JP2008022743A - New spheroid, method for producing spheroid, use of spheroid in medicine screening, toxicity evaluation, and production of animal model of disease, spheroid cell-culturing kit, use for spheroid formation of antibody, use for spheroid formation of lectin, and use for spheroid formation of cell-adhesion molecule - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To produce spheroid in mass by a simple culture technique. <P>SOLUTION: The spheroid and spheroid cell group contain a material present on the cell surface of an antibody, lectin, a cell-adhesion molecule or the like, or a material recognizing cell membrane itself as a cell-coagulating agent. The method for producing the spheroid includes bringing a target cell into contact with the cell-coagulating agent in a medium, and culturing the cell for a prescribed period. The method for screening a medicinal material, the toxicity evaluation method, and the method for producing an animal model of disease, by using the spheroid are also provided. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a spheroid obtained by a novel production method, a method for producing such a spheroid, and use of such spheroid for drug screening, toxicity evaluation, and production of a disease model animal.

  Today, in the development of new pharmaceuticals, cells cultured by cell culture technology are used as simulators for drug pharmacological activity evaluation and toxicity tests.

  However, since cells cultured by conventional cell culture technology are monolayer cells in which the cells spread in a two-dimensional direction, it is not possible to construct a three-dimensional tissue equivalent to that in the living body, There has been a problem that the specific functions possessed cannot be maintained for a long time.

  Therefore, in recent years, spheroid culture, which is a three-dimensional tissue having a function equivalent to that in a living body, has attracted attention. For example, seeding a plurality of single cells in a funnel having a bottom surface and culturing spheroids by aggregating and dividing the single cells on the bottom surface (Patent Document 1), or a cast liquid surface of a predetermined polymer mixture There is a type in which spheroids are cultured on a honeycomb structure obtained by condensing water and evaporating minute water droplets generated by the dew condensation (Patent Document 2).

  Some spheroids produced by such a method have a problem that it is difficult to grow spheroids because some do not have adhesion to the bottom surface of the culture vessel. Even if it exists, there existed a problem that the formation efficiency of a spheroid was low and the desired quantity of spheroid was not obtained.

JP-A-6-327462 JP 2002-335949 A

  That is, it has been difficult to efficiently form a large amount of spheroids by the conventional spheroid formation method. Therefore, for example, spheroids cannot be used for drug screening and safety evaluation in the drug discovery stage, and higher efficiency has been demanded.

  The present inventors have intensively studied to solve the above problems, and found that a large amount of spheroids can be stably and stably formed by contacting a cell aggregating agent and cultured cells in a medium. Thus, the present invention has been completed. That is, the gist of the present invention is as follows.

1. A spheroid fixed to a solid phase, comprising an exogenous cell aggregating agent.
2. 2. The spheroid according to 1, which comprises at least 10 cells.
3. 3. The spheroid according to 1 or 2, which has a diameter of 10 μm or more.
4). 4. The spheroid according to any one of 1 to 3, wherein the cells constituting the spheroid are any of primary hepatocytes, hepatocyte cell lines, primary cancer cells, and cancer cell line cells.
5. The spheroid according to any one of 1 to 4, wherein the cell aggregating agent contains any one of an antibody, a lectin, and a cell adhesion molecule.
6). 5. The spheroid according to any one of 1 to 4, wherein the cell aggregating agent is a lectin.
7). 6. The spheroid according to 6, wherein the lectin is galectin or annexin.
A spheroid cell group in which the cells forming the spheroids described in any one of 8.1 to 7 occupy 30% or more of the total number of cells.
9. A method for producing a spheroid, comprising culturing cells in the presence of an exogenous cell aggregating agent.
10. The manufacturing method of a spheroid including each process of following (a) thru | or (c).
(A) a step of dispersing the cells in a medium in contact with the solid phase;
(B) adding a cell aggregating agent to the medium;
(C) A step of bringing cells into contact with a cell aggregating agent in a medium.
11. The method for producing a spheroid according to 9 or 10, wherein the cell comprises any cell selected from the group consisting of primary hepatocytes, hepatocyte cell lines, primary cancer cells, and cancer cell line cells.
12 The method for producing a spheroid according to any one of 9 to 11, wherein the cell aggregating agent contains any one of an antibody, a lectin, and a cell adhesion molecule.
13. The method for producing a spheroid according to any one of 9 to 11, wherein the cell aggregating agent is a lectin.
14 14. The method for producing a spheroid according to 13, wherein the lectin is galectin or annexin.
15. Use of the spheroid according to any one of 1 to 7 for drug screening.
16. Use of the spheroid according to any one of 1 to 7 for evaluation of drug toxicity.
17. Use of the spheroid according to any one of 1 to 7 for producing a disease state model animal.
18. A spheroid cell culture kit comprising the spheroid according to any one of 1 to 7 and a cell culture container.
19. Use of antibodies for spheroid formation.
20. Use of lectins for spheroid formation.
21. Use of cell adhesion molecules for spheroid formation.

