Cell structure, production method therefor, and method for evaluating hepatotoxicity of substance to be tested

Abstract

To provide a cell structure having improved responsiveness to a substance having hepatotoxicity.SOLUTION: The present invention relates to a cell structure including cells containing at least hepatocytes and vascular endothelial cells, and extracellular matrix component. The extracellular matrix components are disposed between the cells, and a liver sinusoidal network is provided between the cells.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a cell structure, a method for manufacturing the same, and a method for evaluating the hepatotoxicity of a test substance.

Background Art

[0002] Drug-induced liver injury (DILI) is a major cause of drug development / sales suspension. Since the mechanism of action of DILI is complex and diverse, and there are also many toxicities that are expressed by human-specific biological reactions, it is not always easy to predict it in animal tests. Therefore, a liver-like tissue is required as an alternative tool for predicting such human-specific toxicities in advance.

[0003] As a method for producing an artificially biomimetic structure, for example, a method for producing a three-dimensional tissue by culturing a coated cell in which the entire surface of a cultured cell is coated with an adhesion film (Patent Document 1), a method of mixing cells with a cationic substance and an extracellular matrix component to obtain a mixture, collecting cells from the obtained mixture, and forming a cell aggregate on a substrate, a method for producing a three-dimensional cell tissue (Patent Document 2), etc. are known. Further, the present inventors have proposed a method for producing a three-dimensional tissue with a high collagen concentration by bringing cells into contact with endogenous collagen, and preferably further bringing them into contact with fibrous exogenous collagen (Patent Document 3).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0005] An in vitro liver-like tissue that can three-dimensionally reproduce the higher-order tissue morphology such as blood vessel structures and enable accurate toxicity evaluation has not yet been constructed.

[0006] An object of the present invention is to provide a cell structure excellent in responsiveness to a substance having hepatotoxicity and a method for producing the same. [Means for Solving the Problems]

[0007] That is, the present invention relates to, for example, each of the following inventions. [1] A cell structure comprising at least a hepatocyte and an endothelial cell, and an extracellular matrix component, wherein the extracellular matrix component is disposed between the cells, and having a hepatic sinusoidal network between the cells. [2] The cell structure according to [1], wherein the ratio of the number of the hepatocytes to the total number of the cells is 60% or more and 80% or less. [3] The cell structure according to [1] or [2], wherein the ratio of the number of the endothelial cells to the total number of the cells is 5% or more and 35% or less. [4] The cell structure according to any one of [1] to [3], wherein the extracellular matrix component is fibrous. [5] The cell structure according to any one of [1] to [4], wherein the extracellular matrix component contains a collagen component. [6] The cell structure according to any one of [1] to [5], containing a polyelectrolyte. [7] The cell structure according to any one of [1] to [6], wherein the extracellular matrix component contains a fragmented extracellular matrix component. [8] The cell structure according to any one of [1] to [7], wherein the endothelial cell is a sinusoidal endothelial cell. [9] The cell structure according to any one of [1] to [8], further comprising hepatic stellate cells.

[10] A culture step of culturing the cell structure according to any one of [1] to [9] in contact with a test substance. A method for evaluating the hepatotoxicity of a test substance, wherein the presence or degree of hepatotoxicity is evaluated using the cell viability, albumin production, adenosine triphosphate content, or hepatic sinusoidal network of the cell structure after the culture step.

[11] The method for evaluating the hepatotoxicity of a test substance according to

[10] , wherein the culture step is performed by culturing the cell structure in a medium containing the test substance.

[12] A contact step of contacting at least a cell containing hepatocytes and vascular endothelial cells with an extracellular matrix component in an aqueous medium. A culture step of culturing the cell contacted with the extracellular matrix component. The contact step is performed under conditions where aggregation of the extracellular matrix component in the aqueous medium is suppressed. The method for producing a cell structure, wherein the culture step is performed under conditions suitable for culturing non-parenchymal liver cells.

[13] The method for producing a cell structure according to

[12] , wherein the extracellular matrix component is fibrous.

[14] The method for producing a cell structure according to

[12] or

[13] , wherein the contact step is performed by mixing the cell, the extracellular matrix component, and a polyelectrolyte.

[15] The method for producing a cell structure according to

[14] , wherein the polyelectrolyte is heparin.

[16] The method for producing a cell structure according to any one of

[12] to

[15] , wherein the contact step includes accumulating the cell and the extracellular matrix component after contact between the cell and the extracellular matrix component.

[17] The method for producing a cell structure according to any one of

[12] to

[16] , wherein the extracellular matrix component contains a collagen component.

[18] The method for producing a cell structure according to any one of

[12] to

[17] , wherein the extracellular matrix component contains a fragmented extracellular matrix component.

[19] The method for producing a cell structure according to

[18] , wherein the fragmented extracellular matrix component is a defibrated extracellular matrix component.

[20] The method for producing a cell structure according to any one of

[12] to

[19] , wherein the vascular endothelial cells are sinusoidal endothelial cells.

[21] The method for producing a cell structure according to any one of

[12] to

[20] , wherein the cells further contain hepatic stellate cells.

[22] The method for producing a cell structure according to any one of

[12] to

[21] , wherein the culturing step is performed in the presence of an angiogenesis promoting factor. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide a cell structure excellent in responsiveness to a hepatotoxic substance and a method for producing the same.

[0009] The cell structure of the present invention can maintain liver function for a relatively long period (for example, 2 weeks or more). By using the cell structure of the present invention, it is possible to evaluate the hepatotoxicity of a test substance. [Brief Description of the Drawings]

[0010]

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Mode for Carrying Out the Invention

[0011] Hereinafter, the modes for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[0012] [Cell construct] The cell construct according to this embodiment includes at least cells containing hepatocytes and vascular endothelial cells, and an extracellular matrix, and the extracellular matrix is disposed between the cells. The cell construct according to this embodiment can be used as a liver-like tissue (liver model), which is a biological tissue model having a function similar to at least a part of the functions of the liver and / or a structure similar to at least a part of the structure of the liver.

[0013] As used herein, the "cell construct" means an aggregate of cells (lump-like cell population) in which cells are three-dimensionally arranged, and is an aggregate artificially created by cell culture. An extracellular matrix component may be disposed between at least some of the cells. In the cell construct, there may be a portion where the cells are in direct contact with each other.

[0014] The shape of the cell construct is not particularly limited, and examples thereof include a sheet shape, a spherical shape, a substantially spherical shape, an ellipsoidal shape, a substantially ellipsoidal shape, a hemispherical shape, a substantially hemispherical shape, a semi-circular shape, a substantially semi-circular shape, a rectangular parallelepiped shape, a substantially rectangular parallelepiped shape, and the like. Here, the biological tissue includes sweat glands, lymphatic vessels, sebaceous glands, etc., and the composition is more complex than that of the cell construct. Therefore, the cell construct and the biological tissue can be easily distinguished. Further, the cell construct may be an aggregate that is aggregated in a lump in a state of being adhered to a support, or may be an aggregate that is aggregated in a lump in a state of not being adhered to a support.

[0015] (Cell) The cell may be a somatic cell or a germ cell. Further, the cell may be a stem cell, or may be a cultured cell such as a primary cultured cell, a subcultured cell, or a cell line cell. As used herein, the term "stem cell" means a cell having self-renewal ability and pluripotency. Stem cells include pluripotent stem cells capable of differentiating into any cell tumor, and tissue stem cells (also called somatic stem cells) capable of differentiating into a specific cell tumor. Examples of pluripotent stem cells include embryonic stem cells (ES cells), somatic cell-derived ES cells (ntES cells), and induced pluripotent stem cells (iPS cells). Examples of tissue stem cells include mesenchymal stem cells (e.g., bone marrow-derived stem cells), hematopoietic stem cells, and neural stem cells.

[0016] The total number of cells constituting the cell structure according to the present embodiment is not particularly limited, and is appropriately determined in consideration of the thickness and shape of the cell structure to be constructed, the size of the cell culture container used for construction, and the like.

[0017] In the cell structure according to the present embodiment, the cells include at least hepatocytes and vascular endothelial cells.

[0018] Hepatocytes are also referred to as liver parenchymal cells, and are cells having functions such as bile secretion and plasma protein secretion. The hepatocytes constituting the cell structure may be primary hepatocytes collected from the liver of an animal, cells obtained by culturing primary hepatocytes, a cultured cell line obtained by immortalizing primary hepatocytes, or hepatoblasts artificially differentiated from stem cells. Examples of primary hepatocytes include primary human hepatocytes such as PXB Cell. Examples of the cultured cell line include a cell line derived from inactivated liver cancer cells such as HepG2. Examples of stem cells that can be differentiated into hepatoblasts include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and mesenchymal stem cells. The hepatocytes included in the cell structure according to the present embodiment are preferably non-cancerous cells such as primary hepatocytes and hepatoblasts, and PXB Cell is more preferable in terms of ease of handling.

[0019] The hepatocytes contained in the cell construct may be of one type or two or more types. For example, the cell construct may contain a plurality of hepatocytes with different genotypes of proteins involved in liver function. Conversely, all the hepatocytes contained in the cell construct may have the same genotype of proteins involved in liver function. Examples of proteins involved in liver function include drug-metabolizing enzymes.

[0020] The ratio (X1 / X0×100) of the number of hepatocytes (X1) to the total number of cells (X0) in the cell construct may be 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, or 65% or more, and may be 95% or less, 90% or less, 80% or less, or 75% or less. From the perspective of being more suitable as a liver-like tissue, the ratio (X1 / X0×100) of the number of hepatocytes (X1) to the total number of cells (X0) in the cell construct may be 60% or more and 80% or less, or 60% or more and 70% or less.

[0021] Vascular endothelial cells refer to flattened cells that form the surface of the vascular lumen. The vascular endothelial cells may be, for example, sinusoidal endothelial cells, vascular endothelial cells derived from human umbilical vein (HUVEC). Sinusoidal endothelial cells are non-parenchymal liver cells (cells other than hepatocytes among the cells that make up the liver), and have a characteristic morphology different from other vascular endothelial cells, such as having a large number of small pore aggregates (sieve plate structure) in the cytoplasm and lacking a basement membrane. The vascular endothelial cells that make up the cell structure may be primary cells (primary vascular endothelial cells) collected from the liver of an animal (e.g., human), cells obtained by culturing primary cells, cultured cell lines established from primary cells, or cells artificially differentiated from stem cells. Examples of primary vascular endothelial cells include primary sinusoidal endothelial cells such as product model number 5000 manufactured by Sciencell. Examples of cultured cell lines include the cultured cell line of product model number T0056 manufactured by Applied Biological Materials. Examples of stem cells to be differentiated include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), etc. The vascular endothelial cells contained in the cell structure according to this embodiment may be non-cancerous cells.

