Method for producing cell aggregates

Abstract

To provide a method for producing a cell aggregate that can be used as a liver model in a state more similar to that in vivo.SOLUTION: The present invention relates to a method for producing a cell aggregate having a bile canaliculus, the method including: a contact step for bringing heparin and cells, including hepatocytes, into contact with each other in an aqueous medium; and a culturing step for culturing the cells that have been brought into contact with heparin to form a cell aggregate.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing cell aggregates. [Background technology]

[0002] The liver is an organ where toxicity is likely to manifest during the drug development process, and drug-induced liver injury (DILI) is a major reason for the discontinuation of drug development and sales. Compounds that have the potential to cause DILI (hereinafter referred to as DILI compounds) are not necessarily easy to identify in animal experiments, and if their damaging properties are discovered after clinical trials or drug release, significant losses will be incurred. Therefore, an evaluation system that can predict DILI compounds in advance is needed. Artificial structures that mimic biological tissues are useful for evaluation systems that can predict DILI compounds in advance.

[0003] Known techniques for artificially producing structures that mimic biological tissues include, for example, a method for producing a three-dimensional tissue by culturing coated cells whose entire surface is covered with an adhesive film (Patent Document 1), and a method for producing a three-dimensional cellular tissue (Patent Document 2), which includes mixing cells with a cationic substance and an extracellular matrix component to obtain a mixture, collecting the cells from the mixture, and forming a cell aggregate on a substrate. The present inventors have also proposed a method for producing a three-dimensional tissue with a high collagen concentration by contacting cells with endogenous collagen, and preferably further contacting them with fibrous exogenous collagen (Patent Document 3). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2012-115254 [Patent Document 2] International Publication No. 2017 / 146124 [Patent Document 3] International Publication No. 2018 / 143286 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a method for producing a cell aggregate that can be used as a liver model that is closer to the in vivo state. [Means for solving the problem]

[0006] That is, the present invention relates to, for example, the following inventions. [1] 1. A method for producing a cell aggregate having bile canaliculi, comprising: a contacting step of contacting cells including hepatocytes with heparin in an aqueous medium; and culturing the cells contacted with the heparin to form the cell aggregates. [2] The method according to [1], wherein the hepatocytes are mature hepatocytes. [3] The method according to [1] or [2], wherein the heparin concentration when contacted with the cells in the aqueous medium is 0.5 mg / mL or more. [4] The method according to any one of [1] to [3], wherein the ratio of the number of the hepatocytes to the total number of the cells is 65% or more. [5] The method according to any one of [1] to [4], wherein in the contacting step, the cells are further contacted with an extracellular matrix component. [6] The method according to [5], wherein the extracellular matrix component is a collagen component. [7] The method according to any one of [1] to [6], wherein the expression intensity ratio of MRP2 represented by the following formula (1) is 120% or more. MRP2 expression intensity ratio = X / Y × 100 (1) [In formula (1), X represents the expression intensity of MRP2 in the cell aggregates formed in the culture step, Y indicates the expression intensity of control cell aggregates formed by culturing cells including hepatocytes without contact with heparin and extracellular matrix components.] [Effects of the Invention]

[0007] According to the present invention, a method for producing a cell aggregate that can be used as a liver model that is closer to the in vivo state can be provided. [Brief explanation of the drawings]

[0008] [Figure 1] 1 is a micrograph showing the results of observing collagen and heparin deposited on cells. [Figure 2] 1 shows micrographs showing the results of observing the aggregation tendency of cells treated with collagen (col) and heparin (hep). [Figure 3] 10 shows micrographs showing the expression status of MRP2 in cell aggregates after 3 days of culture. [Figure 4] 10 shows micrographs showing the results of observing the expression of MRP2 in cell aggregates after 3 days of culture. [Figure 5] 1 is a graph showing the results of image analysis of the expression intensity ratio of MRP2. [Figure 6] 1 shows micrographs showing the results of a bile acid assay, where (a) is bright field, (b) is fluorescent, and (c) is a composite of (a) and (b). [Figure 7] 1 shows micrographs showing the results of a bile acid assay, where (a) is bright field, (b) is fluorescent, and (c) is a composite of (a) and (b). [Figure 8] 1 shows micrographs showing the results of a bile acid assay, where (a) is bright field, (b) is fluorescent, and (c) is a composite of (a) and (b). [Figure 9] 1 shows micrographs showing the results of a bile acid assay, where (a) is bright field, (b) is fluorescent, and (c) is a composite of (a) and (b). DETAILED DESCRIPTION OF THE INVENTION

[0009] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0010] [Cell aggregate] The method according to this embodiment is a method for producing a cell aggregate having bile canaliculi, and includes a contact step in which cells including hepatocytes are contacted with heparin in an aqueous medium, and a culture step in which the cells contacted with heparin are cultured to form a cell aggregate.

[0011] The cell aggregates obtained by the method of this embodiment can be used as a liver model (hepatoid tissue), which is a biological tissue model having functions similar to at least a portion of the function of the liver and / or a structure similar to at least a portion of the structure of the liver.