  The present invention makes it possible to easily form a large amount of spheroids with extremely high efficiency.

Hereinafter, the present invention will be described in detail according to the best mode for carrying out the invention.
1. The Spheroid of the Present Invention The spheroid of the present invention is a spheroid that contains an exogenous cell aggregating agent and is fixed to a solid phase.

  In the present specification, a cell group in which a plurality of cells form a substantially spherical shape is referred to as a “spheroid”, and a cell that does not form such a spheroid is referred to as a “plate cell”.

  The “exogenous cell aggregating agent” in the spheroid of the present invention means a cell aggregating agent that is not produced by the cells constituting the spheroid, specifically, cells added to the culture medium during the spheroid formation process. Represents a flocculant.

  The cell forming the spheroid of the present invention is not particularly limited as long as it can be grown in a culture solution, but is preferably an animal cell, and more preferably a mammal-derived cell. Examples of mammals include humans, dogs, cats, monkeys, cows, pigs, horses, sheep, goats, hamsters, guinea pigs, rats, mice, etc., but in particular humans, dogs, cats, monkeys, rats, mice. Are particularly preferable from the viewpoint of the ease of formation of spheroids.

  When using cells derived from mammals, the tissue or organ from which the cells are derived is either a tissue-forming cell or a tissue / organ that can be artificially added with a proliferation ability. There is no particular limitation as long as it is an organ. Examples of such tissues / organs include normal liver, cornea, skin, cartilage, large intestine, small intestine, pancreas, stomach, muscle tissue, heart, lung, esophagus, bone marrow, kidney, spleen, testis, ovary, adipose tissue, etc. In addition to tissues and organs, these cells are cancerous (tumorous). Among them, the cells used for the spheroids of the present invention are preferably cells derived from tumors derived from the liver, cartilage, skin, pancreas (α cell, β cell), adipose tissue, and the above tissues / organs, particularly the liver or tumor. It is preferably a cell derived from. Specifically, primary hepatocytes, hepatocyte cell lines, primary cancer cells, and cancer cell line cells are exemplified as preferred cells.

  The “cell aggregating agent” in the spheroid of the present invention is a substance that specifically recognizes and binds to a substance or cell membrane present on the cell surface and forms a cross-linked structure, and has toxicity to cells in cell culture. The substance is not particularly limited as long as the substance is negligible, and examples thereof include antibodies, lectins, ligands, cell adhesion molecules, and the like.

  The “antibody” is preferably IgA or IgM having a plurality of antigen recognition sites, and those skilled in the art can select and use an optimal antibody as appropriate according to the cell used and its origin. For example, an anti-heparan sulfate antibody that recognizes heparan sulfate expressed on the cell surface, an anti-asialoglycoprotein receptor antibody that recognizes an asialoglycoprotein receptor that is a cell surface protein, and the like are exemplified.

  Examples of “lectins” include galectins, C-type lectins, selectins, annexins, legume lectins, etc., but galectins that recognize galactose present on the cell surface in various cells, or similarly various cells Of these, annexins that recognize glycosaminoglycans present on the cell surface are preferred. Malt agglutinin (WGA), which is a lectin that recognizes galactose in the same manner as galectins, can also be used.