[0022] The ratio (X2 / X0×100) of the number of vascular endothelial cells (X2) to the total number of cells (X0) in the cell structure may be 5% or more, 10% or more, 12% or more, 14% or more, 15% or more, 20% or more, or 25% or more, and may be 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, or 18% or less. From the viewpoint of being more suitable as a liver-like tissue, the ratio (X2 / X0×100) of the number of vascular endothelial cells (X2) to the total number of cells (X0) in the cell structure may be 5% or more and 40% or less, 5% or more and 35% or less, 10% or more and 35% or less, 10% or more and 25% or less, or 12% or more and 20% or less. The number of sinusoidal endothelial cells may be within the above range with respect to the total number of cells in the cell structure.

[0023] In the cell structure according to this embodiment, the cells may further contain hepatic stellate cells from the viewpoint of being more suitable as a hepatocyte-like tissue. Hepatic stellate cells are non-parenchymal liver cells (cells other than hepatocytes among the cells constituting the liver, which have functions such as storing vitamin A and are present in the Disse space, which is the region between hepatocytes and sinusoids in the liver. Examples of hepatic stellate cells may include primary cells (primary hepatic stellate cells) collected from the liver of an animal (e.g., a human), cells obtained by culturing primary cells, cultured cell lines established from primary cells, or cells artificially differentiated from stem cells. Examples of primary hepatic stellate cells include primary hepatic stellate cells of model number 5300 manufactured by Sciencell. Examples of cultured cell lines include cultured cell lines such as LX-2. Examples of stem cells to be differentiated include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), mesenchymal stem cells, and the like. The hepatic stellate cells contained in the cell structure according to this embodiment may be non-cancerous cells.

[0024] The ratio (X3 / X0×100) of the number of hepatic stellate cells (X3) to the total number of cells (X0) in the cell structure may be 1% or more, 2% or more, 3% or more, 4% or more, or 5% or more, and may be 20% or less, 15% or less, 14% or less, 13% or less, 12% or less, or 11% or less. From the viewpoint of being more suitable as a hepatocyte-like tissue, the ratio (X3 / X0×100) of the number of hepatic stellate cells (X3) to the total number of cells (X0) in the cell structure may be 1% or more and 15% or less, or 3% or more and 12% or less.

[0025] In the present embodiment, the cells may contain other cells in addition to hepatocytes, vascular endothelial cells, and hepatic stellate cells. The other cells may be, for example, mature somatic cells or undifferentiated cells such as stem cells. Specific examples of somatic cells include, for example, nerve cells, dendritic cells, immune cells, lymphatic endothelial cells, fibroblasts, epithelial cells (excluding hepatocytes), cardiomyocytes, pancreatic islet cells, smooth muscle cells, bone cells, alveolar epithelial cells, spleen cells, and the like. Examples of stem cells include ES cells, iPS cells, mesenchymal stem cells, and the like. The other cells may be normal cells or 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 that has acquired the ability to proliferate infinitely.

[0026] The cells in the cell structure may or may not contain mesenchymal stem cells as other cells. The ratio (X4 / X0 × 100) of the number of mesenchymal stem cells (X4) to the total number of cells (X0) in the cell structure may be 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, or 45% or more, and may be 80% or less, 70% or less, 60% or less, or 55% or less.

[0027] The origin of the hepatocytes, vascular endothelial cells, hepatic stellate cells, or the above-mentioned other cells contained in the cells is not particularly limited, and may be, for example, cells derived from mammalian animals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, rats, and the like.

[0028] The cells in the cell structure according to the present embodiment do not necessarily contain cells differentiated from induced pluripotent stem cells (iPS cells). When the cells in the cell structure according to the present embodiment do not contain cells differentiated from iPS cells, it becomes easier to grasp the degree of differentiation from iPS cells, the ratio of differentiated cells to the whole cells, and as a result, it becomes even easier to grasp the content rate of each cell constituting the cell structure.

[0029] The cell structure according to this embodiment has blood vessels formed between at least some of the cells. "Blood vessels are formed between the cells" means that the tubular structure formed by vascular endothelial cells has a structure extending between cells. The cell structure may have gaps where no blood vessels are formed between the cells. The gap may be an empty space with nothing in it, or it may be filled with extracellular matrix components or the like.

[0030] It is difficult to maintain an artificially produced thick cell structure in a state where no blood vessels are present, and it is said that it is necessary to supply oxygen and the like from the outside. In contrast, since the cell structure according to this embodiment has blood vessels formed between the cells like a living tissue, it is expected to be maintained for a long time. It is also expected to be easily engrafted when transplanted into mammals or the like.

[0031] The cell structure according to this embodiment preferably has a hepatic sinusoidal network between the cells. "Having a hepatic sinusoidal network" means that the tubular structure formed by vascular endothelial cells has a structure formed in a mesh-like pattern so as to branch and surround the cells. The blood vessel structure and hepatic sinusoidal network between the cells can be confirmed by immunohistochemical staining. For example, when the cell structure is observed under a microscope from above, it may have a hepatic sinusoidal network to such an extent that it has a plurality of branch points and is observed to have a mesh structure.

[0032] (Extracellular matrix component) The cell structure according to this embodiment contains extracellular matrix components. The extracellular matrix components are arranged between at least some of the cells.

[0033] As used herein, the "extracellular matrix component" is an aggregate of extracellular matrix molecules formed by a plurality of extracellular matrix molecules. The extracellular matrix refers to substances existing outside cells in an organism. As the extracellular matrix, any substance can be used as long as it does not adversely affect cell growth and the formation of cell aggregates. Specific examples include, but are not limited to, collagen, elastin, proteoglycan, fibronectin, hyaluronic acid, laminin, vitronectin, tenascin, entactin, fibrillin, and cadherin. The extracellular matrix component may be used alone or in combination. The extracellular matrix component may, for example, contain a collagen component or may be a collagen component. When the extracellular matrix component is a collagen component, the collagen component functions as a scaffold for cell adhesion, and the formation of a three-dimensional cell structure is further promoted. The extracellular matrix component in the present embodiment is preferably a substance existing outside animal cells, that is, an extracellular matrix component of an animal. Note that the extracellular matrix molecule may be a modified form or variant of the above-described extracellular matrix molecule, or a polypeptide such as a chemically synthesized peptide, as long as it does not adversely affect cell growth and the formation of cell aggregates.

[0034] The extracellular matrix component may have a repetition of a sequence represented by Gly-X-Y characteristic of collagen. Here, Gly represents a glycine residue, and X and Y each independently represent any amino acid residue. The plurality of Gly-X-Y may be the same or different from each other. By having a repetition of the sequence represented by Gly-X-Y, the binding to the arrangement of molecular chains is less, and the function as a scaffold material becomes even more excellent. In the extracellular matrix component having a repetition of the sequence represented by Gly-X-Y, the ratio of the sequence represented by Gly-X-Y may be 80% or more, preferably 95% or more, of the entire amino acid sequence. Further, the extracellular matrix component may have an RGD sequence. The RGD sequence refers to a sequence represented by Arg-Gly-Asp (arginine residue - glycine residue - aspartic acid residue). When the extracellular matrix component has an RGD sequence, cell adhesion is further promoted, and it becomes even more suitable as a scaffold material. The extracellular matrix components containing the sequence represented by Gly-X-Y and the RGD sequence include collagen, fibronectin, vitronectin, laminin, cadherin, and the like.

[0035] Examples of the shape of the extracellular matrix component include fibrous. Fibrous means a shape composed of filamentous extracellular matrix components or a shape formed by cross-linking filamentous extracellular matrix components between molecules. At least a part of the extracellular matrix component may be fibrous. The shape of the extracellular matrix component is the shape of a lump of extracellular matrix components (aggregate of extracellular matrix components) observed when observed under a microscope, and the extracellular matrix component preferably has a size of an average diameter and / or an average length described later. The fibrous extracellular matrix component includes a thin filament (fine fiber) formed by the aggregation of a plurality of filamentous extracellular matrix molecules, a filament formed by further aggregation of the fine fibers, and those obtained by defibrating these filaments. When the extracellular matrix component having a fibrous shape is included, the RGD sequence is preserved without being destroyed in the fibrous extracellular matrix component, and it can function more effectively as a scaffold material for cell adhesion.

[0036] The extracellular matrix component may include fragmented extracellular matrix components. "Fragmentation" means making an aggregate of extracellular matrix components into a smaller size. The fragmented extracellular matrix component may include fibrillated extracellular matrix components. The fibrillated extracellular matrix component is a component obtained by fibrillating the above-described extracellular matrix component by applying a physical force. For example, fibrillation is performed under conditions that do not cleave the bonds within the extracellular matrix molecule.

[0037] The fragmented extracellular matrix component can be produced, for example, by a method including a step of fragmenting the extracellular matrix component (fragmentation step).

[0038] The method for fragmenting extracellular matrix components is not particularly limited and may be fragmented by applying physical force. Unlike enzymatic treatment, the extracellular matrix components fragmented by applying physical force usually do not change in molecular structure before and after fragmentation (the molecular structure is maintained). The method for fragmenting extracellular matrix components may be, for example, a method of finely crushing massive extracellular matrix components. The extracellular matrix components may be fragmented in a solid phase or in an aqueous medium. For example, the extracellular matrix components may be fragmented by applying physical force such as an ultrasonic homogenizer, a stirring homogenizer, and a high-pressure homogenizer. When using a stirring homogenizer, the extracellular matrix components may be homogenized as they are or in an aqueous medium such as physiological saline. Also, by adjusting the homogenization time, number of times, etc., it is possible to obtain extracellular matrix components fragmented into millimeter-sized or nanometer-sized fragments. When fragmenting extracellular matrix components in an aqueous medium, the fragmented extracellular matrix components can be produced by, for example, a method including a step of fragmenting the extracellular matrix components in an aqueous medium and a step of removing the aqueous medium from the liquid containing the fragmented extracellular matrix components and the aqueous medium (removing step). The removing step may be carried out, for example, by freeze-drying. "Removing the aqueous medium" does not mean that no moisture adheres to the fragmented extracellular matrix components at all, but means that the moisture adheres to such an extent that it can be reached by common sense by the above-described general drying methods.