[0012] As used herein, the term "cell aggregate" refers to a collection of cells (a mass of cells) that is artificially produced by cell culture. The cell aggregate may be a structure in which cells are arranged three-dimensionally (a three-dimensional tissue), or a structure in which cells are arranged two-dimensionally.

[0013] The shape of the cell aggregate is not particularly limited, and examples thereof include sheet-like, spherical, approximately spherical, ellipsoidal, approximately ellipsoidal, hemispherical, approximately hemispherical, semicircular, approximately semicircular, rectangular, and approximately rectangular. Here, biological tissue includes sweat glands, lymphatic vessels, sebaceous glands, and the like, and has a more complex structure than cell aggregates. Therefore, cell aggregates and biological tissues can be easily distinguished. Furthermore, cell aggregates may be aggregates in a mass adhered to a support, or may be aggregates in a mass not adhered to a support.

[0014] (cell) The cells may be somatic cells or germ cells. Furthermore, the cells may be stem cells or cultured cells such as primary cultured cells, subcultured cells, and cell line cells. As used herein, "stem cells" refer to cells with self-renewal and pluripotency. Stem cells include pluripotent stem cells, which have the ability to differentiate into any cell type, and tissue stem cells (also called somatic stem cells), which have the ability to differentiate into specific cell types. 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.

[0015] In the cell aggregate of this embodiment, the cells include hepatocytes. Hepatocytes, also known as hepatocyte parenchymal cells, are cells that have functions such as secreting bile and plasma proteins. The hepatocytes may be mature hepatocytes or other liver cells. Mature hepatocytes may be, for example, primary hepatocytes collected from an animal liver, cultured primary hepatocytes, or cultured cell lines established from primary hepatocytes. Other hepatocytes may be, for example, hepatoblasts artificially differentiated from stem cells. Examples of primary hepatocytes include primary human hepatocytes such as PXB cells. Examples of cultured cell lines include cell lines derived from inactivated hepatoma 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 contained in the cell aggregate of this embodiment are preferably non-cancerous cells, such as primary hepatocytes and hepatoblasts. The hepatocytes may be mature hepatocytes, and PXB cells are more preferred because of their ease of handling.

[0016] The hepatocytes contained in the cell aggregate may be of one type or two or more types. For example, the cell aggregate may contain a plurality of hepatocytes having different genotypes for proteins involved in liver function. Conversely, all the hepatocytes contained in the cell aggregate may have the same genotype for proteins involved in liver function. Examples of proteins involved in liver function include drug-metabolizing enzymes.

[0017] The total number of cells constituting the cell aggregate in this embodiment is not particularly limited and is determined appropriately taking into consideration the thickness and shape of the cell aggregate to be constructed, the size of the cell culture vessel to be used for construction, etc.

[0018] The ratio of the number of hepatocytes to the total number of cells in the cell aggregate (number of hepatocytes / total number of cells × 100) 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 enabling the formation of a liver model that is closer to the in vivo state, the ratio of the number of hepatocytes to the total number of cells in the cell aggregate may be 60% or more and 80% or less, or 60% or more and 70% or less.

[0019] The cells may further include vascular endothelial cells, from the viewpoint of enabling the formation of a liver model that is closer to the in vivo state. Vascular endothelial cells refer to flat cells that constitute the surface of the vascular lumen. Vascular endothelial cells may be, for example, sinusoidal endothelial cells or human umbilical vein-derived vascular endothelial cells (HUVECs). Sinusoidal endothelial cells are non-parenchymal hepatic cells (cells that constitute the liver other than hepatocytes) and have a characteristic morphology that differs from other vascular endothelial cells, such as a collection of numerous small pores (cribriform structure) in the cytoplasm and the lack of a basement membrane. The vascular endothelial cells that constitute the cell aggregates may be primary cells (primary vascular endothelial cells) collected from the liver of an animal (e.g., human), cells cultured from primary cells, a cultured cell line 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 number 5000 manufactured by Sciencell. An example of the cultured cell line is the cultured cell line manufactured by Applied Biological Materials, product number T0056. 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 aggregate according to this embodiment may be non-cancerous cells.

[0020] The ratio of the number of vascular endothelial cells to the total number of cells in the cell aggregate (number of vascular endothelial cells / total number of cells × 100) may be 5% or more, 10% or more, 15% or more, 20% or more, or 25% or more, or may be 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, or 25% or less. From the viewpoint of enabling the formation of a liver model that is closer to the in vivo state, the ratio of the number of vascular endothelial cells to the total number of cells in the cell aggregate may be 5% or more and 40% or less, 5% or more and 35% or less, 10% or more and 35% or less, or 10% or more and 30% or less. The ratio of sinusoidal endothelial cells to the total number of cells in the cell aggregate may be within the above range.