  Examples of the “cell adhesion molecule” include molecules that promote adhesion between cells (cadherin superfamily (E cadherin, p cadherin, N cadherin, etc.), catenin (α catenin, β catenin, p120 catenin, etc.), integrin superfamily, Examples include immunoglobulin superfamily (NCAM, L1, ICAM family, nectin), claudin family, desmoglein, neuroligin, neulexin) and molecules that promote adhesion between substrate and cells (fibronectin, laminin) It is preferable to use the former.

  The flocculant used in the spheroid of the present invention is not limited to those exemplified above, as long as it is a substance that can specifically bind to a plurality of receptors that are known to be expressed on the cell surface to be cultured. For example, an artificially synthesized substance (such as a substance in which a ligand, an antibody, a cell adhesion molecule, a lectin, a lipid, or the like is bonded to both ends of a molecule such as polyethylene glycol) can be used. Furthermore, it is also possible to use an existing substance such as chymotrypsin that is known to exhibit an aggregating action on cancer cells.

  These aggregating agents can be selectively used depending on the cells that form the desired spheroid of the present invention. For example, when primary hepatocytes or hepatocyte cell lines having galactose on the cell surface are used for spheroid formation, galectins are used. Or malt agglutinin can be used. On the other hand, when primary cancer cells or cancer cell line cells are used for spheroid formation, chymotrypsin, antitumor antibodies, and the like can also be used. Further, when a nerve cell is used as the cell, NCAM or the like can be used.

  Such a cell agglutinating agent can be selected as appropriate by those skilled in the art from the existing information such as a substance exhibiting specific binding properties of each substance and proteins and sugar chains existing on the cell surface. It is possible to use.

  The spheroid of the present invention is preferably formed of 10 or more cells, more preferably 20 or more, and most preferably 25 or more. This is because the spheroid formed by such a plurality of cells has a different external environment and internal environment, and can more realistically reproduce the internal state of the tissue. For example, after the spheroid number is visually counted in advance, the spheroids are trypsinized to disperse the cells, and the total number of cells is measured. Based on the cell number and the spheroid number, In addition, it can be performed by various known methods such as calculating the average value of the number of cells forming one spheroid.

  The major axis of the spheroid of the present invention is 10 μm or more, more preferably 20 μm or more. The measurement of the major axis of such spheroids can be performed visually under a microscope.

  The spheroid of the present invention is characterized by being fixed to a solid phase. The “solid phase” in the spheroid of the present invention refers to a culture vessel or bead made of a material that does not dissolve in the culture solution (polystyrene, polyimide, cyclic olefin-based thermoplastic resin, aluminum oxide, glass, quartz glass, silicon, etc.). The surface of such a solid phase may be flat, but preferably a one-dimensional line having a line width of 1 μm or less and a depth of 10 nm or more, or a two-dimensional shape (polygon such as a rectangle or hexagon, a circle, or the like) It is more preferable to have a non-circular shape. This is because by providing such a structure on the solid surface, spheroids partially adhered to the solid phase are efficiently formed (more than 30% of the cells added at the start of culture form spheroids).

  Such spheroids of the present invention are (1) screening of substances that become drugs at the stage of drug discovery, etc., (2) toxicity tests of substances, and (3) various pathological models by transplanting the spheroids of the present invention. Can be used for animal preparation.

  For example, (1) when the spheroid of the present invention is used for drug screening, the following method can be used. That is, a medium in which substances to be screened are dissolved at various test concentrations is added to the spheroids of the present invention fixed to a culture vessel (multiplate or culture dish), and after culturing for a certain period of time, The morphological change, biochemical change, and histological change of invention spheroids are confirmed by applying existing methods. By this method, by selecting a screening target substance that is excellent in the effect of the screening target substance on the spheroid of the present invention, unlike a monolayer culture, for a cell group (spheroid) having a three-dimensional structure closer to a tissue It is possible to screen for substances that are expected to be effective.