[0039] The diameter and length of the fragmented extracellular matrix components can be determined by analyzing each fragmented extracellular matrix component with an electron microscope.

[0040] The average length of the fragmented extracellular matrix components may be 100 nm or more and 400 μm or less, and may be 100 nm or more and 200 μm or less. In one embodiment, the average length of the fragmented extracellular matrix components may be 5 μm or more and 400 μm or less, may be 10 μm or more and 400 μm or less, and may be 100 μm or more and 400 μm or less, from the viewpoint of facilitating the formation of thick cell structures. In other embodiments, the average length of the fragmented extracellular matrix components may be 100 μm or less, may be 50 μm or less, may be 30 μm or less, may be 15 μm or less, may be 10 μm or less, may be 1 μm or less, and may be 100 nm or more. It is preferable that the average length of most of the fragmented extracellular matrix components among all the fragmented extracellular matrix components is within the above numerical range. Specifically, it is preferable that the average length of 50% or more of the fragmented extracellular matrix components among all the fragmented extracellular matrix components is within the above numerical range, and it is more preferable that the average length of 95% of the fragmented extracellular matrix components is within the above numerical range. The fragmented extracellular matrix components are preferably fragmented collagen components having an average length within the above range.

[0041] The average diameter of the fragmented extracellular matrix components may be 50 nm to 30 μm, may be 4 μm to 30 μm, and may be 5 μm to 30 μm. The fragmented extracellular matrix components are preferably fragmented collagen components having an average diameter within the above range.

[0042] The average length and average diameter of the fragmented extracellular matrix components can be determined by measuring individual fragmented extracellular matrix components with an optical microscope or the like and performing image analysis. In this specification, "average length" means the average value of the lengths in the longitudinal direction of the measured sample, and "average diameter" means the average value of the lengths in the direction perpendicular to the longitudinal direction of the measured sample.

[0043] The fragmented collagen component is also referred to as the "fragmented collagen ingredient". The "fragmented collagen ingredient" means a fragmented collagen component such as a fibrous collagen component, which maintains a triple helix structure. The average length of the fragmented collagen component is preferably 100 nm to 200 μm, more preferably 22 μm to 200 μm, and even more preferably 100 μm to 200 μm. The average diameter of the fragmented collagen component is preferably 50 nm to 30 μm, more preferably 4 μm to 30 μm, and even more preferably 20 μm to 30 μm.

[0044] At least a part of the extracellular matrix component may be cross-linked intermolecularly or intramolecularly. The extracellular matrix component may be cross-linked intermolecularly or intramolecularly of the extracellular matrix molecules constituting the extracellular matrix component. When the extracellular matrix component contains a fragmented extracellular matrix component, at least a part of the fragmented extracellular matrix component may be cross-linked intermolecularly or intramolecularly.

[0045] The extracellular matrix component cross-linked at least in part intermolecularly or intramolecularly can be produced, for example, by a method including a step of cross-linking the extracellular matrix component (cross-linking step). The extracellular matrix component can contain, for example, a fragmented and cross-linked extracellular matrix component. The fragmented and cross-linked extracellular matrix component can be produced, for example, by a method comprising a step of fragmenting the extracellular matrix component and a step of cross-linking the fragmented extracellular matrix component in this order, or a method comprising a step of cross-linking the extracellular matrix component and a step of fragmenting the cross-linked extracellular matrix component in this order.

[0046] Examples of the cross-linking method include physical cross-linking by application of heat, ultraviolet rays, radiation, etc., chemical cross-linking by cross-linking agents, enzyme reactions, etc., but the method is not particularly limited. From the viewpoint of not interfering with cell growth, physical cross-linking is preferred. The cross-linking (physical cross-linking and chemical cross-linking) may be cross-linking via a covalent bond.

[0047] When the extracellular matrix component contains a collagen component, crosslinking may be formed between collagen molecules (triple helix structure) or between collagen fibrils formed by collagen molecules. The crosslinking may be crosslinking by heat (thermal crosslinking). Thermal crosslinking can be carried out, for example, by performing heat treatment under reduced pressure using a vacuum pump. When performing thermal crosslinking of the collagen component, the extracellular matrix component may be crosslinked by the amino group of the collagen molecule forming a peptide bond (-NH-CO-) with the carboxyl group of the same or another collagen molecule.

[0048] The extracellular matrix component can also be crosslinked by using a crosslinking agent. The crosslinking agent may be, for example, one capable of crosslinking a carboxyl group and an amino group, or one capable of crosslinking amino groups. As the crosslinking agent, for example, aldehyde-based, carbodiimide-based, epoxide-based, and imidazole-based crosslinking agents are preferable from the viewpoints of economy, safety, and operability. Specifically, water-soluble carbodiimides such as glutaraldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide sulfonate can be mentioned.

[0049] Quantification of the degree of crosslinking can be appropriately selected according to the type of extracellular matrix component, the means of crosslinking, etc. The degree of crosslinking may be 1% or more, 2% or more, 4% or more, 8% or more, or 12% or more, and may also be 30% or less, 20% or less, or 15% or less. When the degree of crosslinking is within the above range, the extracellular matrix molecules can be appropriately dispersed, and the redispersibility after dry storage is good.

[0050] When amino groups in extracellular matrix components are used for crosslinking, the degree of crosslinking can be quantified based on the TNBS (2,4,6-trinitrobenzenesulfonic acid) method described in Acta Biomaterialia, 2015, vol. 25, pp. 131-142, etc. The degree of crosslinking by the TNBS method may be within the above-mentioned range. The degree of crosslinking by the TNBS method is the ratio of the amino groups used for crosslinking among the amino groups possessed by the extracellular matrix. When the extracellular matrix component contains a collagen component, it is preferable that the degree of crosslinking measured by the TNBS method is within the above range.

[0051] The degree of crosslinking may also be calculated by quantifying carboxyl groups. For example, in the case of extracellular matrix components that are insoluble in water, it may be quantified by the TBO (toluidine blue O) method. The degree of crosslinking by the TBO method may be within the range described above.

[0052] In the crosslinking step, the temperature (heating temperature) and time (heating time) when heating the extracellular matrix component can be determined appropriately. The heating temperature may be, for example, 100 °C or higher, 200 °C or lower, or 220 °C or lower. Specifically, the heating temperature may be, for example, 100 °C, 110 °C, 120 °C, 130 °C, 140 °C, 150 °C, 160 °C, 170 °C, 180 °C, 190 °C, 200 °C, or 220 °C, etc. The heating time (the time held at the above heating temperature) can be set appropriately according to the heating temperature. The heating time may be, for example, 6 hours or more and 72 hours or less, more preferably 24 hours or more and 48 hours or less when heating at 100 °C to 200 °C. In the crosslinking step, heating may be performed in the absence of a solvent, or heating may also be performed under reduced pressure conditions.

[0053] The content of the extracellular matrix component in the cell construct may be 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 0.5% by mass or more, 1% by mass or more, 2% by mass or more, 3% by mass or more, 4% by mass or more, 5% by mass or more, 6% by mass or more, 7% by mass or more, 8% by mass or more, 9% by mass or more, 10% by mass, 15% by mass or more, 20% by mass or more, 25% by mass or more, or 30% by mass or more, and may be 90% by mass or less, 80% by mass or less, 70% by mass or less, 60% by mass or less, 50% by mass or less, 30% by mass or less, 20% by mass or less, or 15% by mass or less, based on the dry weight of the cell construct. The content of the extracellular matrix component in the cell construct may be 0.01 to 90% by mass, 10 to 90% by mass, 10 to 80% by mass, 10 to 70% by mass, 10 to 60% by mass, 1 to 50% by mass, 10 to 50% by mass, 10 to 30% by mass, or 20 to 30% by mass, based on the dry weight of the cell construct.

[0054] Here, the "extracellular matrix component in the cell construct" means the extracellular matrix component constituting the cell construct, and may be derived from an endogenous extracellular matrix component or an exogenous extracellular matrix component.

[0055] The "endogenous extracellular matrix component" means the extracellular matrix component produced by extracellular matrix-producing cells. Examples of extracellular matrix-producing cells include mesenchymal cells such as the above-described fibroblasts, chondrocytes, and osteoblasts. The endogenous extracellular matrix component may be fibrous or non-fibrous.

[0056] "Exogenous extracellular matrix component" means an extracellular matrix component supplied from the outside. The cell structure according to this embodiment includes fragmented extracellular matrix components that are exogenous extracellular matrix components. The exogenous extracellular matrix component may be the same as or different from the endogenous extracellular matrix component in terms of the animal species from which it is derived. Examples of the animal species from which it is derived include humans, pigs, cows, etc. Further, the exogenous extracellular matrix component may be an artificial extracellular matrix component.

[0057] When the extracellular matrix component is a collagen component, the exogenous extracellular matrix component is also referred to as an "exogenous collagen component", and the "exogenous collagen component" that means a collagen component supplied from the outside is an aggregate of collagen molecules formed by a plurality of collagen molecules, and specifically includes fibrous collagen, non-fibrous collagen, etc. The exogenous collagen component is preferably fibrous collagen. The above fibrous collagen means a collagen component that is the main component of collagen fibers, and examples thereof include type I collagen, type II collagen, and type III collagen. The above fibrous collagen may be commercially available collagen, and a specific example thereof is porcine skin-derived type I collagen manufactured by Nippon Ham Foods Co., Ltd. Examples of the exogenous non-fibrous collagen include type IV collagen.

[0058] In the exogenous extracellular matrix component, the animal species from which it is derived may be different from the cells. Further, when the cells include extracellular matrix-producing cells, in the exogenous extracellular matrix component, the animal species from which it is derived may be different from the extracellular matrix-producing cells. That is, the exogenous extracellular matrix component may be a heterologous extracellular matrix component.

[0059] That is, when the cell construct contains an endogenous extracellular matrix component and a fragmented extracellular matrix component, the extracellular matrix component content rate constituting the cell construct means the total amount of the endogenous extracellular matrix component and the fragmented extracellular matrix component. The extracellular matrix content rate can be calculated from the volume of the obtained cell construct and the mass of the decellularized cell construct.