[0021] In the cell aggregate according to this embodiment, the cells may further include hepatic stellate cells, from the viewpoint of enabling the formation of a liver model that is closer to the in vivo state. Hepatic stellate cells are non-parenchymal hepatic cells (cells other than hepatocytes among the cells that make up the liver). They have functions such as storing vitamin A and are present in the space of Disse, which is the region between hepatocytes and sinusoids in the liver. Hepatic stellate cells may be, for example, primary cells (primary hepatic stellate cells) collected from the liver of an animal (e.g., human), cells obtained by culturing primary cells, a cultured cell line established from primary cells, or cells artificially differentiated from stem cells. An example of primary hepatic stellate cells is primary hepatic stellate cells manufactured by Sciencell, model number 5300. An example of a cultured cell line is a cultured cell line such as LX-2. Examples of stem cells to be differentiated include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and mesenchymal stem cells. The hepatic stellate cells contained in the cell aggregate according to this embodiment may be non-cancerous cells.

[0022] The ratio of the number of hepatic stellate cells to the total number of cells in the cell aggregate (number of hepatic stellate cells / total number of cells × 100) may be 1% or more, 2% or more, 3% or more, 4% or more, or 5% or more, or 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 enabling the formation of a liver model that is closer to the in vivo state, the ratio of the number of hepatic stellate cells to the total number of cells in the cell aggregate may be 1% or more and 15% or less, or 3% or more and 12% or less.

[0023] In this embodiment, the cells may include cells other than 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 nerve cells, dendritic cells, immune cells, lymphatic endothelial cells, fibroblasts, epithelial cells (excluding hepatocytes), cardiac myocytes, pancreatic islet cells, smooth muscle cells, bone cells, alveolar epithelial cells, and spleen cells. Examples of stem cells include ES cells, iPS cells, and mesenchymal stem cells. The other cells may be normal cells or cells with enhanced or suppressed cell functions, such as cancer cells. "Cancer cells" are cells that are derived from somatic cells and have the ability to proliferate indefinitely.

[0024] The origin of the hepatocytes, vascular endothelial cells, hepatic stellate cells, or other cells contained therein is not particularly limited, but may be, for example, cells derived from mammals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, and rats.

[0025] The cells in the cell aggregate according to this embodiment may not include cells induced to differentiate from induced pluripotent stem cells (iPS cells). That is, the cells used in the contacting step may be mature cells. When the cells in the cell aggregate according to this embodiment do not include cells induced to differentiate from iPS cells, it becomes easier to grasp the degree of differentiation from iPS cells, the proportion of differentiated cells relative to the total cells, and the like, and as a result, it becomes even easier to grasp the content of each cell constituting the cell aggregate.

[0026] In the contacting step, cells including hepatocytes are contacted with heparin in an aqueous medium. Methods for contacting heparin with cells include, for example, suspending cells in a solution containing heparin, adding heparin or a solution containing heparin to a cell-containing liquid containing cells, or adding heparin or a solution containing heparin and cells or a cell-containing liquid containing cells to a previously prepared aqueous medium. The added heparin may be in the form of a salt.

[0027] The heparin concentration when contacting cells in an aqueous medium may be 0.1 mg / mL or more, 0.2 mg / mL or more, 0.3 mg / mL or more, 0.4 mg / mL or more, or 0.5 mg / mL or more, based on the total volume of the aqueous medium. The heparin concentration when contacting cells in an aqueous medium may be 12.0 mg / mL or less, 10.0 mg / mL or less, 8.0 mg / mL or less, 6.0 mg / mL or less, 5.0 mg / mL or less, 3.0 mg / mL or less, 1.0 mg / mL or less, 0.8 mg / mL or less, or 0.6 mg / mL or less, based on the total volume of the aqueous medium. When the heparin concentration when contacting cells in an aqueous medium is within the above-mentioned range, a liver model that is more similar to a living body can be formed.

[0028] The amount of heparin in the contacting step may be, for example, 0.001 mg or more, or 0.01 mg or more, and 1 mg or less, or 0.1 mg or less, per 300,000 cells (the number of cells to be contacted with heparin).

[0029] The origin of heparin is not particularly limited, but it may be derived from mammals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, and rats.

[0030] The term "aqueous medium" refers to a liquid medium containing water as an essential component. The aqueous medium may be an aqueous medium containing a cationic substance. Examples of aqueous media containing a cationic substance include cationic buffers such as Tris-hydrochloric acid buffer, Tris-maleic acid buffer, Bis-Tris buffer, and HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). The aqueous medium containing a cationic substance may be a medium containing water and a cationic compound such as ethanolamine, diethanolamine, triethanolamine, polyvinylamine, polyallylamine, polylysine, polyhistidine, or polyarginine. A culture medium can also be used as the aqueous medium. Examples of culture media include liquid media such as Dulbecco's Modified Eagle Medium (DMEM) and vascular endothelial cell-specific medium (EGM2). The liquid medium may also be a mixed medium obtained by mixing two types of media.

[0031] The concentration and pH of the cationic substance (e.g., Tris in Tris-HCl buffer) in the aqueous medium containing the cationic substance are not particularly limited, as long as they do not adversely affect cell growth and the formation of cell aggregates. For example, the concentration of the cationic substance may be 10 to 200 mM, 30 to 150 mM, or 50 to 120 mM, based on the total amount of the aqueous medium containing the cationic substance. The pH of the aqueous medium (e.g., cationic buffer) may be 6.0 to 8.0, 6.8 to 7.8, 6.8 to 7.6, or 7.2 to 7.6.

