cell mixture containing stromal cells and the like, and then the cell mixture is formed on the cell layer. A cell layer can be formed by placing a semipermeable membrane and laminating a cell mixture containing hepatocytes on the semipermeable membrane. As a result, a vascular network structure is formed in the entire cell layer including stromal cells, and a cell structure in which hepatocytes exist only in a layer separated from the stromal cells by a semipermeable membrane is constructed. it can.

  Examples of a method for constructing two or more cell layers at a time include the method described in Japanese Patent No. 5850419. In this method, the entire cell surface is previously coated with a polymer containing an arginine-glycine-aspartic acid (RGD) sequence to which integrin binds and a polymer interacting with the polymer containing the RGD sequence. This is a method for constructing a three-dimensional liver tissue structure composed of multiple cell layers by storing coated cells coated with an adhesive film in a cell culture container and then accumulating the coated cells by centrifugation or the like. For example, when performing the method, a coated cell prepared by preparing a cell mixture in which all the cells constituting the three-dimensional liver tissue structure are mixed and adding an adhesive component to the cell mixture. Is used. Thus, a three-dimensional liver tissue structure in which hepatocytes are scattered throughout the entire structure can be constructed by a single centrifugation process. In addition, for example, coated cells coated with mesenchymal stem cells, coated cells coated with endothelial cells, coated cells coated with fibroblasts, and a group of cells contained in liver tissue collected from animals The coated cells were prepared separately, and a multi-layer consisting of fibroblast-coated cells was formed, and then a single layer consisting of endothelial cell-coated cells was laminated thereon, and fibroblasts were further formed thereon. A multi-layer consisting of coated cells is laminated, and a single layer consisting of coated cells coated with mesenchymal stem cells is laminated thereon, and a single layer consisting of coated cells of cells containing hepatocytes is further laminated thereon. Let As a result, a three-dimensional liver tissue structure having a vascular network structure sandwiched between thick fibroblast layers and a layer containing hepatocytes on the top surface in contact with mesenchymal stem cells can be constructed. .

  In addition, for example, coated cells coated with endothelial cells, coated cells coated with fibroblasts, coated cells coated with mesenchymal stem cells, and coated cells coated with hepatocytes are prepared separately, After a multilayer is formed from fibroblast-coated cells, a single layer formed from endothelial cell-coated cells is stacked thereon, and a multilayer formed from fibroblast-coated cells is further stacked thereon. Further, a layer formed from a mesenchymal stem cell-coated cell is further laminated thereon, a semipermeable membrane is further placed thereon, and a hepatocyte-containing cell-coated cell is formed on the semipermeable membrane. One layer can also be laminated. Thus, a three-dimensional structure comprising a vascular network structure sandwiched between thick fibroblast layers and a layer containing hepatocytes in a state of being separated from a fibroblast layer or a mesenchymal stem cell layer by a semipermeable membrane. A typical liver tissue structure can be constructed.

The three-dimensional liver tissue structure according to the present invention can also be constructed by a method having the following steps (a) to (c). By this method, a three-dimensional liver tissue structure can be constructed easily and stably.
(A) mixing a cell and an extracellular matrix component in a cationic buffer to obtain a mixture;
(B) seeding the mixture obtained by the step (a) in a cell culture vessel;
(C) A step of removing a liquid component from the cell mixture in the cell culture vessel after the step (b) to obtain a cell structure in which cells are laminated in the cell culture vessel.

  In the step (a), a cell is mixed with a buffer solution containing a cationic substance (cationic buffer solution) and an extracellular matrix component, and a cell aggregate is formed from the cell mixture, whereby a large void is formed inside. Less steric cellular tissue can be obtained. In addition, since the obtained three-dimensional cell tissue is relatively stable, it can be cultured for at least several days, and the tissue is difficult to collapse even when the medium is changed. In the present invention, in the step (b), the cell mixture seeded in the cell culture container may be precipitated in the cell culture container. In sedimentation of the cell mixture, cells may be actively sedimented by centrifugation or the like, or may be spontaneously sedimented.