[0060] For example, when the extracellular matrix component contained in the cell construct is a collagen component, examples of the method for quantifying the amount of the collagen component in the cell construct include, for example, the following method for quantifying hydroxyproline. Hydrochloric acid (HCl) is mixed with the lysate obtained by lysing the cell construct, incubated at a high temperature for a predetermined time, then returned to room temperature, and the supernatant obtained by centrifugation is diluted to a predetermined concentration to prepare a sample. After treating the hydroxyproline standard solution in the same manner as the sample, it is serially diluted to prepare a standard. Each of the sample and the standard is subjected to predetermined treatment with a hydroxyproline assay buffer and a detection reagent, and the absorbance at 570 nm is measured. The amount of the collagen component is calculated by comparing the absorbance of the sample with that of the standard. Note that the cell construct may be directly suspended in high-concentration hydrochloric acid, the lysate obtained by dissolution is centrifuged, and the supernatant may be recovered and used for quantification of the collagen component. Also, the cell construct to be lysed may be in the state as recovered from the culture solution, or may be dried after recovery to remove the liquid component and then lysed. However, when the cell construct in the state as recovered from the culture solution is lysed for quantification of the collagen component, it is expected that the measured value of the cell construct weight will vary due to the medium components absorbed by the cell construct and the remaining influence of the medium due to experimental technique problems. Therefore, from the viewpoint of stably measuring the weight of the tissue and the amount of the collagen component per unit weight, it is preferable to use the weight after drying as a reference.

[0061] More specifically, examples of the method for quantifying the amount of the collagen component include, for example, the following method. (Preparation of sample) Mix the entire amount of the freeze-dried cell structure with 6 mol / L HCl, incubate in a heat block at 95 °C for 20 hours or more, and then return to room temperature. After centrifuging at 13000 g for 10 minutes, collect the supernatant of the sample solution. Appropriately dilute with 6 mol / L HCl so that the results fall within the calibration curve range in the measurements described below, and then prepare the sample by diluting 200 μL with 100 μL of ultrapure water. Use 35 μL of the sample.

[0062] (Preparation of Standard) Add 125 μL of the standard solution (1200 μg / mL in acetic acid) and 125 μL of 12 mol / l HCl to a screw-cap tube, mix, incubate in a heat block at 95 °C for 20 hours, and then return to room temperature. After centrifuging at 13000 g for 10 minutes, dilute the supernatant with ultrapure water to prepare S1 at 300 μg / mL, and serially dilute S1 to prepare S2 (200 μg / mL), S3 (100 μg / mL), S4 (50 μg / mL), S5 (25 μg / mL), S6 (12.5 μg / mL), and S7 (6.25 μg / mL). Also prepare S8 (0 μg / mL) with only 90 μL of 4 mol / l HCl.

[0063] (Assay) Add 35 μL of the standard and the sample respectively to a plate (attached to the QuickZyme Total Collagen Assay kit, QuickZyme Biosciences). Add 75 μL of the assay buffer (attached to the above kit) to each well. Close the plate with a seal and incubate at room temperature for 20 minutes with shaking. Remove the seal, and add 75 μL of the detection reagent (reagent A:B = 30 μL:45 μL, attached to the above kit) to each well. Close the plate with a seal, mix the solution by shaking, and incubate at 60 °C for 60 minutes. Cool sufficiently on ice, remove the seal, and measure the absorbance at 570 nm. Calculate the amount of collagen component by comparing the absorbance of the sample with that of the standard.

[0064] The collagen component in the cell structure may be defined by its area ratio or volume ratio. "Defining by area ratio or volume ratio" means, for example, after distinguishing the collagen component in the cell structure from other tissue components by a known staining method (such as immunostaining using an anti-collagen antibody or Masson's trichrome staining), calculating the ratio of the area of the region where the collagen component exists in the whole cell structure using naked-eye observation, various microscopes, image analysis software, etc. When defining by area ratio, there is no limitation on which cross-section or surface in the cell structure is used to define the area ratio. For example, when the cell structure is a spheroid, etc., it may be defined by a cross-sectional view passing through its approximate center.

[0065] For example, when the collagen component in the cell structure is defined by area ratio, the ratio of the area is usually 0.01 - 99%, and may be 1 - 99%, 5 - 90%, 7 - 90%, 20 - 90%, 30 - 90%, or 50 - 90% based on the total area of the above cell structure. Regarding the "collagen component in the cell structure", it is as described above. The ratio of the area of the collagen component constituting the cell structure means the ratio of the combined area of the endogenous collagen component and the exogenous collagen component. The ratio of the area of the collagen component can be calculated, for example, by staining the obtained cell structure with Masson's trichrome and taking the ratio of the area of the blue-stained collagen component to the total area of a cross-section passing through the approximate center of the cell structure.

[0066] (Polyelectrolyte) The cell structure according to one embodiment may further contain a polyelectrolyte. A polyelectrolyte is a polymer compound having the properties of an electrolyte. Examples of polyelectrolytes include glycosaminoglycans such as heparin, chondroitin sulfate (e.g., chondroitin 4-sulfate, chondroitin 6-sulfate), heparan sulfate, dermatan sulfate, keratan sulfate, hyaluronic acid; dextran sulfate, laminaran sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, polyacrylamide-2-methylpropanesulfonic acid, and polyacrylic acid, or derivatives thereof, etc., but are not limited thereto. The polyelectrolyte may consist of one of the above-mentioned substances, or may contain a combination of two or more thereof.

[0067] The polyelectrolyte is preferably a glycosaminoglycan, more preferably contains at least one selected from the group consisting of heparin, dextran sulfate, chondroitin sulfate, and dermatan sulfate, and still more preferably is heparin. When the cell structure contains a polyelectrolyte, excessive aggregation of extracellular matrix components can be more effectively suppressed, and as a result, a cell structure excellent in responsiveness to hepatotoxic substances can be more easily obtained. When the cell structure contains heparin, the effect becomes even more remarkable.

[0068] The ratio (C2 / C1) of the mass C2 of the polyelectrolyte to the mass C1 of the extracellular matrix component may be 1 / 100 to 100 / 1, 1 / 10 to 10 / 1, 1 / 5 to 5 / 1, or 1 / 2 to 2 / 1, and may be 1 / 1.5 to 1.5 / 1.

[0069] (Cell structure) The thickness of the above cell structure may be 10 μm or more, 30 μm or more, 50 μm or more, 100 μm or more, 300 μm or more, or 1000 μm or more. Such a cell structure has a structure closer to that of a living tissue and is suitable as a substitute for experimental animals and a transplantation material. The upper limit of the thickness of the cell structure is not particularly limited, but may be, for example, 10 mm or less, 3 mm or less, 2 mm or less, 1.5 mm or less, or 1 mm or less.

[0070] Here, the "thickness of the cell structure" means the distance between both ends in the direction perpendicular to the main surface when the cell structure is sheet-like or rectangular parallelepiped-like. When there are irregularities on the main surface, the thickness means the distance at the thinnest part of the main surface.

[0071] When the cell structure is spherical or substantially spherical, the thickness of the cell structure means the diameter of the cell structure. When the cell structure is ellipsoidal or substantially ellipsoidal, the thickness of the cell structure means the minor axis of the cell structure. When the cell structure is substantially spherical or substantially ellipsoidal and there are irregularities on the surface, the thickness of the cell structure means the shortest distance between two points where a straight line passing through the center of gravity of the cell structure intersects the above surface.

[0072] The cell structure preferably contains fragmented extracellular matrix components and / or polyelectrolytes. In this case, the cell structure becomes even more suitable as a tissue model for evaluating hepatotoxicity and the like.

[0073] (Fibrin) The cell structure according to this embodiment may contain fibrin. Fibrin is a component generated by the action of thrombin on fibrinogen to release chains A and B from the N-termini of chains Aα and Bβ. Fibrin is a polymer and is generally insoluble in water. Fibrin is formed by bringing fibrinogen into contact with thrombin.

[0074] The cell structure according to this embodiment is constructed in a cell culture container. The cell culture container is not particularly limited as long as it enables the construction of the cell structure and the culture of the constructed cell structure. Specifically, examples of the cell culture container include dishes, cell culture inserts (e.g., Transwell (registered trademark) inserts, Netwell (registered trademark) inserts, Falcon (registered trademark) cell culture inserts, Millicell (registered trademark) cell culture inserts, etc.), tubes, flasks, bottles, plates, and the like. In constructing the cell structure, a dish or various cell culture inserts are preferred from the viewpoint of enabling more appropriate evaluation using the cell structure.

[0075] [Method for producing cell structure] The method for producing a cell structure according to this embodiment includes a contact step of bringing a cell containing at least a hepatocyte and a vascular endothelial cell into contact with an extracellular matrix component in an aqueous medium, and a culture step of culturing the cell brought into contact with the extracellular matrix component. As for the cell and the extracellular matrix component, those as described above can be used. In the method for producing a cell structure according to this embodiment, the contact step may be performed under conditions where aggregation of the extracellular matrix component in the aqueous medium is suppressed, and the culture step may be performed under conditions suitable for culturing non-parenchymal liver cells.

[0076] (Contact step) In the contact step, a cell containing at least a hepatocyte and a vascular endothelial cell is brought into contact with an extracellular matrix component in an aqueous medium. It is expected that obtaining a three-dimensional cell structure will be facilitated by bringing the cell into contact with the extracellular matrix component.

[0077] The contact between the cells and the extracellular matrix components may be carried out, for example, under conditions where the aggregation of the extracellular matrix components in an aqueous medium is suppressed. When the contact between the cells and the extracellular matrix components is carried out under such conditions, a hepatic sinusoid network is more likely to be formed in the cell structure. In a cell structure, when the cells contain hepatocytes, since the hepatocytes are likely to adhere to each other, the gaps between the cells are likely to become small, and as a result, it is considered that a tubular structure such as a hepatic sinusoid network is less likely to be formed. When the cells and the extracellular matrix components are brought into contact with each other under conditions where excessive aggregation of the extracellular matrix components in an aqueous medium is suppressed, the distribution of the extracellular matrix components and the cells in the cell structure becomes more uniform, and as a result, it is presumed that a hepatic sinusoid network is more likely to be formed, and a cell structure excellent in responsiveness to a hepatotoxic substance is likely to be obtained.