[0032] In the contacting step, the cells may be further contacted with an extracellular matrix component. When the cells are further contacted with an extracellular matrix component, the cells are more likely to aggregate. Furthermore, when the cells are further contacted with an extracellular matrix component, the cells are more likely to form cell aggregates arranged in three dimensions. In addition, when the cells are further contacted with an extracellular matrix component (particularly when a collagen component is contacted), the expression intensity ratio of MRP2, which will be described later, tends to be higher, making it easier to obtain a liver model that is more similar to the in vivo state. Furthermore, when the cells are further contacted with an extracellular matrix component in the contacting step, it is easier to obtain a thicker structure (three-dimensional structure).

[0033] Examples of methods for further contacting cells with extracellular matrix components include suspending cells in a solution containing extracellular matrix components and heparin, adding a solution containing extracellular matrix components and heparin to a cell-containing solution containing cells, and adding heparin or a solution containing heparin, cells or a cell-containing solution, and extracellular matrix components or a solution containing extracellular matrix components to a previously prepared aqueous medium.

[0034] As used herein, the term "extracellular matrix component" refers to an assembly of extracellular matrix molecules formed by multiple extracellular matrix molecules. Extracellular matrix refers to a substance present outside cells in an organism. Any substance can be used as the extracellular matrix as long as it does not adversely affect cell growth and cell aggregate formation. 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, it further facilitates cell aggregation. When the extracellular matrix component is a collagen component, the collagen component functions as a scaffold for cell adhesion, further promoting the formation of three-dimensional cell aggregates. In this embodiment, the extracellular matrix component is preferably a substance present outside animal cells, i.e., an animal extracellular matrix component. The extracellular matrix molecule may be a modified or variant of the above-mentioned extracellular matrix molecule, or may be a polypeptide such as a chemically synthesized peptide, as long as it does not adversely affect cell growth and cell aggregate formation.

[0035] The extracellular matrix component may have repeats of a Gly-XY sequence, which is characteristic of collagen. Here, Gly represents a glycine residue, and X and Y each independently represent any amino acid residue. Multiple Gly-XY residues may be the same or different. Having repeats of a Gly-XY sequence reduces constraints on the molecular chain arrangement, resulting in improved scaffold function. In an extracellular matrix component having repeats of a Gly-XY sequence, the proportion of Gly-XY sequences in the total amino acid sequence may be 80% or more, preferably 95% or more. The extracellular matrix component may also have an RGD sequence. The RGD sequence refers to a sequence represented by Arg-Gly-Asp (arginine residue-glycine residue-aspartic acid residue). When an extracellular matrix component has an RGD sequence, cell adhesion is further promoted, making it even more suitable as a scaffold. Extracellular matrix components containing a sequence represented by Gly-XY and an RGD sequence include collagen, fibronectin, vitronectin, laminin, cadherin, and the like.

[0036] The concentration of the extracellular matrix component when contacted with cells in an aqueous medium may be 0.001 mg / mL or more, 0.01 mg / mL or more, 0.025 mg / mL or more, 0.05 mg / mL or more, 0.1 mg / mL or more, 0.2 mg / mL or more, 0.3 mg / mL or more, 0.4 mg / mL or more, or 0.5 mg / mL or more, based on the total volume of the aqueous medium. The concentration of the extracellular matrix component when contacted with cells in an aqueous medium may be, for example, 10.0 mg / mL or less, 8.0 mg / mL or less, 6.0 mg / mL or less, 5.0 mg / mL or less, 3.0 mg / mL or less, 1.0 mg / mL or less, 0.8 mg / mL or less, or 0.6 mg / mL or less, based on the total volume of the aqueous medium.

[0037] The amount of extracellular matrix components in the contacting step may be 0.001 mg or more, or 0.01 mg or more, and 1 mg or less, or 0.1 mg or less, per 300,000 cells (the number of cells when contacted with the extracellular matrix components).

[0038] The order in which the cells are contacted with the components is not particularly limited. For example, some of the cells may be contacted with the components, and then the remaining cells may be contacted with the components, or all of the cells may be contacted with the components simultaneously or approximately simultaneously.

[0039] The aqueous medium may be mixed by stirring or the like after the addition of each of the above components, or may not be mixed. The contacting step may include incubating for a certain period of time after contacting each of the above components with the cells. Examples of the certain period of time include 1 minute, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, or 3 hours. The incubation time may be, for example, 1 minute to 3 hours, or 1 minute to 15 minutes.

[0040] The contacting step may include adding fibrinogen and thrombin to the culture medium after the contacting step or after the culturing step. For example, fibrinogen and thrombin may be added simultaneously, or one may be added first and then the other. In the contacting step, for example, a fibrinogen-containing solution and a thrombin-containing solution may be mixed simultaneously or separately with a cell-containing material containing cells and heparin. For example, fibrinogen and thrombin may be added by using equal or approximately equal amounts of a 20 U / mL thrombin solution and a 10 mg / mL fibrinogen solution.