  For example, when the spheroid of the present invention is used for (2) substance toxicity test, the following method can be used. That is, to the spheroids of the present invention fixed to a culture vessel (multiplate or culture dish), a medium containing various test concentrations of a target substance for toxicity test is added and cultured continuously for a certain period of time. The morphological change, biochemical change, and histological change of invention spheroids are confirmed by applying existing methods. This method enables a more reliable toxicity test that is closer to an animal test than a basic toxicity test that has been performed with conventional monolayer culture cells. It can be obtained using technology.

  For example, (3) the following method can be used when the spheroid of the present invention is used for the preparation of a disease state model animal. For example, when preparing a tumor model mouse, the spheroid of the present invention prepared from tumor cells used for modeling is prepared. Such spheroids of the present invention are released from the solid phase by shaking or treating with a very small amount of trypsin, and then the released spheroids are administered into the abdominal cavity, muscle, vena cava, pulmonary artery, hepatic artery, etc. of the mouse. Thereafter, by continuing the breeding of the animal, the disease state model mouse can be easily prepared as compared with the conventional mouse.

  The spheroids of the present invention are characterized by the nature of the cells that form them (in the case of cells or cell lines obtained from a pathological tissue, the pathological state, the cells obtained from normal tissues or cells of a cell line are normal tissues) Therefore, the target disease state model animal can be prepared at a higher frequency than the method of preparing the model animal by the conventional method of administering cells.

2. The present invention cell group The present invention cell group is characterized in that cells forming the present spheroids occupy 30% or more of the total number of cells.

  In the cell group of the present invention, the cells forming the spheroid of the present invention occupy 30% or more of the total number of cells. Such a cell group is formed as a cell group existing in the culture system when the spheroid of the present invention is produced, for example, by the production method of the present invention described later, but can also be prepared by the following method.

  That is, a culture vessel containing the spheroid of the present invention is prepared. For such a culture vessel, cells that do not form the spheroids of the present invention (may be the same cells as the cells that form the spheroids of the present invention, or may be different cells, and a mixture of these cells) The cell group of the present invention can be obtained. The cell group of the present invention thus obtained can achieve the effect of reproducing the in vivo environment similar to co-culture. Such a combination of cells can be easily selected by those skilled in the art, and examples thereof include combinations of vascular endothelial cells and fibroblasts. In this case, vascular endothelial cells may be spheroids, and fibroblasts may be spheroids.

3. Production method of the present invention The production method of the present invention is a method for producing a spheroid, characterized in that cells are cultured in the presence of an exogenous cell aggregating agent.

  The “cell” in the production method of the present invention is a cell that forms a spheroid, and is not particularly limited as long as it can be grown in a culture solution. In particular, animal cells are preferable, and mammal-derived cells are more preferable among them. Examples of mammals include humans, dogs, cats, monkeys, cows, pigs, horses, sheep, goats, hamsters, guinea pigs, rats, mice, etc., but in particular humans, dogs, cats, monkeys, rats, mice. Are particularly preferable from the viewpoint of the ease of formation of spheroids. A person skilled in the art can appropriately select and use a preferable medium according to the cells to be used.

  When using cells derived from mammals, the tissue or organ from which the cells are derived is either a tissue-forming cell or a tissue / organ that can be artificially added with a proliferation ability. There is no particular limitation as long as it is an organ. Examples of such tissues / organs include normal liver, cornea, skin, cartilage, large intestine, small intestine, pancreas, stomach, muscle tissue, heart, lung, esophagus, bone marrow, kidney, spleen, testis, ovary, adipose tissue, etc. In addition to tissues and organs, these cells are cancerous (tumorous). Among them, cells used in the production method of the present invention are particularly preferably liver, cartilage, skin, pancreatic cells (α cells, β cells), adipose tissue, and tumor-derived cells derived from the respective tissues / organs. Or it is preferable that it is a cell derived from a tumor. Specifically, primary hepatocytes, hepatocyte cell lines, primary cancer cells, and cancer cell line cells are exemplified as preferred cells.