[0078] The extracellular matrix components can suppress aggregation in an aqueous medium, for example, by causing a component that suppresses the aggregation of the extracellular matrix components (for example, the above-described polyelectrolyte) to be present together with the extracellular matrix components, and / or by using fragmented extracellular matrix components as at least a part of the extracellular matrix components. The conditions under which the aggregation of the extracellular matrix components in an aqueous medium is suppressed may be, for example, conditions where a polyelectrolyte is present and / or conditions that include fragmented extracellular matrix in which the extracellular matrix components are fragmented.

[0079] "Aqueous medium" means a liquid having water as an essential constituent. As the aqueous medium, for example, an aqueous medium containing a cationic substance may be used. The aqueous medium containing a cationic substance may be, for example, a cationic buffer such as Tris-HCl buffer, Tris-maleic acid buffer, Bis-Tris buffer, or HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), or a medium containing a cationic compound such as ethanolamine, diethanolamine, triethanolamine, polyvinylamine, polyallylamine, polylysine, polyhistidine, polyarginine and water as the cationic substance. Further, as the above aqueous medium, a culture medium can also be used. Examples of the culture medium include liquid media such as Dulbecco’s Modified Eagle Medium (DMEM) and Endothelial Cell Growth Medium-2 (EGM2). The liquid medium may be a mixed medium in which two kinds of media are mixed.

[0080] The concentration and pH of the cationic substance (for example, Tris in Tris-HCl buffer) in the aqueous medium containing a cationic substance are not particularly limited as long as they do not adversely affect cell growth and the construction of cell structures. For example, the concentration of the cationic substance may be 10 to 100 mM, or 40 to 70 mM, or 50 mM based on the total amount of the aqueous medium containing the cationic substance. The pH of the aqueous medium (for example, cationic buffer) may be 6.0 to 8.0, 6.8 to 7.8, or 7.2 to 7.6.

[0081] Examples of the contacting step include a method of mixing an aqueous medium containing an extracellular matrix component and a culture solution containing cells under conditions where aggregation of the extracellular matrix component in the aqueous medium is suppressed, a method of adding an aqueous medium containing an extracellular matrix component to a culture solution containing cells, a method of adding cells to an aqueous medium containing an extracellular matrix component, and a method of adding an extracellular matrix component and cells to a previously prepared aqueous medium, respectively.

[0082] The order of bringing the above cells into contact with the extracellular matrix component is not particularly limited. For example, after bringing some cells into contact with the extracellular matrix component, the remaining cells may be brought into contact with the extracellular matrix component, or all the cells may be brought into contact with the extracellular matrix component simultaneously or substantially simultaneously.

[0083] The aqueous medium containing cells and the extracellular matrix may or may not be mixed by stirring or the like after the addition of each substance. The contact step may include incubating for a certain period of time after bringing the cells and the extracellular matrix component into contact.

[0084] The contact step may be carried out after accumulating the cells in an aqueous medium. That is, the contact step may be carried out by bringing the extracellular matrix component into contact after accumulating the cells in an aqueous medium. By bringing the accumulated cells into contact with the extracellular matrix component, it becomes easier to produce a cell structure having a high cell density in the lower layer. The cells can be accumulated by methods such as centrifugation and natural sedimentation.

[0085] In the contact step, the ratio (X1 / X0×100) of the number of hepatocytes to the total number of cells (X0) may be 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, or 65% or more, and may be 95% or less, 90% or less, 80% or less, or 75% or less. From the viewpoint of being more suitable as a liver-like tissue, the ratio (X1 / X0×100) of the number of hepatocytes (X1) to the total number of cells (X0) in the contact step may be 60% or more and 80% or less, or 60% or more and 70% or less.

[0086] In the contacting step, the ratio (X2 / X0×100) of the number of vascular endothelial cells (X2) to the total number of cells (X0) may be 5% or more, 10% or more, 12% or more, 14% or more, 15% or more, 20% or more, or 25% or more, and may be 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, or 18% or less. From the viewpoint of being more suitable as a hepatocyte-like tissue, the ratio (X2 / X0×100) of the number of vascular endothelial cells (X2) to the total number of cells (X0) in the cell construct may be 5% or more and 40% or less, 5% or more and 35% or less, 10% or more and 35% or less, 10% or more and 25% or less, or 12% or more and 20% or less.

[0087] When the cells contain hepatic stellate cells, in the contacting step, the ratio (X3 / X0×100) of the number of hepatic stellate cells (X3) to the total number of cells (X0) may be 1% or more, 2% or more, 3% or more, 4% or more, or 5% or more, and may be 20% or less, 15% or less, 14% or less, 13% or less, 12% or less, or 11% or less. From the viewpoint of being more suitable as a hepatocyte-like tissue, the ratio (X3 / X0×100) of the number of hepatic stellate cells (X3) to the total number of cells (X0) in the cell construct may be 1% or more and 15% or less, or 3% or more and 12% or less.

[0088] In the contacting step, the ratio (X5 / X0×100) of the number of cells other than hepatocytes, vascular endothelial cells, and hepatic stellate cells (X5) to the total number of cells (X0) may be 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, or 45% or more, and may be 80% or less, 70% or less, 60% or less, or 55% or less. The ratio of the number of mesenchymal stem cells (X4) to the total number of cells in the contacting step may also be within the above range.

[0089] Preferably, the above cells are not derived from iPS cells. When using cells not derived from iPS cells, the types and contents of cells in the cell construct are more easily specified, and as a result, it becomes a more suitable tissue model for evaluating hepatotoxicity.

[0090] In the contacting step, the concentration of the extracellular matrix component can be appropriately determined according to the shape, thickness of the target cell structure, the size of the culture vessel, etc. For example, the concentration of the extracellular matrix component in the aqueous medium in the contacting step may be 0.1 to 90% by mass, or may be 1 to 30% by mass.

[0091] The amount of the extracellular matrix component in the contacting step is, for example, 0.1 to 100 mg, 0.5 to 50 mg, 0.8 to 25 mg, 1.0 to 10 mg, 1.0 to 5.0 mg, 1.0 to 2.0 mg, or 1.0 to 1.8 mg with respect to 1.0×10 6 cells, and may be 0.7 mg or more, 1.1 mg or more, 1.2 mg or more, 1.3 mg or more, or 1.4 mg or more, and may be 7.0 mg or less, 3.0 mg or less, 2.3 mg or less, 1.8 mg or less, 1.7 mg or less, 1.6 mg or less, or 1.5 mg or less. The amount of the fragmented extracellular matrix component in the contacting step may be within the above range.

[0092] In the contacting step, the mass ratio of the extracellular matrix component to the cells (extracellular matrix component / cells) is preferably 1 / 1 to 1000 / 1, more preferably 9 / 1 to 900 / 1, and even more preferably 10 / 1 to 500 / 1.

[0093] In the first embodiment, the contacting step may be, for example, a step of mixing the above cells, an extracellular matrix component, and a polyelectrolyte in an aqueous medium (contacting step A).

[0094] As the polyelectrolyte, those described above can be used. For example, the concentration of the polyelectrolyte may be more than 0 mg, 0.001 mg or more, 0.005 mg or more, 0.01 mg or more, 0.025 mg or more, 0.05 mg or more, or 0.075 mg or more with respect to 1 mL of the total amount of the aqueous medium, and may be less than 1.0 mg, 0.5 mg or less, or 0.1 mg or less.

[0095] The first contacting step may be performed, for example, by mixing a cell extracellular matrix component-containing liquid containing a cell extracellular matrix component and a first aqueous medium, a polyelectrolyte-containing liquid containing a polyelectrolyte and a second aqueous medium, and cells. In the cell extracellular matrix component-containing liquid, the cell extracellular matrix component may be dissolved or dispersed in the first aqueous medium. In the polyelectrolyte-containing liquid, the polyelectrolyte may be dissolved in the second aqueous medium. The first aqueous medium and the second aqueous medium may be the same type of aqueous medium or different types of aqueous media.

[0096] The order of mixing the cell extracellular matrix component-containing liquid, the polyelectrolyte-containing liquid, and the cells is not particularly limited, and they may be mixed in any order. For example, a mixed liquid prepared by previously mixing the cell extracellular matrix component-containing liquid and the polyelectrolyte-containing liquid may be prepared, and the mixed liquid and the cells may be mixed. Alternatively, all of them may be mixed substantially simultaneously.

[0097] In the cell extracellular matrix component-containing liquid, the content of the cell extracellular matrix component may be 0.001 to 1.5 mg / mL, 0.05 to 1.5 mg / mL, or 0.1 to 1.0 mg / mL with respect to the total amount of the cell extracellular matrix component-containing liquid.

[0098] In the polyelectrolyte-containing liquid, the content of the polyelectrolyte may be 0.001 to 10.0 mg / mL, 0.05 to 5.0 mg / mL, or 0.1 to 1.0 mg / mL with respect to the total amount of the polyelectrolyte-containing liquid.

[0099] The ratio (A1:A2) of the mass A1 of the polyelectrolyte to the mass A2 of the cell extracellular matrix component in the contacting step may be 1:200 to 200:1, 1:100 to 100:1, 1:10 to 10:1, 1:5 to 5:1, 1:2 to 2:1, 1:1.5 to 1.5:1, or 1:1.

[0100] In the second embodiment, the contacting step may be a step of bringing the fragmented extracellular matrix component into contact with the cells (contacting step B). As the fragmented extracellular matrix component, those described above can be used. The second contacting step may be performed by using the fragmented extracellular matrix component for part or all of the extracellular matrix component.

[0101] The contacting step, or after the contacting step and before the culturing step, may include adding fibrinogen and / or thrombin. Fibrinogen and thrombin may be added, for example, simultaneously, or one of them may be added first and then the other. In the contacting step, for example, a first liquid containing an extracellular matrix component, an aqueous medium, and fibrinogen may be mixed with a second liquid containing cells, an aqueous medium, and thrombin. By adding fibrinogen and / or thrombin, shrinkage that may occur in the culturing step described later is more easily suppressed, and the shape and size of the cell construct can be easily controlled. In addition, since the suspension of the cells and the extracellular matrix component can be gelled, it becomes easier to maintain a state in which each cell and the extracellular matrix component are uniformly mixed and in a state in which the cells and the extracellular matrix component are in proximity to each other.