[0041] The addition of fibrinogen and thrombin makes it easier to suppress shrinkage that may occur during the culture process described below, making it easier to control the shape and size of the cell aggregates. Furthermore, by gelling the cell suspension, it becomes easier to maintain a uniform mixture of the cells and the above-mentioned components, and to maintain the cells and the above-mentioned components in close proximity. When fibrinogen and thrombin are added during the contacting process, or after the contacting process and before the culture process, cell dispersion in the medium during cell seeding is further suppressed, making it possible to form cell aggregates with a smaller amount of cells.

[0042] After contacting the cells with the above components, the method may include concentrating the cells and the above components (heparin and / or extracellular matrix components), removing the aqueous components, and adding an aqueous medium to the aggregate of cells and the above components obtained by the concentrating to obtain a cell suspension again. The cell suspension obtained in this manner may be subjected to the culture step described below. An example of a method for concentrating the aqueous medium is centrifuging the entire aqueous medium. The aggregate of cells and the above components may be, for example, a viscous body. An example of an aqueous medium for resuspension is a culture medium. By forming an aggregate of cells and the above components, the cells can be surrounded by the above components (i.e., buried). By resuspending the aggregate of cells and the above components in the medium, the cells can be transferred to the culture step while still in a state of being somewhat buried. By resuspending the aggregate of cells and the above components, the viscosity is sufficiently reduced, making it easier to handle using a pipette, etc.

[0043] In the culturing step, the cells contacted with heparin are cultured to form cell aggregates having bile canaliculi. The cell culture is carried out under conditions that allow the cells to grow. When cells contacted with an extracellular matrix component in addition to heparin are cultured, cell aggregates can be formed in which the extracellular matrix component is arranged between at least some of the cells in the cell aggregates.

[0044] The medium used in the culturing step may not contain insulin or transferrin, which are proteins secreted from the liver. Examples of the medium used in the culturing step include vascular endothelial medium (e.g., EGM2 (manufactured by Lonza), EGM2-MV (manufactured by Lonza), Endothelial Cell Growth Medium 2 (manufactured by Promocell), Endothelial Cell Growth Medium MV 2 (manufactured by Promocell), and ECM (manufactured by Sciencell). The medium may be a serum-supplemented medium or a serum-free medium. The medium may also be a mixed medium obtained by mixing two types of medium. For example, the medium may be a mixed medium obtained by mixing a vascular endothelial medium and a medium for growing mesenchymal stem cells.

[0045] The culture temperature in the culture 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 culture time may be one day or more, or one day to two weeks, or one week to two weeks.

[0046] The culture vessel (support) is not particularly limited and may be, for example, a dish, a well insert, a low-adhesion plate, or a plate with a U-shaped or V-shaped bottom. The cells may be cultured while attached to the support, or may be cultured without being attached to the support, or may be detached from the support during the culture process and then cultured. When culturing the cells without being attached to the support, or when culturing the cells after being detached from the support during the culture process, it is preferable to use a plate with a U-shaped or V-shaped bottom that inhibits cell adhesion to the support, or a low-adhesion plate.

[0047] The cell density in the medium in the culture step can be appropriately determined depending on the shape and thickness of the target cell aggregate, the size of the culture vessel, etc. For example, the cell density in the medium in the culture step can be set to 1 to 10 8 cells / mL, and 3 ~10 7The 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.

[0048] After the above-mentioned culture step (hereinafter also referred to as the "first culture step"; the initial contact step is also referred to as the "first contact step"), a step of contacting the cells (second contact step) and a step of culturing the cells (second culture step) may be included. The cells in the second contact step and second culture step may be of the same species as the cells used in the first contact step and first culture step, or may be of a different species. A two-layer structured cell aggregate can be produced by the second contact step and second culture step. Furthermore, by repeatedly including the contact step and culture step, a multi-layer cell aggregate can be produced, and more complex tissues closer to those of a living organism can also be produced.

[0049] The cell aggregate according to this embodiment has bile canaliculi. Bile canaliculi are spaces located between cells, including hepatocytes, particularly spaces located between contacting hepatocytes. It is known that bile acids taken up by hepatocytes in vivo are excreted into the bile canaliculi. Whether a cell aggregate has bile canaliculi can be confirmed, for example, by the expression of MRP2 (multidrug resistance-associated protein 2), a transporter on the bile canaliculi. The expression of MRP2 can be confirmed by the method described in the Examples below. Whether a cell aggregate has bile canaliculi can also be confirmed, for example, by contacting the cell aggregate with optically detectable labeled bile acids and detecting bile acids retained within the cells or bile canaliculi. The retention of bile acids within the cells or bile canaliculi can be confirmed by the method described in the Examples below.