  In step (a), it is preferable to further mix the cells with a strong electrolyte polymer. This is a case where the cells are naturally precipitated by mixing cells with a cationic substance, a strong electrolyte polymer and an extracellular matrix component without requiring a process such as centrifugation to actively collect cells in step (b). Even so, a thick three-dimensional cellular tissue can be obtained.

  Examples of the cationic buffer include tris-hydrochloric acid buffer, tris-maleic acid buffer, bis-tris-buffer, and HEPES. The concentration and pH of the cationic substance (for example, Tris in Tris-HCl buffer) in the cationic buffer are not particularly limited as long as they do not adversely affect cell growth and cell structure construction. For example, the concentration of the cationic substance in the cationic buffer can be 10 to 100 mM, preferably 40 to 70 mM, and more preferably 50 mM. Further, the pH of the cationic buffer solution can be 6.0 to 8.0, preferably 6.8 to 7.8, and more preferably 7.2 to 7.6. .

  Examples of the strong electrolyte polymer include heparin, chondroitin sulfate (eg, chondroitin 4-sulfate, chondroitin 6-sulfate), heparan sulfate, dermatan sulfate, keratan sulfate, hyaluronic acid and other glycosaminoglycans; dextran sulfate, , Rhamnan sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, polyacrylamide-2-methylpropane sulfonic acid, and polyacrylic acid, or derivatives thereof, but are not limited thereto. In the mixture prepared in the step (a), only one type of strong electrolyte polymer may be mixed, or two or more types may be combined and mixed. In the construction of the three-dimensional liver tissue structure according to the present invention, the polyelectrolyte is preferably a glycosaminoglycan. More preferably, at least one of heparin, dextran sulfate, chondroitin sulfate, and dermatan sulfate is used. More preferably, the strong electrolyte polymer used in the present invention is heparin. The amount of the strong electrolyte polymer mixed with the cationic buffer is not particularly limited as long as it does not adversely affect the cell growth and the construction of the cell structure. For example, the concentration of the strong electrolyte polymer in the cationic buffer can be greater than 0 mg / mL (higher than 0 mg / mL) and less than 1.0 mg / mL, and is 0.025 to 0.1 mg / mL. It is preferably 0.05 to 0.1 mg / mL. In the present invention, the cell structure can also be constructed by adjusting the mixture without mixing the strong electrolyte.

  Examples of the extracellular matrix component include collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, fibrillin, proteoglycan, or a variant or variant thereof. Examples of proteoglycans include chondroitin sulfate proteoglycan, heparan sulfate proteoglycan, keratan sulfate proteoglycan, dermatan sulfate proteoglycan and the like. In the mixture prepared in the step (a), only one type of extracellular matrix component may be mixed, or two or more types may be mixed and mixed. In the construction of the three-dimensional liver tissue structure according to the present invention, collagen, laminin, and fibronectin are preferably used, and collagen is more preferably used. As long as the growth of cells and the formation of cell structures are not adversely affected, the above-described extracellular matrix component variants and variants may be used. The amount of the extracellular matrix component mixed with the cationic buffer is not particularly limited as long as it does not adversely affect cell growth and cell structure construction. For example, the concentration of extracellular matrix components in the cationic buffer can be greater than 0 mg / mL (greater than 0 mg / mL) and less than 1.0 mg / mL, and is 0.025 to 0.1 mg / mL. It is preferably 0.05 to 0.1 mg / mL.

  The compounding ratio of the strong electrolyte polymer and the extracellular matrix component mixed in the cationic buffer is 1: 2 to 2: 1. In the construction of the three-dimensional liver tissue structure according to the present invention, the compounding ratio of the strong electrolyte polymer and the extracellular matrix component is preferably 1: 1.5 to 1.5: 1, and 1: 1. More preferably.