[0102] The contacting step may include accumulating the cells and the extracellular matrix component after bringing the cells into contact with the extracellular matrix component. By accumulating these components, the distribution of the extracellular matrix component and the cells in the cell construct becomes more uniform. Examples of the method for accumulating the cells and the extracellular matrix component include a method of centrifuging a culture solution containing the extracellular matrix component and the cells, and a method of natural sedimentation.

[0103] (Culturing step) In the culturing step, cells in contact with extracellular matrix components are cultured. The culturing of cells in contact with the extracellular matrix is carried out under conditions suitable for culturing non-parenchymal liver cells. Conditions suitable for culturing non-parenchymal liver cells mean conditions under which cells other than hepatocytes (e.g., sinusoidal endothelial cells) are more likely to grow compared to hepatocytes. The culturing step may be carried out, for example, by culturing the above cells in a medium containing a medium for non-parenchymal liver cells, not containing a medium for hepatocytes, and containing a medium for non-parenchymal liver cells.

[0104] The medium used in the culturing step does not have to contain insulin and transferrin, which are proteins secreted from the liver. Examples of the medium used in the culturing step include media for vascular endothelial cells (e.g., EGM2 (manufactured by Lonza), EGM2-MV (Lonza), Endothelial Cell Growth Medium 2 (Promocell), Endothelial Cell Growth Medium MV 2 (Promocell), ECM (Sciencell)). The medium may be a medium supplemented with serum or a serum-free medium. The medium may also be a mixed medium obtained by mixing two types of media. For example, it may be a mixed medium obtained by mixing a medium for vascular endothelial cells and a medium for mesenchymal stem cell proliferation.

[0105] The culturing step may be carried out in the presence of an angiogenesis promoting factor. As the medium for culturing the above cells, a medium containing an angiogenesis promoting factor may be used. Examples of the angiogenesis promoting factor include vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF).

[0106] The culturing temperature in the culturing step may be, for example, 20°C to 40°C or 30°C to 37°C. The pH of the medium may be 6 to 8 or 7.2 to 7.4. The culturing time may be 1 day to 2 weeks or 1 week to 2 weeks.

[0107] The incubator (support) is not particularly limited and may be, for example, a dish, a well insert, a low-adhesion plate, a plate having a bottom shape such as U-shaped or V-shaped. The above cells may be cultured while adhered to the support, may be cultured without adhering to the support, or may be cultured after being detached from the support during the culture. When culturing the above cells without adhering them to the support, or when culturing them after detaching them from the support during the culture, it is preferable to use a plate having a bottom shape such as U-shaped or V-shaped that inhibits the adhesion of the cells to the support, or a low-adsorption plate.

[0108] The cell density in the medium in the culturing step can be appropriately determined according to the shape, thickness of the target cell structure, the size of the incubator, etc. For example, the cell density in the medium in the culturing step may be 1 to 10 8 cells / mL, and may be 10 3 to 10 7 cells / mL. Also, the cell density in the medium in the culturing step may be the same as the cell density in the aqueous medium in the contacting step.

[0109] After the above culturing step (hereinafter, also referred to as the "first culturing step". Also, the first contacting step is also referred to as the "first contacting step"), a step of further contacting the cells (second contacting step) and a step of culturing the cells (second culturing step) may be included. The above cells in the second contacting step and the second culturing step may be of the same type or different types from the cells used in the first contacting step and the first culturing step. By the second contacting step and the second culturing step, a cell structure having a two-layer structure can be produced. Further, by repeatedly including the contacting step and the culturing step, a cell structure having a plurality of layers can be produced, and a tissue closer to a more complex living body can also be produced.

[0110] According to the manufacturing method according to the present embodiment, a cell structure in which blood vessels are formed between cells and a cell structure having a hepatic sinusoid network between cells can be preferably manufactured.

[0111] [Use of cell structure] The cell construct according to this embodiment can be applied as a substitute for experimental animals, a transplantation material, etc. As a specific example, it can be applied to tissue reconstruction, pathological in vitro models, screening of pharmaceuticals (evaluation of drugs), assay screening of cosmetics, etc.

[0112] [Method for Evaluating Hepatotoxicity of Test Substance] Since the cell construct according to this embodiment is excellent in responsiveness to substances having hepatotoxicity, it can be suitably used as a tool for evaluating the presence or degree of hepatotoxicity of a test substance. By using the cell construct according to this embodiment, a more reliable evaluation of the hepatotoxicity of a test substance can be easily obtained. By using the method for evaluating hepatotoxicity of a test substance using the cell construct according to this embodiment, screening for substances having hepatotoxicity can also be performed.

[0113] The method for evaluating hepatotoxicity of a test substance according to this embodiment includes a culturing step of culturing the above-described cell construct in a state of being in contact with the test substance. The method for evaluating hepatotoxicity is not particularly limited. For example, the number of viable cells of the cell construct, ATP content, albumin production amount, other assay methods (MTT assay, MTS assay, etc.) capable of obtaining a value correlated with the number of living cells, quantification of the amount of bile acid uptake by hepatocytes, glutathione quantification, or using markers used clinically to evaluate the presence or absence of liver damage such as ALT and bilirubin, etc., can be appropriately selected according to the mechanism of toxicity to be focused on.

[0114] In the method for evaluating hepatotoxicity of a test substance according to this embodiment, for example, the presence or degree of hepatotoxicity may be evaluated using, as an index, the number of viable cells, albumin production amount, or adenosine triphosphate (ATP) content of the cell construct after the culturing step.

[0115] In the evaluation step, the presence or degree of hepatotoxicity may be evaluated using, as an index, the hepatic sinusoidal network (vascular network) of the cell construct after the culturing step. When using the hepatic sinusoidal network as an index, specifically, for example, the degree of fragmentation of the hepatic sinusoidal network and / or the total vascular length of the hepatic sinusoidal network may be used as an index.

[0116] In the culturing process, the contact between the cell construct and the test substance can be carried out, for example, by adding the test substance to the culture medium of the cell construct.

[0117] The test substance may be, for example, a drug suspected of having hepatotoxicity.

[0118] The test substance to be evaluated may be one type or two or more types. When evaluating two or more compounds as test substances, each compound may be evaluated by contacting it with the cell construct, or a plurality of compounds may be simultaneously contacted with the cell construct for evaluation.

[0119] The culturing process may be carried out by culturing the cell construct in a medium containing the test substance. The time for culturing the cell construct in the medium containing the test substance is not particularly limited, but may be, for example, 24 to 96 hours, 48 to 96 hours, or 48 to 72 hours. Within the limit of not significantly changing the culturing environment, hydrodynamic additions such as reflux can be applied as necessary.

[0120] A method for evaluating the presence or degree of hepatotoxicity using the number of viable cells of the cell construct after the culturing process as an index can be carried out, for example, by the following method.

[0121] When the number of viable hepatocytes in the cell construct is less (the survival rate is lower) compared to the case where the cell construct is cultured in the absence of the test substance, it is evaluated that the test substance is toxic to the hepatocytes contained in the cell construct, that is, there is hepatotoxicity. The greater the decrease in the survival rate of hepatocytes compared to the case in the absence of the test substance, the stronger the hepatotoxicity can be evaluated. On the other hand, when the number of viable hepatocytes is about the same or significantly more (the survival rate is about the same or higher) compared to the case of culturing in the absence of the test substance, it is evaluated that the test substance has no hepatotoxicity.

[0122] The viable cell count of hepatocytes can be evaluated using a signal correlated with the viable hepatocytes or their abundance. It is only necessary to be able to measure the viable cell count of hepatocytes at the time of evaluation, and it is not necessarily required to measure in a living state. For example, hepatocytes can be labeled to distinguish them from other cells, and the signal from the label can be used as an indicator for examination. For example, after fluorescently labeling hepatocytes, by determining the viability of the cells, the viable hepatocytes in the cell structure can be directly counted. At this time, image analysis technology can also be utilized. The determination of cell viability can be performed by known cell viability determination methods such as trypan blue staining and PI (Propidium Iodide) staining. The fluorescent labeling of hepatocytes can be performed by known methods such as an immunostaining method using an antibody against a substance specifically expressed on the cell surface of hepatocytes as a primary antibody and a fluorescent-labeled secondary antibody that specifically binds to the primary antibody. The determination of cell viability and the measurement of the viable cell count may be performed in the state of the cell structure, or may be performed in a state where the cell structure is disrupted to the single-cell level. For example, after disrupting the three-dimensional structure of the cell structure after labeling hepatocytes and dead cells, only the viable hepatocytes at the time of evaluation can be directly counted by FACS (fluorescence activated cell sorting) or the like using the label as an indicator.

[0123] The viable cell count of hepatocytes in the cell structure can also be measured over time by labeling the hepatocytes in the cell structure in a living state and detecting the signal from the label over time. The hepatocytes in the cell structure may be labeled after constructing the cell structure, or the hepatocytes may be labeled in advance before constructing the cell structure. In addition, when using hepatocytes that constantly express a fluorescent dye, the viable cell count of hepatocytes can also be evaluated by measuring the fluorescence intensity of the lysate obtained by lysing the cell structure with a microplate reader or the like.

[0124] A method for evaluating the presence or degree of hepatotoxicity using the albumin production amount of the cell structure as an indicator after the culture process can be performed, for example, by the following method.

[0125] When the amount of albumin produced in the cell construct is low (the ability to produce albumin is low) or the ATP content in the cell construct is low compared to the case of culturing in the absence of the test substance, the test substance is evaluated as having toxicity to the hepatocytes contained in the cell construct at that concentration, that is, having hepatotoxicity. The greater the decrease in the amount of albumin produced or the decrease in the ATP content compared to the case in the absence of the test substance, the stronger the hepatotoxicity can be evaluated. On the other hand, when the amount of albumin produced is comparable to or significantly higher (the ability to produce albumin is comparable to or higher) or the ATP content is comparable to or significantly higher compared to the case of culturing in the absence of the test substance, the test substance is evaluated as having no hepatotoxicity at that concentration.

[0126] The amount of albumin produced can be evaluated, for example, by measuring albumin in the culture supernatant using the ELISA method.

[0127] It is preferable that the hepatocytes contained in the cell construct share the genotype of at least one or more drug-metabolizing enzymes, and it is more preferable that the genotypes of all drug-metabolizing enzymes are common. Since hepatotoxicity tends to depend on drug-metabolizing enzymes, hepatotoxicity can be evaluated more accurately by using a cell construct containing hepatocytes with a homozygous genotype of drug-metabolizing enzymes.