[0050] The cell aggregate according to this embodiment may have an MRP2 expression intensity ratio represented by the following formula (1) of more than 100%. MRP2 expression intensity ratio = X / Y × 100 (1)

[0051] In formula (1), X represents the expression intensity of MRP2 in the cell aggregates formed in the culture step, and Y represents the expression intensity of a control cell aggregate formed by culturing cells, including hepatocytes, without contacting them with heparin and extracellular matrix components. When the cells are not contacted with an extracellular matrix component in the contact step, the control cell aggregate is a cell aggregate produced under the same conditions as the cell aggregates (test cell aggregates) formed in the culture step, except that heparin is not contacted with the cells. When the cells are contacted with an extracellular matrix component in the contact step, the control cell aggregate is a cell aggregate produced under the same conditions as the test cell aggregate, except that heparin is not contacted with the cells and extracellular matrix components are not contacted with the cells. Specific conditions may be as described in the Examples below. The culture period for the test cell aggregates and the control cell aggregates may be one day or more, or three days or more, or may be three days.

[0052] The MRP2 expression intensity ratio of the cell aggregate according to this embodiment may be 105% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, or 180% or more. The MRP2 expression intensity ratio of the cell aggregate according to this embodiment may be, for example, 300% or less, or 250% or less.

[0053] The cell aggregate according to this embodiment can be suitably used as a model for evaluating the hepatotoxicity of drugs, tissue for transplantation into non-human animals, and the like. [Example]

[0054] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to these examples.

[0055] 1. Experimental Procedure [Materials, supplies and equipment used] The following cultured cells, materials, supplies, and equipment were prepared. (cultured cells) [Table 1]

[0056] (Reagents and consumables) [Table 2]

[0057] (1) Cell counting device ·Invitrogen Countess(R) II FL (2) Microscope Yokogawa Electric CQ1 Confocal Microscope (3) Other Image analysis software: Image J (Fiji) Individual plugin: Mexican hat filter

[0058] <Test Example 1: Confirmation of the aggregation behavior of cells treated with heparin and collagen> The aggregation behavior of cells was examined when cells were treated with heparin and collagen, either alone or simultaneously.

[0059] [Treatment of cells with heparin and collagen] The cells used were PXB cells, SEC, and LX2.

[0060] Process 1-1 (Recovery of cells other than PXB cells) Frozen stocks of Lx2 and SEC cells were put to sleep and cultured without passage according to the manufacturer's recommended protocol. After recovering the cells from the culture flasks and petri dishes using trypsin according to standard methods, the cell mass of each cell was measured.

[0061] Process 1-2 (Recovery of PXB cells) The cells were recovered and the cell mass was measured according to the following procedure. PXB cells were washed with PBS, 0.25% trypsin-EDTA was added to the washed PXB cells, and the cells were incubated in an incubator. The number of collected PXB cells was measured using a cell counter.

[0062] Process 1-3 The collected cells were mixed at the following ratios and cell amounts to obtain a cell mixture. Total cell count: 300,000 cells ·Tissue cell ratio: ·PXB cell:65% ·SEC:25% ·LX2:10%

[0063] Process 1-4 100 μL of each of the following solutions was added to the cell mixture at room temperature, and the mixture was suspended until the cells were no longer visible. The mixture was then centrifuged (400 g x 2 min) to obtain a viscous mass. It was confirmed in advance that the pH of each solution was approximately 7.4, regardless of the presence or absence of heparin and collagen. (1)100mM Tris-HCl buffer (2) Collagen-containing solution (0.2 mg / mL collagen in 100 mM Tris-HCl buffer*) (3) Collagen and heparin-containing solution (0.2 mg / mL collagen and 0.5 mg / mL heparin in 100 mM Tris-HCl buffer) *Since collagen gels quickly in a neutral solution at room temperature, equal amounts of pre-prepared 0.4 mg / mL collagen in 5 mM acetate buffer and 200 mM Tris-HCl buffer were mixed with each of the above solutions.

[0064] Process 1-5 After removing the supernatant from the solution containing the viscous bodies, HCM was added to bring the final volume of the solution to 100 μL to obtain a cell suspension.

[0065] Process 1-6 The cell suspension was seeded at 10 μL / well into a 96-well microplate to which 190 μL of HCM had been added in advance, and suspended in the wells.

[0066] [Live cell observation using confocal microscope] A 96-well microplate containing seeded and suspended cells was placed inside a confocal microscope with the temperature inside the device set to 37°C and 5% CO2 supplied, and the aggregation state was observed over time from 30 minutes to 6 hours after seeding.

[0067] <Test Example 2: Organization of cells treated with heparin and collagen in fibrin gel> The hepatotoxicity models treated with each solution were evaluated to see if there were any differences in the state of adhesion between hepatocytes.

[0068] [Construction and culture of hepatotoxicity model] Process 2-1 A cell mixture having the following ratio and cell amount was obtained by the same procedure as steps 1-1 to 1-3 of Test Example 1. Total cell count: 300,000 cells ·Tissue cell ratio: ·PXB cell:65% ·SEC:25% ·LX2:10%

[0069] Process 2-2 100 μL of each of the following solutions was added to the cell mixture at room temperature, and the mixture was suspended until the cells were no longer visible. The mixture was then centrifuged (400 g x 2 min) to obtain a viscous mass. The pH of each solution was confirmed to be approximately 7.4, regardless of the presence or absence of heparin and collagen. (1)100mM Tris-HCl buffer (2) Collagen-containing solution (0.2 mg / mL collagen in 100 mM Tris-HCl buffer*) (3) Heparin-containing solution (0.5 mg / mL heparin in 100 mM Tris-HCl buffer) (4) Collagen and heparin-containing solution (0.2 mg / mL collagen and 0.5 mg / mL heparin in 100 mM Tris-HCl buffer) *Since collagen gels quickly in a neutral solution at room temperature, equal amounts of pre-prepared 0.4 mg / mL collagen in 5 mM acetate buffer and 200 mM Tris-HCl buffer were mixed with each of the above solutions.