  Steps (a) to (c) are repeated. Specifically, after seeding the mixture prepared in step (a) as step (b) on the cell structure obtained in step (c), By repeating the step (c), a cell structure having a sufficient thickness can be constructed. The cell composition of the mixture newly seeded on the cell structure obtained in the step (c) may be the same as or different from the cell composition constituting the already constructed cell structure. .

  By adjusting the number of cells seeded in the step (b), the thickness of the cell layer laminated in the step (c) can be adjusted. The greater the number of cells seeded in step (b), the greater the number of cell layers stacked in step (c).

For example, first, in step (a), a mixture containing only vascular endothelial cells is prepared as cells, and steps (b) and (c) are performed to form a cell structure comprising five vascular endothelial cell layers in a cell culture container. Get. Next, as a step (a), a mixture containing only hepatocytes as a cell is prepared, and steps (b) and (c) are performed to laminate five hepatocyte layers on the vascular endothelial cell layer in the cell culture container. Let Further, as a step (a), a mixture containing only mesenchymal stem cells is prepared as a cell, and steps (b) and (c) are performed to form a two-layer mesenchymal layer on the hepatocyte layer in the cell culture container. Stack stem cells. As a result, a cell structure in which the vascular endothelial cell layer 5 layers−the hepatocyte layer 5 layers−the mesenchymal stem cell layer 2 layers and the cell types stacked in order for each cell type can be constructed.
Further, in step (a), a mixture is prepared by mixing all vascular endothelial cells for five vascular endothelial cell layers and two mesenchymal stem cells for two mesenchymal stem cell layers, and steps (b) and (c). The hepatocytes for 5 hepatocyte layers prepared in the same manner are laminated on the multilayer structure thus formed, so that it has a thickness of 12 layers and the vascular network structure is inside the structure. A cell structure in which a hepatocyte layer is laminated on a structure scattered in the cell can be constructed. Furthermore, in the step (a), a mixture obtained by mixing all the vascular endothelial cells for the five vascular endothelial cell layers, the mesenchymal stem cells for the two mesenchymal stem cell layers, and the hepatocytes for the five hepatocyte layers is mixed. By preparing and carrying out steps (b) and (c), it has a thickness of 12 layers, and both hepatocytes, mesenchymal stem cells and vascular network structures are scattered independently inside the structure. Cell structures can be constructed.

  When the steps (a) to (c) are repeated, the obtained cell structure may be cultured after the step (c) and before the step (b). The culture conditions such as the composition of the culture medium used for the culture, the culture temperature, the culture time, and the atmospheric composition during the culture are those suitable for culturing the cells constituting the cell structure. Examples of the culture medium include D-MEM, E-MEM, MEMα, RPMI-1640, Ham's F-12, and the like.

  After step (a), (a′-1) removing the liquid portion from the obtained mixture to obtain cell aggregates, and (a′-2) suspending the cell aggregates in the solution are performed. The process may proceed to step (b). Although a desired tissue body can be obtained by performing the above-mentioned steps (a) to (c), (a′-1) and (a′-2) are performed after the step (a), and the steps By carrying out (b), a more homogeneous tissue can be obtained.

Further, after the step (a), the following steps (b′-1) and (b′-2) may be performed instead of the step (b). In this embodiment and the present specification, “cell viscous body” refers to a gel-like cell aggregate as described in Non-Patent Document 3.
(B'-1) After seeding the mixture obtained in step (a) in a cell culture container, removing the liquid component from the mixture to obtain a cell viscous body;
(B′-2) A step of suspending a cell viscous body in a cell culture vessel in a solvent.

  The solvent for preparing the cell suspension is not particularly limited as long as it is not toxic to cells and does not impair the growth and function, and water, buffer solution, cell culture medium, etc. can be used. . Examples of the buffer include phosphate physiological saline (PBS), HEPES, Hanks buffer, and the like. Examples of the culture medium include D-MEM, E-MEM, MEMα, RPMI-1640, Ham's F-12, and the like.

Instead of the step (c), the following step (c ′) may be performed.
(C ′) removing liquid components from the seeded mixture to form a cell layer on the substrate.