[0128] A method for evaluating the presence or degree of hepatotoxicity using the hepatic sinusoidal network of the cell construct as an index after the culturing step can be carried out, for example, by a method including a step of quantifying the total vascular length of the hepatic sinusoidal network in the cell construct after the culturing step. As a method for quantifying the total vascular length of the hepatic sinusoidal network, it can be carried out, for example, by the method described in the examples below. Since the method for evaluating the hepatotoxicity of the test substance according to this embodiment can evaluate the hepatotoxicity using the hepatic sinusoidal network in the cell construct as an index, it is possible to evaluate the presence and degree of hepatotoxicity of the test substance with higher sensitivity. The method for evaluating the hepatotoxicity of the test substance according to this embodiment is particularly effective when evaluating a test substance whose hepatic sinusoidal network is presumed to be affected.

Examples

[0129] Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

[0130] To prepare a cell construct, the following cells and media were prepared. (Cells) Human hepatocytes: PXB cell from Phoenix Bio Human mesenchymal stem cells (MSC): Product model number PT-2501 manufactured by Lonza Liver sinusoidal endothelial cells (SEC): Product model number 5000 manufactured by Sciencell Hepatic stellate cells (Lx2): Product model number SCC064 manufactured by Merck Millipore (Media) Medium for mesenchymal stem cell proliferation: Product name MSCGM2, product code C28009, manufactured by Promocell Vascular endothelial medium 1: Product name ECM, product code 1001, manufactured by Sciencell Vascular endothelial medium 2: Product name EGM2-MV, product code CC-3202, manufactured by Lonza Medium for hepatocytes: Product name d-HCGM, product code PPC-M200, manufactured by Phoenix Bio

[0131] <Test Example 1: Preparation of Cell Construct 1> Heparin (manufactured by Sigma) was dissolved in 20 mM Tris-HCl buffer (pH 7.4) to obtain a heparin solution. Based on the total mass of the heparin solution, the heparin content was 1.0 mg / mL. Collagen (type I collagen, manufactured by Nippi) was dissolved in 5 mM acetic acid solution to obtain a collagen solution. Based on the total mass of the collagen solution, the collagen content was 0.4 mg / mL.

[0132] 100 μL of heparin solution and 100 μL of collagen solution were mixed in a microtube, and the mixture, PXB cells, SEC, Lx2, and, if necessary, MSCs were placed and suspended. The number of cells of each type was adjusted to the ratios shown in Table 1. The total number of cells per well was set to approximately 100,000 cells.

[0133] The obtained suspension was centrifuged at 25 °C and 400 × g for 1 minute. This formed a viscous substance with collagen and heparin attached to the cell surface. After stretching, the supernatant was removed, and 250 μL of a mixed medium (mass ratio 1:1) of medium 1 for vascular endothelium (ECM) and medium for mesenchymal stem cell proliferation (MSCGM2) was added into the microtube. The obtained suspension was seeded into a 96-well cell culture insert (Roche Inc, E-Plate Insert 16). After seeding, it was cultured in a CO2 incubator (37 °C, 5% CO2) for a predetermined period to obtain a cell construct.

[0134]

Table 1

[0135] Figures 1 to 3 are photographs showing the observation results of the cell constructs of Examples 1 to 3. Figure 1 shows the observation results of the sample of Example 1 immunostained with CD31 and albumin after being fixed on the 7th day from the start of culture. Figure 2 shows the observation results of the sample of Example 2 immunostained with CD31 and albumin after being fixed on the 8th day from the start of culture. Figure 3 shows the observation results of the sample of the Example immunostained with CD31 and albumin after being fixed on the 8th day from the start of culture. The cell constructs shown in Figures 1 to 3 had hepatic sinusoidal networks.

[0136] <Test Example 2: Preparation of Cell Construct 2> [Preparation of Dissolved Collagen Component] 100 mg of a type I collagen sponge fragment derived from porcine skin (manufactured by Nippon Ham Co., Ltd.) was heated at 200°C for 24 hours to obtain a collagen component (crosslinked collagen component) in which at least a part was crosslinked. Note that no significant change in appearance was confirmed in the collagen before and after heating at 200°C. 50 mg of the crosslinked collagen component was placed in a 15 mL tube, 5 mL of ultrapure water was added, and it was homogenized for 6 minutes using a homogenizer (AS ONE VH-10).

[0137] The aqueous solution containing the homogenized crosslinked collagen component was centrifuged at 10,000 rpm for 10 minutes under the condition of 21°C. The supernatant was aspirated, and the collagen pellet was mixed with 5 mL of new ultrapure water to prepare a collagen solution. While maintaining the tube containing the collagen solution on ice, it was sonicated at 100 V for 20 seconds using a sonicator (Sonics and Materials VC50). After taking out the sonicator, the tube containing the collagen solution was cooled on ice for 10 seconds, and this was repeated 100 times. After performing sonication 100 times, the collagen solution was filtered through a filter with a pore size of 40 μm to obtain a dispersion containing the defibrated collagen component (sCMF). The dispersion was freeze-dried by a conventional method to obtain the defibrated collagen component (sCMF) as a dried product. The average length of sCMF was 14.8 ± 8.2 μm (N = 20).

[0138] [Preparation of cell constructs] A dispersion A was obtained by dispersing the above defibrated collagen component in a medium (DMEM) containing serum in which 10 mg / mL of fibrinogen (manufactured by Sigma) was dissolved so that the concentration became 30 mg / mL. Also, a dispersion B was obtained by dispersing cells in a medium (DMEM) containing serum in which 10 U / mL of thrombin (manufactured by Sigma) was dissolved so that the total number of cells per well became 30,000 cells. A suspension obtained by mixing the obtained dispersion A (defibrated collagen component) and dispersion B at a ratio of 1:1 was added to a 48-well plate at 10 μL per well. The observation results of the cells and the culture containing the defibrated collagen component at the time point when 6 days had passed since the start of culture are shown in FIGS. 4 to 6.

[0139] As the culture medium, a mixed medium (mass ratio: 1:1) of endothelial cell medium 1 (ECM) and mesenchymal stem cell growth medium (MSCGM2), endothelial cell medium 1 (ECM), or endothelial cell medium 1 (ECM) and hepatocyte medium (d-HCGM) was used.

[0140]

Table 2

[0141] When a medium suitable for non-parenchymal liver cells was used, it was confirmed that fenestrated networks were formed in the cell structures (Examples 4 to 5). On the other hand, when the hepatocyte medium was used, hepatic fenestrated networks were not formed in the obtained cell structures (Comparative Example 1).

[0142] <Test Example 3: Preparation of Cell Structure 3> Into the microtube, a mixed solution of 100 μL of heparin solution and 100 μL of collagen solution, PXB cell, SEC and Lx2, and, if necessary, MSC were placed and suspended. The cell numbers of various cells were adjusted to the ratios shown in Table 3. The total cell number was set to about 30,000 cells.

[0143] The obtained suspension was centrifuged at 25 °C and 400 × g for 1 minute. Thereby, a viscous body with collagen and heparin attached to the cell surface was formed. After stretching, the supernatant was removed, and 250 μL of a mixed medium (mass ratio 1:1) of endothelial cell medium 1 (ECM) and mesenchymal stem cell growth medium (MSCGM2) was added into the microtube, and the obtained suspension was seeded into a 96-well cell culture insert (Roche Inc, E-PlateInsert 16). After seeding, it was cultured in a CO2 incubator (37 °C, 5% CO2) for a predetermined period to obtain cell structures.

[0144]

Table 3

[0145] As shown in FIGS. 7 to 8, it was confirmed that hepatic sinusoid networks were formed in the obtained cell constructs even when the ratio of the number of cells was different from that in Examples 1 to 5.

[0146] <Test Example 4: Preparation of Cell Construct 4> A cell construct of Example 8 was prepared in the same manner as Condition 1 of Test Example 1, except that the medium was changed to endothelial cell medium 2 (EGM2MV) and fixation was performed on the 7th day of the culture period. The results are shown in FIG. 9. It was also confirmed that hepatic sinusoid networks were formed in the cell constructs obtained using different media.

[0147] <Test Example 5: Measurement of Albumin Secretion Amount> In the cell construct prepared by the method of Example 1, the albumin secretion amount was measured by quantifying the albumin amount in the culture supernatant during the culture period by ELISA. The medium was changed on the day before the supernatant collection day, and the culture supernatant for 24 hours was used as a sample for ELISA measurement. As a negative control, a cell construct prepared in the same manner as Example 1 except that it did not contain PXB Cell was prepared.

[0148] The measurement results of the albumin secretion amount are shown in FIG. 10. At the time point 15 days after the start of the culture, significant maintenance of albumin secretion was confirmed compared to the negative control.

[0149] <Test Example 6: Evaluation of Hepatotoxicity> For the hepatotoxicity evaluation test, cell constructs prepared by the methods of Example 5 and Comparative Example 1 at the time point 7 days after the start of the culture were used as evaluation cell constructs. Nefazodone and Troglitazone, which are drugs known to have hepatotoxicity, were used for the evaluation of hepatotoxicity.

[0150] At the time point 7 days after the start of the culture, the medium was changed to a medium in which the drug (Nefazodone or Troglitazone) was dissolved. At the time point 10 days after the start of the culture, the medium was changed again to the medium in which the drug was dissolved, and the cell viability was quantified by ATP assay at the time point 14 days after the start of the culture. The evaluation results of hepatotoxicity are shown in FIGS. 11 to 12.

[0151] It was confirmed that a cell structure having a hepatic sinusoidal network is superior in responsiveness to a hepatotoxic substance compared to a cell structure not having a hepatic sinusoidal network.

[0152] <Test Example 7: Monocrotaline Administration Experiment> [Preparation of Liver Model (Hepatotoxicity Model)] (Recovery of Cells Other than PXB cells) The cryopreserved stocks of Lx2, SEC, and MSC cells were thawed, cultured without subculture based on the manufacturer's recommended protocol, and recovered from the culture flasks and dishes with trypsin based on the usual method. Then, the cell amounts were measured.

[0153] (Recovery of PXB cells) The cells were recovered by the following procedure, and the cell amount was measured. The PXB cells were washed with PBS. 2 mL of 0.25% trypsin-EDTA was added to the PXB cells and incubated in an incubator. Then, HCGM was added to the PXB cells and pipetting was performed to recover the PXB cells, which were counted with a cell counter.