[0070] Process 2-3 After removing the supernatant from the solution containing the viscous bodies obtained in step 2-2, 20 U / mL thrombin solution (solvent: HCM) was added so that the final volume of the solution was the number of wells to be seeded x 2 μL, and a cell suspension was obtained.

[0071] Process 2-4 2 μL of 10 mg / mL fibrinogen solution was seeded onto a 48-well microplate to form droplets, and then each cell suspension obtained in step 2-3 was added to the droplets. The droplets were then left to stand in an incubator for 1 hour to form fibrin gels.

[0072] Process 2-5 To each well in which the fibrin gel had formed, 0.5 mL of HCM (containing Endothelial Cell Growth Supplement) was added.

[0073] Process 2-6 After HCM was added to each well in which a fibrin gel had formed, the culture was continued for 3 days.

[0074] [Fixation, immunostaining, and microscopic imaging of hepatotoxicity model] Live cell observation was performed in the same manner as in Test Example 1, and then the culture was continued for 3 days. Each sample was then evaluated as a hepatotoxicity model using the following procedure in this order: fixation, permeabilization, blocking, primary antibody treatment, secondary antibody treatment, photography, area calculation, and evaluation.

[0075] (fixation processing) After 3 days of incubation, the 48-well microplate was removed from the incubator, the medium was removed, and the plate was washed with PBS. 300 μL of 4% paraformaldehyde in phosphate buffer (PFA) was then added to each well to fix the 3D hepatotoxicity model. The PFA was then thoroughly washed away.

[0076] (Permeabilization and Blocking) 100 μL of 0.2 (v / v)% TRITON / 1 (w / v)% BSA PBS solution (hereinafter referred to as BSA solution) was added to each well, and the plate was left to stand at room temperature for 2 hours.

[0077] (Primary antibody treatment) The anti-MRP2 antibody was diluted 100-fold with BSA solution to obtain a primary antibody solution. 100 μL of the primary antibody solution was added to each well and allowed to stand at 4°C for 24 hours. The primary antibody solution was then thoroughly washed away.

[0078] (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 to each well and allowed to stand at room temperature for 1 hour in the dark. The secondary antibody solution was then thoroughly washed away, and 100 μL of PBS was added to each well.

[0079] (Fluorescence microscopy) Images were taken using a confocal microscope under the following conditions: Lens used: 10x lens Shooting mode: confocal Filter: 647(Ex) / 678(Em) Z-axis position: 0-100μm / 5μm increments

[0080] The intensities of the captured images were summed to obtain fluorescence observation images of each hepatotoxicity model (3D model).

[0081] [Image analysis of observed images (MRP2)] For the immunostained images of MRP2 in each of the observed hepatotoxicity models, the area of approximately 1.2 mm from the center 2Regions were cropped and image analysis was performed with ImageJ as described below. (1) Image > Type > 8-bit (2) Process > Subtract Background> Sliding Paraboloid (20 pixels) (3) Image > Adjust > Threshold (Range: 25 - 255) (4) Analyze > Set measurements >area (5) Analyze > tools > ROI manager > Select all > add > measure

[0082] <Test Example 3: Confirmation of bile canaliculus formation by bile acid assay> A hepatotoxicity model was prepared in the same manner as in Test Examples 1 and 2. However, the collagen concentration was 0.3 mg / mL, and the heparin concentrations were 0.05 mg / mL, 0.15 mg / mL, 0.5 mg / mL, or 5.0 mg / mL (all final concentrations).

[0083] After 6 days of culture, the following procedure was performed (this assay was performed while the cells were still viable): the medium was replaced with 300 μL of medium containing 5 μM CLF (FITC-labeled bile acid CORNING #451041). The cells were left in an incubator for 20 minutes, then washed five times with PBS. The medium was replaced and the cells were observed under a fluorescence microscope.

[0084] 2. Experimental Results and Discussion <Test Example 1: Confirmation of the aggregation behavior of cells treated with heparin and collagen> Cells were treated in advance with collagen or heparin modified with FITC, and the fluorescence images confirming the deposition of these molecules on the cells are shown in Figure 1. The scale bar in Figure 1 indicates 100 μm.

[0085] Although there was a difference in the tendency for collagen to be deposited relatively on the surface of the cells, whereas heparin was deposited inside the cells, the deposition of molecules due to the treatment was confirmed.

[0086] Although not shown, when collagen and heparin not labeled with FITC were used, almost no fluorescence was observed under the same contrast, confirming that the fluorescence in Figure 1 was derived from FITC.