  The term “exogenous cell aggregating agent” in the production method of the present invention means a cell aggregating agent that is not produced by the cells that constitute the spheroid, and specifically added to the culture medium during the spheroid formation process. Represents a cell aggregating agent.

  The “cell aggregating agent” in the production method of the present invention is a substance that specifically recognizes and binds to a substance or cell membrane present on the cell surface and forms a cross-linked structure, and has toxicity to cells in cell culture. The substance is not particularly limited as long as the substance is negligible, and examples thereof include antibodies, lectins, ligands, cell adhesion molecules, and the like.

  The “antibody” is preferably IgA or IgM having a plurality of antigen recognition sites, and those skilled in the art can select and use an optimal antibody as appropriate according to the cell used and its origin. Examples thereof include an anti-heparan sulfate antibody that recognizes heparan sulfate expressed on the cell surface, and an anti-asialoglycoprotein receptor antibody that recognizes an asialoglycoprotein receptor that is a cell surface protein.

  Examples of “lectins” include galectins, C-type lectins, selectins, annexins, legume lectins, etc., but galectins that recognize galactose present on the cell surface in various cells, or similarly various cells Of these, annexins that recognize glycosaminoglycans present on the cell surface are preferred. Malt agglutinin (WGA), which is a lectin that recognizes galactose in the same manner as galectins, can also be used.

  Examples of the “cell adhesion molecule” include molecules that promote adhesion between cells (cadherin superfamily (E cadherin, p cadherin, N cadherin, etc.), catenin (α catenin, β catenin, p120 catenin, etc.), integrin superfamily, Examples include immunoglobulin superfamily (NCAM, L1, ICAM family, nectin), claudin family, desmoglein, neuroligin, neulexin) and molecules that promote adhesion between substrate and cells (fibronectin, laminin) It is preferable to use the former.

  The flocculant used in the production method of the present invention is not limited to those exemplified above as long as it is a substance that can specifically bind to a plurality of receptors that are known to be expressed on the surface of cells to be cultured. For example, an artificially synthesized substance (such as a substance in which a ligand, an antibody, a cell adhesion molecule, a lectin, a lipid, or the like is bonded to both ends of a molecule such as polyethylene glycol) can be used. Furthermore, it is also possible to use an existing substance such as chymotrypsin that is known to exhibit an aggregating action on cancer cells.

  These aggregating agents can be selectively used depending on the cells that form the desired spheroid of the present invention. For example, when primary hepatocytes or hepatocyte cell lines having galactose on the cell surface are used for spheroid formation, galectins are used. Or malt agglutinin can be used. On the other hand, when primary cancer cells or cancer cell line cells are used for spheroid formation, chymotrypsin, antitumor antibodies, and the like can also be used. Further, when a nerve cell is used as the cell, NCAM or the like can be used.

  Such a cell agglutinating agent can be selected as appropriate by those skilled in the art from the existing information such as a substance exhibiting specific binding properties of each substance and proteins and sugar chains existing on the cell surface. It is possible to use.

  Spheroids are produced by culturing cells in the presence of a cell aggregating agent. The coexistence conditions of the cells and the cell aggregating agent can be formed by, for example, a method of culturing the cells until the spheroids are formed by adding the cell aggregating agent to the medium in the middle of culturing the cells. It can be formed by a method of culturing until the desired cells are added to the medium and spheroids are formed. In addition, after the cells and the cell aggregating agent are contacted for 1 hour or longer, preferably 3 hours or longer, more preferably 10 hours or longer, particularly preferably 20 hours or longer, the culture fluid is replaced with a culture fluid that does not contain the cell aggregating agent. Thus, spheroids can also be produced by continuing cell culture.

That is, the manufacturing method of the present invention includes the following steps.
(A) Step of dispersing cells in medium in contact with solid phase (b) Step of adding cell aggregating agent to medium (c) Step of contacting cells and cell aggregating agent in medium The following (a) to ( The explanation will be divided into c).