  The method for removing the liquid component in the steps (c) and (c ′) is not particularly limited as long as it does not adversely affect the growth of the cell and the construction of the cell structure. From the suspension of the liquid component and the solid component. As a method of removing the liquid component, it can be appropriately performed by a method known to those skilled in the art. Examples of the technique include a centrifugal separation process, a magnetic separation process, and a filtration process. For example, when a cell culture insert is used as a cell culture container, the liquid component can be removed by subjecting the cell culture insert seeded with the mixture to a centrifugation treatment at 10 ° C. and 400 × g for 1 minute. it can.

  The three-dimensional liver tissue structure according to the present invention is superior in liver function to a hepatocyte layer cultured in a single layer because hepatocytes are stacked in the same manner as liver tissue in vivo. In particular, even when the three-dimensional liver tissue structure produced by the production method including the steps (a) to (c) has a thick structure, it has few voids between cell layers and is stably cultured for a long period of time. It is a possible structure. For this reason, the three-dimensional liver tissue structure according to the present invention is superior in liver function to a tissue structure constructed by another three-dimensional tissue creation method such as spheroids. In particular, a three-dimensional liver tissue structure containing mesenchymal stem cells can maintain liver functions including albumin production for a longer period of time. The amount of albumin produced by the three-dimensional liver tissue structure according to the present invention is preferably 100 ng / mL or more, more preferably 250 ng / mL or more at the time of the ninth day after culturing after the construction of the cell structure. More preferably, it is more preferably 500 ng / mL or more. In addition, the albumin production amount of the three-dimensional liver tissue structure according to the present invention is preferably 100 ng / mL or more at the time of the 11th day after culturing after the construction of the cell structure, and 250 ng / mL. More preferably, it is more preferably 500 ng / mL or more. The amount of albumin produced by the three-dimensional liver tissue structure according to the present invention is determined, for example, by measuring the amount of albumin in the supernatant of the culture medium of the three-dimensional liver tissue structure by a known method such as an ELISA method. Is required.

<Method for evaluating hepatotoxicity of compound>
Since the three-dimensional liver tissue structure according to the present invention can maintain a good liver function for a relatively long period of time, it is suitable as a tool for evaluating the presence and strength of a compound's liver toxicity. By using the three-dimensional liver tissue structure according to the present invention, it is possible to make a more reliable evaluation of the hepatotoxicity of the compound. In addition, a compound having hepatotoxicity can also be screened using the method for evaluating the hepatotoxicity of a compound using this three-dimensional liver tissue structure.

  Specifically, the method for evaluating hepatotoxicity of a compound according to the present invention (hereinafter sometimes referred to as “evaluation method according to the present invention”) is a method in which the three-dimensional liver tissue structure is contacted with a test compound. The presence or absence of hepatic toxicity of the test compound is evaluated using as an index the culture step of culturing in a state and the survival rate or albumin producing ability of hepatocytes in the three-dimensional liver tissue structure after the culture step. The contact between the three-dimensional liver tissue structure and the test compound can be performed by adding the test compound to the culture medium of the three-dimensional liver tissue structure.

  The test compound to be evaluated in the evaluation method according to the present invention may be one type or two or more types. Further, when evaluating two or more kinds, each test compound may be evaluated by contacting with a three-dimensional liver tissue structure, and a plurality of test compounds may be simultaneously contacted with a three-dimensional liver tissue structure. You may evaluate. When evaluating hepatotoxicity when two or more compounds are administered in combination, the compound to be administered in combination is simultaneously contacted with the three-dimensional liver tissue structure, and the hepatocytes in the three-dimensional liver tissue structure are contacted. Assess liver toxicity based on survival.

  The culture time for culturing the three-dimensional liver tissue structure in a medium containing a test compound is not particularly limited, and can be, for example, 24 to 96 hours, 48 to 96 hours. It is preferable that it is 48 to 72 hours. In addition, hydrodynamic addition such as reflux can be given as necessary within a limit that does not significantly change the culture environment.