[0087] In Test Example 1, cells treated with each solution were seeded onto a 96-well microplate, and live cell observation was carried out up to 6 hours after seeding. The results are shown in FIG.

[0088] From the results in Figure 2, it was confirmed that when cells were treated with a solution containing collagen (solution (2) in Test Example 1) and a solution containing collagen and heparin (solution (3) in Test Example 1), cell aggregation was significantly promoted at the stage of 2 hours or more after seeding, for example, in areas such as the dashed frame in Figure 2.

[0089] If one attempts to prepare a neutral solution containing collagen alone, it quickly gels at room temperature, making it difficult to handle as a liquid. In this experiment, adding Tris-HCl buffer to the collagen-containing acetic acid solution immediately before use to adjust the pH to 7.4 allowed it to be handled as a solution, but gelation is occurring at the microscopic level within the solution, and the state is thought to be incompletely stable. Therefore, there is concern that this method may result in errors between experimental runs. Adding heparin beforehand prevents gelation and maintains a stable solution even over long periods at room temperature, allowing for stable experiments.

[0090] These results confirmed that heparin-collagen treatment promoted cell aggregation.

[0091] <Test Example 2: Organization of cells treated with heparin and collagen in fibrin gel> The results of staining with MRP2 for the hepatotoxicity models prepared under each condition are shown in Figure 3. The scale bars in the bright field and fluorescence images in Figure 3 indicate 500 μm, and the scale bars in the enlarged images indicate 300 μm.

[0092] The results in Figure 3 show that treatments containing collagen alone tended to result in excessive cell aggregation, resulting in a significant decrease in MRP2 expression. While the differences between the other conditions were not particularly significant, the heparin-treated and heparin-collagen-treated conditions showed an overall increase in fluorescence compared to the control condition. Therefore, the center of the image was cropped, and the area of the signal exceeding the background level was calculated by image analysis to compare the different conditions.

[0093] Figure 4 is a photograph showing the results of fluorescent observation of MRP2 expression under various treatment conditions. The scale bar in Figure 4 indicates 300 μm. Figure 5 is a graph showing the expression intensity ratio per area (expression area ratio) calculated based on image analysis of the hepatotoxicity model shown in Figure 4.

[0094] Figure 5 shows the results quantitatively supporting the finding that the hepatotoxicity models obtained through heparin treatment and heparin-collagen treatment had a higher MRP2 expression intensity ratio compared to the control.

[0095] <Test Example 3: Confirmation of bile canaliculus formation by bile acid assay> The results of fluorescence observation of various hepatotoxicity models created with varying heparin concentrations after incubation in medium containing CLF are shown in Figures 6, 7, 8, and 9. Figure 6 shows the results of fluorescence observation of the control hepatotoxicity model. The scale bar in each figure represents 500 μm. In normal liver tissue with well-formed bile canaliculi, hepatocytes export bile acids taken up from outside the liver tissue into the bile canaliculi, and it is thought that there will be little FITC-derived fluorescence remaining within the cells after CLF treatment.

[0096] In Figures 7, 8, and 9, where the heparin concentrations used to create the hepatotoxicity models were 0.15 mg / mL, 0.5 mg / mL, or 5.0 mg / mL, there was little intracellular FITC-derived fluorescence, while there was a significant amount of extracellular FITC-derived fluorescence. This confirmed that CLF taken up into hepatocytes was efficiently excreted through the bile canaliculi, further suppressing its retention within hepatocytes. Furthermore, as the heparin concentration increased during hepatotoxicity model creation, the intracellular FITC-derived fluorescence decreased, while dot-like or linear fluorescence around the cells increased. This confirms that the use of heparin during hepatotoxicity model creation facilitates the excretion of CLF taken up into hepatocytes into the bile canaliculi.

Claims

1. 1. A method for producing a cell aggregate having bile canaliculi, comprising: a contacting step of contacting cells including hepatocytes with heparin in an aqueous medium; a culturing step of culturing the cells contacted with the heparin to form the cell aggregates, the heparin concentration when contacted with the cells in the aqueous medium is 0.5 mg / mL or more based on the total amount of the aqueous medium; A method wherein the contacting step further comprises contacting the cells with a collagen component.

2. The method of claim 1 , wherein the hepatocytes are mature hepatocytes.

3. 3. The method according to claim 1, wherein the heparin concentration when contacted with the cells in the aqueous medium is 0.5 mg / mL or more and 12.0 mg / mL or less, based on the total volume of the aqueous medium.

4. The method according to any one of claims 1 to 3, wherein the ratio of the number of hepatocytes to the total number of cells is 65% or more.

5. The method according to any one of claims 1 to 4, wherein the expression intensity ratio of MRP2 in the cell aggregate, represented by the following formula (1), is 120% or more: MRP2 expression intensity ratio = X / Y × 100 (1) [In formula (1), X represents the expression intensity of MRP2 in the cell aggregates formed in the culture step, Y indicates the expression intensity of control cell aggregates formed by culturing cells including hepatocytes without contacting them with heparin and extracellular matrix components.]

Images