(A) Step of dispersing cells in medium in contact with solid phase “Solid phase” in this step is a solid phase to which the formed spheroids adhere, and specifically, a culture vessel, beads, or the like is added. It is a culture container. These solid phases are made of a material that does not dissolve in the culture medium (polystyrene, polyimide, cyclic olefin-based thermoplastic resin, aluminum oxide, glass, quartz glass, silicon, etc.). The surface of such a solid phase may be flat, but preferably a one-dimensional line having a line width of 1 μm or less and a depth of 10 nm or more, or a two-dimensional shape (polygon such as a rectangle or hexagon, a circle, or the like) It is more preferable to have a non-circular shape. This is because by providing such a structure on the solid surface, spheroids partially adhered to the solid phase are efficiently formed (more than 30% of the cells added at the start of culture form spheroids).

“Cells” and “medium” in this step have the same meaning as described above.
The method for dispersing the cells in this step in the medium is not particularly limited.

(B) Step of adding a cell aggregating agent to the medium The “cell aggregating agent” in this step has the same meaning as described above.

  The method for adding the cell aggregating agent to the medium is not particularly limited, and the cell aggregating agent may be added to the medium, the medium may be added to the cell aggregating agent, or the container may be added to the container. On the other hand, the culture medium and the cell aggregating agent may be added simultaneously.

(C) Step of contacting cells and cell aggregating agent in medium Since spheroids are formed when cells come into contact with cell aggregating agents, it is necessary to contact cells and cell aggregating agents in the medium. .

  The contact condition between the cells and the cell aggregating agent is, for example, a method in which the cells are cultured until the spheroids are formed by adding the cell aggregating agent to the medium while the cells are being cultured (ie, after the step (a) In addition to the method of (b) and mixing, a method of culturing until a spheroid is formed by adding a target cell to a medium containing a cell aggregating agent (that is, after the step (b) above, A method of mixing the medium, the medium and the cell aggregating agent at the same time (ie, the process of (a) and the process of (b) are performed simultaneously). (Method). In addition, after the cells and the cell aggregating agent are contacted for 1 hour or longer, preferably 3 hours or longer, more preferably 10 hours or longer, particularly preferably 20 hours or longer, the culture fluid is replaced with a culture fluid that does not contain the cell aggregating agent. Thus, spheroids can also be produced by continuing cell culture.

  The concentration of the cell aggregating agent in the medium in the production method of the present invention can be adjusted as appropriate, but is 1 μg / ml or more, preferably 10 μg / ml or more, particularly 50 μg / ml or more. It is preferable that it is 100 μg / ml or more. This is because when the concentration is 10 μg / ml or more, a spheroid formation rate of 70% or more can be achieved, and when the concentration is 50 μ / ml or more, a spheroid formation rate of 90% or more can be achieved. The “spheroid formation rate” is a spheroid with respect to the number of cell groups existing in the culture system (a spheroid is counted as one, and a plate cell is counted as a group of cells (colony) generated from one clone). The number is expressed as a percentage and is an index serving as an index of the efficiency of spheroid formation.

  Spheroids can be produced by culturing the cells by standing at 37 ° C. under conditions of 0.3% carbon dioxide / air under such concentration conditions. It should be noted that the carbon dioxide concentration and temperature conditions during the culture can be changed as appropriate as long as they are within the range of conditions for normal culture.

4). Present Spheroid Cell Culture Kit The present spheroid cell culture kit includes the present spheroid and a cell culture container.

  The cell culture container in the spheroid cell culture kit of the present invention is the culture container when the spheroid of the present invention is fixed to the culture container, and the person in charge when the spheroid is fixed to a carrier such as a bead instead of the culture container. Means a separate culture vessel.

  With the spheroid cell culture kit in which such a culture container and the spheroid of the present invention are set, spheroids that have been difficult to adjust and could not be used in large quantities can be used by a simple culture operation.

  In addition to the above-described configuration, the spheroid cell culture kit of the present invention may further contain a medium suitable for cells that form spheroids and a reagent for performing physiological analysis. The cell aggregating agent may be contained. This is because such a configuration enables spheroid culture with easier operation.