  Evaluation of the hepatotoxicity of the test compound using the survival rate of hepatocytes in the three-dimensional liver tissue structure after culture as an index can be performed, for example, as follows. When the number of living hepatocytes in the three-dimensional liver tissue structure is small (the survival rate is low) compared to the case where the test compound is cultured in the absence of the test compound, the test compound is It is evaluated that it is toxic to the hepatocytes contained in the liver tissue structure, that is, has liver toxicity. It can be evaluated that the hepatotoxicity is stronger as the decrease in the survival rate of the hepatocytes is larger than in the absence of the test compound. On the other hand, when the number of living hepatocytes is the same or significantly higher (survival rate is the same or higher) as compared to the case of culturing in the absence of the test compound, the test compound is Evaluate that there is no hepatotoxicity.

  In addition, when the amount of albumin produced in the three-dimensional liver tissue structure is low (albumin-producing ability is low) compared to the case where the test compound is cultured in the absence, the test compound is used as the three-dimensional liver. It is evaluated that it is toxic to the hepatocytes contained in the tissue structure, that is, hepatotoxic. It can be evaluated that the hepatotoxicity is stronger as the decrease in albumin production is larger than in the absence of the test compound. On the other hand, when the amount of albumin produced is the same or significantly higher (albumin-producing ability is the same or higher) as compared with the case of culturing in the absence of the test compound, the test compound is hepatotoxic. Evaluate that there is no.

  The number of living hepatocytes can be evaluated using a signal correlated with the number of living hepatocytes or the abundance thereof. It is only necessary to be able to measure the number of cancer cells at the time of evaluation, and it is not always necessary to measure in a living state. For example, hepatocytes can be labeled so as to be distinguished from other cells, and the signal from the label can be examined as an indicator. For example, live liver cells in a cell structure can be directly counted by fluorescently labeling hepatocytes and then determining whether the cells are alive or dead. At this time, an image analysis technique can also be used. Cell viability can be determined by a known cell viability determination method such as trypan blue staining or PI (Propidium Iodide) staining. The fluorescent labeling of hepatocytes is performed by immunostaining using, for example, an antibody against a substance specifically expressed on the cell surface of hepatocytes as a primary antibody and a fluorescently labeled secondary antibody that specifically binds to the primary antibody. It can carry out by well-known methods, such as a method. The determination of the viability of a cell and the measurement of the number of living cells may be performed in the state of a cell structure, or may be performed in a state where the cell structure is destroyed to a single cell level. For example, after destroying the three-dimensional structure of the cell structure after labeling hepatocytes and dead cells, only live cells at the time of evaluation are directly counted by FACS (fluorescence activated cell sorting) using the label as an index You can also

  It is also possible to measure the number of living hepatocytes in the cell structure over time by labeling the hepatocytes in the cell structure alive and detecting the signal from the label over time. it can. After constructing the cell structure, hepatocytes in the cell structure may be labeled, or hepatocytes may be labeled in advance before constructing the cell structure. In addition, when hepatocytes in which fluorescent dyes are constantly expressed are used, the fluorescence intensity of the lysate obtained by dissolving the cell structure can also be measured with a microplate reader or the like. Viable cell count can be assessed.

  In general, the hepatotoxicity of a substance depends on the effect of the substance on drug metabolizing enzymes in liver tissue. For this reason, it is preferable that all hepatocytes contained in the three-dimensional liver tissue structure used in the evaluation method according to the present invention have at least one or more types of drug metabolizing enzyme genotypes in common. More preferably, the drug-metabolizing enzymes have a common genotype. By using a three-dimensional liver tissue structure in which the genotype of the drug metabolizing enzyme is homozygous, the influence of the test compound on the drug metabolizing enzyme can be more accurately evaluated.

  The present invention will be described in more detail and specifically with reference to the following examples. However, the examples do not limit the scope of the present invention.

  In the following examples, collagen I was used as collagen unless otherwise specified.