[0154] PXB cells, SEC, and LX2 were mixed to obtain the following ratio and cell amount, thereby obtaining a cell mixture. ·Total cell amount per well: 30,000 cells ·Ratio of cells in the tissue: PXB cells: 65%, SEC: 25%, LX2: 10%

[0155] A heparin-collagen solution was prepared by mixing an equal volume of a 1.0 mg / mL heparin solution (buffer: 100 mM Tris-HCL) and a 0.3 mg / mL collagen solution (buffer: 5 mM acetate).

[0156] 100 μL of the heparin-collagen solution was added to the cell mixture, and the mixture was suspended until the cells were no longer visible, followed by centrifugation (400 g × 2 min) to form a viscous substance in the solution.

[0157] After removing the supernatant from the solution containing the viscous substance, a 20 U / mL thrombin solution (solvent: HCM (culture medium for hepatocytes)) was added so that the final volume of the solution became the solution volume of "the number of wells scheduled for seeding" × 2 μL, and a cell suspension was obtained.

[0158] A 10 mg / mL fibrinogen solution was placed on a 48-well plate to form droplets. The cell suspension was added inside the droplets, and then left standing in an incubator for 40 minutes to form a fibrin gel.

[0159] For each well in which a fibrin gel was formed, 0.5 mL of HCM (containing Endothelial Cell Growth Supplement) was added to obtain a liver model as a cell construct.

[0160] [Drug administration to the liver model] From the start of culturing the liver model, on the 1st day (Day1) and the 4th day (Day4), drug administration was carried out by replacing the medium of the liver model with a medium containing monocrotaline at a concentration of 2000 μM, 666 μM, 222 μM, or 74 μM. The compound used was previously dissolved at a high concentration in DMSO and stored. At the time of drug administration, DMSO was included in the medium at a concentration of 1%. Since 1% DMSO was included in each drug administration condition, a medium exchange with a medium containing only 1% DMSO was also carried out simultaneously as a negative control.

[0161] [ATP assay of the liver model] On the 7th day (Day7) after the start of culturing the liver model, an ATP assay was carried out using the CellTiter-Glo® 3D Cell Viability Assay kit as follows.

[0162] The medium containing the compound was removed from each well of the 48-well plate, 100 μL of DMEM at room temperature was added, and then 100 μL of the ATP assay reagent included in the kit was added at room temperature. The 48-well plate to which the ATP assay reagent was added was shaken for 5 minutes using a thermostatic shaker (1000 rpm, room temperature). Subsequently, the 48-well plate was allowed to stand at room temperature for 25 minutes.

[0163] The total volume of the mixture of DMEM and the ATP assay reagent was transferred from each well to a 96-well plate for luminescence measurement, and the luminescence intensity was measured using a plate reader.

[0164] [Fixation, Immunostaining, and Microscopic Imaging of Liver Models] For each sample administered with the drug, fixation treatment, permeabilization treatment, blocking, primary antibody treatment, secondary antibody treatment, imaging, area calculation, and evaluation were performed in this order. Hereinafter, the procedures from fixation treatment to evaluation are described.

[0165] (Fixation Treatment) Six days after the start of culturing the liver model (day6), the 48-well plate was taken out of the incubator, the medium was removed and washed with PBS, and then 300 μL of 4% paraformaldehyde-phosphate buffer (hereinafter, PFA) was added to each well to fix the liver model. Thereafter, the PFA was thoroughly washed away.

[0166] (Permeabilization Treatment and Blocking) 100 μL of 0.2 (v / v) % TRITON / 1 (w / v) % BSA PBS solution (hereinafter, BSA solution) was added into the insert in each well and allowed to stand at room temperature for 2 hours.

[0167] (Primary Antibody Treatment) The mouse-derived anti-CD31 antibody was diluted 100-fold with the BSA solution to obtain a primary antibody solution. 100 μL of the primary antibody solution was added into the insert in each well and allowed to stand at 4°C for 24 hours. Thereafter, the primary antibody solution was thoroughly washed away.

[0168] (Secondary Antibody Treatment) The secondary antibody was diluted 200-fold with BSA solution to obtain a secondary antibody solution. 100 μL of the secondary antibody solution was added into the insert of each well, and it was allowed to stand at room temperature for 1 hour in the dark. Then, the secondary antibody solution was thoroughly washed away, and 100 μL of PBS was added to each well.

[0169] (Fluorescence microscopy imaging) Imaging was performed using a confocal microscope. The imaging conditions were as follows. · Lens used: 4x lens · Imaging mode: non-confocal · Filter: 647 (Ex) / 512 (Em) · Z-axis position: 0 - 100 μm in 10-μm increments

[0170] The Intensity of the images taken by the above method was summed up to obtain fluorescence observation images of each liver model.

[0171] For each of the taken images, image analysis was performed using Image J according to the following steps (1) to (10). As a result, a skeleton image extracted from the original image was obtained. The area of the hepatic sinusoidal network (blood vessels) in the skeleton image was extracted by image outer product. (1) Image > Type > 8-bit (2) Process > Smooth (3) Process > Subtract Background > Sliding Paraboloid (20 pixels) (4) Process > Enhance contrast (Saturated pixels: 10.0%, Normalize) (5) Process > Math > Subtract (value: 100) (6) Plugins > Mexican hat filter (Radius: 5.0) (7) Plugins > Skelton > Skeletonize (2D / 3D) (8) Image > Adjust > Threshold (Threshold setting: 1 - 255) (9)Process > Analyze particle (Size: >250μm) (10)Analyze > Analyze Skeleton > Analyze Skeleton(2D / 3D)

[0172] Figure 13 shows the CD31 immunostaining results 7 days (Day7) after the start of culturing of the liver model administered with monocrotaline at a predetermined concentration. Figure 14 is a photograph of a portion of approximately 1 m at the approximate center of the liver model. As shown in Figures 13 to 14, it was observed that as the monocrotaline concentration increased, the hepatic sinusoidal network became fragmented. 2 As shown in Figures 13 to 14, it was observed that as the monocrotaline concentration increased, the hepatic sinusoidal network became fragmented.

[0173] Figure 15 shows the quantification results of the total vessel length of the hepatic sinusoidal network. Figure 16 shows the results of the ATP assay. As shown in Figures 15 to 16, when the hepatic sinusoidal network was quantified (when the amount of damage to the vascular network was quantified), the drug efficacy could be evaluated with higher sensitivity than the ATP assay.

[0174] <Test Example 8: Toxicity Evaluation Experiment of 18 Compounds> The toxicity evaluation of the compounds was carried out using a cell structure having a hepatic sinusoidal network prepared using CMF (Example A) and a cell structure having a hepatic sinusoidal network prepared using a heparin and collagen solution (Example B). The toxicity evaluation was performed by administering the compound and conducting an ATP assay in the same procedure as in Test Example 6 for the compound to be evaluated.

[0175] The cell structure of Example A was obtained by the method described in Test Example 6.

[0176] The cell structure of Example B was obtained by the method described in Test Example 7.

[0177] The toxicity evaluation was carried out based on the IC50 and MOS value. The MOS value is an index used for toxicity evaluation as a value considering the maximum concentration in blood (Cmax).

[0178] Reference Example A and Reference Example B are literature values based on Proctor WR et al., Arch Toxicol, 2017, 91, 2849-2863 and the Supplementary information of the literature. Reference Example A is 2D PHH (Two-dimensional primary human hepatocytes) in the above literature, and Reference Example B is 3D hLiMT (3D human liver microtissues) in the above literature. The IC50 values of Reference Examples A and B are also shown in Tables 4 to 5. It was shown that the cell constructs produced in this test example can reproducibly evaluate those reported to be toxic.

[0179] The following table also shows the drug-induced liver injury severity category (DILI severity category, LKBT). In LKBT, III indicates high concern for DILI (most DILI concern), II indicates low concern for DILI (less DILI concern), and I indicates no concern for DILI.

[0180] [Table 4] [Table 5]

Claims

1. The method comprises the steps of: (a) providing a method for producing a blood vessel comprising the steps of: (a) providing a blood vessel having a vascular endothelial cell; the extracellular matrix components are disposed between the cells; A hepatic sinusoidal network is present between the cells, a cell aggregate containing the cells is encapsulated in the fibrin; The cell structure, wherein the hepatocyte is at least one selected from the group consisting of primary hepatocytes, cultured cells of the primary hepatocytes, and cultured cell lines established from the primary hepatocytes.

2. The cell structure according to claim 1 , wherein the ratio of the number of said hepatocytes to the total number of said cells is 60% or more and 80% or less.

3. The cell structure according to claim 1 or 2, wherein the ratio of the number of vascular endothelial cells to the total number of cells is 5% or more and 35% or less.

4. The cell structure according to any one of claims 1 to 3, further comprising a polyelectrolyte.

5. The cell structure of claim 4, wherein the polyelectrolyte is heparin.

6. The cell structure according to any one of claims 1 to 5, wherein the extracellular matrix component is fibrous.

7. The cell structure according to any one of claims 1 to 6, wherein the extracellular matrix component comprises a collagen component.

8. The cell structure according to any one of claims 1 to 7, wherein the extracellular matrix component comprises a fragmented extracellular matrix component.

9. The cell structure according to any one of claims 1 to 8, wherein the vascular endothelial cells are sinusoidal endothelial cells.

10. The cell structure according to any one of claims 1 to 9, wherein the cells further comprise hepatic stellate cells.

11. The method comprises a culturing step of culturing the cell structure according to any one of claims 1 to 10 in contact with a test substance, A method for evaluating the hepatotoxicity of a test substance, comprising evaluating the presence or absence or the degree of hepatotoxicity using as indicators the number of surviving cells, the amount of albumin produced, or the adenosine triphosphate content of the cell structure after the culture step.

12. The method comprises a culturing step of culturing the cell structure according to any one of claims 1 to 10 in contact with a test substance, A method for evaluating the hepatotoxicity of a test substance, comprising using the hepatic sinusoidal network of the cell structure after the culture step as an indicator to evaluate the presence or absence or the degree of hepatotoxicity.

13. The method for evaluating the hepatotoxicity of a test substance according to claim 11 or 12, wherein the culturing step is carried out by culturing the cell structure in a medium containing the test substance.

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