[Example 1]
The albumin producing ability of a three-dimensional liver tissue structure constructed by stacking hepatocytes after coating with heparin and collagen was examined.
Specifically, the cells were suspended in a mixture of a heparin / 50 mM Tris-HCl buffer (pH 7.4) solution and an equal amount of collagen / 50 mM Tris-HCl buffer (pH 7.4) solution. Table 1 shows the structure of the cells subjected to the experiment. Human hepatocytes were PXB cells from Phoenix Bio, and human umbilical vein endothelial cells (HUVEC) (Lonza, product number: CC-2517A) were used as vascular endothelial cells. Further, human mesenchymal stem cells obtained from Lonza (product model number: PT-2501) and cultured using MSCGM (Mesenchymal Stem Cell Growth Medium) BulletKit (registered trademark) (product model number: PT-3001) Used.

The obtained suspension was seeded in 96-well cell culture insert (Roche Inc, E-Plate Insert 16), and centrifuged at 10 ° C. and 400 × g for 1 minute. Thereby, a cell layer was formed on the cell culture insert, and a cell structure was constructed. Next, 250 μL of Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) was added to the cell culture insert, and this cell structure was added to a CO ₂ incubator (37 ° C., 5% CO ₂ ) for a predetermined period. And cultured for a total of 15 days. During the culture period after cell structure construction, the whole culture supernatant was collected every 2 days, and the amount of albumin (ng / mL) in the culture supernatant was measured using an ELISA kit.

  After the culture, the constructed cell structure was collected and a paraffin-embedded section was prepared. A paraffin-embedded section was prepared according to a known method. The prepared sections were stained with hematoxylin and eosin (HE staining). HE staining followed a known method.

  The results of HE staining on the 15th day of culture are shown in FIG. In the cell structures of conditions A, B, and D, tissues with a uniform thickness of 150 μm or more were observed. In the cell structures of conditions C and E, a tissue body having a non-uniform thickness was obtained.

  In the transmitted light image of each cell structure on the 11th day of culture, a difference in tissue was observed. A comparison between tissues in which MSC coexists (cell structures of conditions A, B, and D) and tissues that do not contain mesenchymal stem cells (cell structures of conditions C and E). In contrast, the PXB cell alone (Condition E cell structure) or PXB / HUVEC (Condition C cell structure) showed uniformity in the cell structure. It decreased (FIG. 2).

  The results of measuring the amount of albumin (ng / mL) in each culture supernatant collected on days 1, 3, 5, 7, 9, 11, 13, and 15 from the start of the culture by ELISA are shown in FIG. In the cell structure treated with heparin / collagen, the albumin producing ability of PXB cell tended to be high. In the cell structure containing mesenchymal stem cells and vascular endothelial cells, the albumin producing ability is lower than that of PXB cell alone, but only when mesenchymal stem cells are added, albumin production after the ninth day of culture is produced. It was found that the performance declined gradually and the production ability was maintained for a long time. Moreover, the albumin production amount of these cell structures was comparable to the structure of Non-Patent Document 2 using the human hepatocellular carcinoma-derived cell line HepG2. This result suggests that a sufficiently thick cell structure can provide high albumin producing ability even when non-cancerous hepatocytes are used, as in the case of using hepatoma cells. It was done.

  According to the present invention, a thicker three-dimensional liver tissue structure can be produced quickly and stably. Moreover, the obtained liver tissue structure can maintain liver function for a long period of time. Therefore, the present invention is useful in the field of regenerative medicine. The present invention is also useful for evaluation of hepatotoxicity in drug development and the like.

Claims (20)

A method for producing a three-dimensional liver tissue structure comprising at least hepatocytes, wherein two or more cell layers are laminated in the thickness direction,
A method for producing a three-dimensional liver tissue structure, comprising producing a three-dimensional liver tissue structure in which 40% or more of all cells constituting the structure are hepatocytes and the thickness is 150 μm or more. (A) a step of mixing a cell containing at least hepatocytes, a strong electrolyte polymer, and an extracellular matrix component in a cationic buffer solution to obtain a mixture;
(B) seeding the mixture obtained by the step (a) in a cell culture vessel;
(C) After the step (b), the liquid component is removed from the cell mixture in the cell culture container, and a cell structure in which cells containing at least hepatocytes are laminated in a multilayer structure is three-dimensionally formed in the cell culture container. Obtaining a typical liver tissue structure;
The manufacturing method of the three-dimensional liver tissue structure of Claim 1 containing this.   The strong electrolyte polymer is selected from the group consisting of heparin, glycosaminoglycan, dextran sulfate, rhamnan sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, polyacrylamide-2-methylpropane sulfonic acid, and polyacrylic acid 1 The manufacturing method of the three-dimensional liver tissue structure of Claim 2 which is a seed | species or more.   The three-dimensional liver tissue according to claim 2 or 3, wherein the extracellular matrix component is one or more selected from the group consisting of collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, fibrillin, and proteoglycan. Manufacturing method of structure.   The three-dimensional liver tissue structure according to any one of claims 2 to 4, wherein the cationic buffer is Tris-HCl buffer, Tris-maleic acid buffer, bis-Tris-buffer, or HEPES. Manufacturing method.   The method for producing a three-dimensional liver tissue structure according to any one of claims 1 to 5, wherein the hepatocytes are non-cancerous cells.   The manufacturing method of the three-dimensional liver tissue structure as described in any one of Claims 1-6 whose thickness of the obtained three-dimensional liver tissue structure is 500 micrometers or less.   The manufacturing method of the three-dimensional liver tissue structure as described in any one of Claims 1-7 in which the said cell in the said process (a) contains a mesenchymal stem cell further.   The manufacturing method of the three-dimensional liver tissue structure as described in any one of Claims 1-8 in which the said cell in the said process (a) contains the cell which comprises stroma further.   The three-dimensional structure according to claim 9, wherein the cells in the step (a) further include one or more selected from the group consisting of vascular endothelial cells and lymphatic endothelial cells as cells constituting the stroma. A method for producing a liver tissue structure.   A three-dimensional liver tissue structure characterized in that it is a cell structure comprising at least hepatocytes and having two or more cell layers laminated in the thickness direction and having a thickness of 150 to 500 µm.   The three-dimensional liver tissue structure according to claim 11, wherein 40% or more of all the cells constituting the three-dimensional liver tissue structure are hepatocytes.   The three-dimensional liver tissue structure according to claim 11 or 12, further comprising a mesenchymal stem cell as a cell constituting the three-dimensional liver tissue structure.   The three-dimensional liver tissue structure according to any one of claims 11 to 13, further comprising cells constituting the stroma as cells constituting the three-dimensional liver tissue structure.   The three-dimensional liver tissue according to claim 14, wherein the three-dimensional liver tissue structure further includes at least one selected from the group consisting of vascular endothelial cells and lymphatic endothelial cells as cells constituting the stroma. Organizational structure. The three-dimensional liver tissue structure according to any one of claims 11 to 15, wherein the total number of cells constituting the three-dimensional liver tissue structure is 1.2 × 10 ⁵ or more.   The three-dimensional liver tissue structure according to any one of claims 11 to 16, wherein the amount of albumin produced by the three-dimensional liver tissue structure is 500 ng / mL or more as of the ninth day of culture.   The three-dimensional liver tissue structure according to any one of claims 11 to 17, wherein the amount of albumin produced by the three-dimensional liver tissue structure is 500 ng / mL or more at the time of culture on the 11th day after the structure construction. . A culture step of culturing the three-dimensional liver tissue structure according to any one of claims 11 to 18 in a state in which the test compound is contacted;
Evaluation of the hepatotoxicity of the compound, wherein the presence or absence of hepatic toxicity of the test compound is evaluated using the survival rate or albumin producing ability of the hepatocytes in the three-dimensional liver tissue structure after the culturing step as an index. Method.   The method for evaluating hepatotoxicity of a compound according to claim 19, wherein all hepatocytes contained in the three-dimensional liver tissue structure have at least one genotype of at least one drug metabolizing enzyme.