  The spheroid cell culture kit of the present invention can be used for various pathological models by screening a substance that becomes a drug in the stage of drug discovery or the like already described in the spheroid of the present invention, testing the toxicity of the substance, and transplanting the spheroid of the present invention. Can be used for animal preparation.

  Hereinafter, the present invention will be described more specifically with reference to examples.

  In order to confirm the improvement of spheroid formation ability by the cell aggregating agent, spheroid culture of HepG2 cells was performed using malt agglutinin.

  In other words, HepG2 cells (RCB1648, a cultured cell line derived from human liver cancer) were suspended at 100,000 cells / ml in an alpha MEM medium (manufactured by Dainippon Pharmaceutical Co., Ltd.) to which an aggregating agent was added to a concentration shown in Table 1 below. , Seeded in a culture dish (96-well polystyrene multiwell plate: manufactured by SCIVAX Co., Ltd. having a honeycomb pattern with a width of 250 nm, a height of 380 nm and a side of 1 μm on the bottom), 2% carbon dioxide / air, 37 ° C. Cultured for 1 day under. Thereafter, the medium was replaced with a medium not containing malt agglutinin and cultured for 3 days.

  As a result, the spheroid formation rate was 68% (256/379) in the control (FIG. 1), whereas 73% (270/372) (FIG. 2) in sample 1 and 100% (279/279) in sample 2 (FIG. 1). 3) It was also revealed that the spheroid formation rate was improved to 100% (110/110) (FIG. 4) in Sample 3 as well.

  When the same test was performed on a dish with a flat bottom and no processing, the control was 0%, the sample 1 was 70%, the sample 2 was 85%, and the sample 3 was 93%. It turns out that it is improving.

  Since the present invention enables the formation of excellent spheroids, it can be widely used in the biotechnology field, and is particularly useful for evaluating the effects of new substances for pharmaceuticals, cosmetics and foods, and for evaluating safety.

It is the photograph observed by culturing HepG2 cells without adding a cell aggregating agent. It is the photograph observed by culturing and observing HepG2 cells by adding a cell aggregating agent to the medium at 1 μg / ml. It is the photograph observed by culturing and observing HepG2 cells by adding a cell aggregating agent to the medium at 10 μg / ml (Sample 2). It is the photograph observed by adding a cell aggregating agent to the medium at 100 μg / ml and culturing HepG2 cells (Sample 3).

Claims (21)

A spheroid fixed to a solid phase, comprising an exogenous cell aggregating agent. The spheroid according to claim 1, comprising at least 10 cells. The spheroid according to claim 1 or 2, which has a diameter of 10 µm or more. The spheroid according to any one of claims 1 to 3, wherein the cells constituting the spheroid are any of primary hepatocytes, hepatocyte cell lines, primary cancer cells, and cancer cell line cells. The spheroid according to any one of claims 1 to 4, wherein the cell aggregating agent contains any one of an antibody, a lectin, and a cell adhesion molecule. The spheroid according to any one of claims 1 to 4, wherein the cell aggregating agent is a lectin. The spheroid according to claim 6, wherein the lectin is galectin or annexin.
A spheroid cell group in which the cells that form the spheroid according to any one of claims 1 to 7 occupy 30% or more of the total number of cells. A method for producing a spheroid, comprising culturing cells in the presence of an exogenous cell aggregating agent. The manufacturing method of a spheroid including each process of following (a) thru | or (c).
(A) a step of dispersing the cells in a medium in contact with the solid phase;
(B) adding a cell aggregating agent to the medium;
(C) A step of bringing cells into contact with a cell aggregating agent in a medium. The method for producing a spheroid according to claim 9 or 10, wherein the cell comprises any cell selected from the group consisting of primary hepatocytes, hepatocyte cell lines, primary cancer cells, and cancer cell line cells. The method for producing a spheroid according to any one of claims 9 to 11, wherein the cell aggregating agent contains any one of an antibody, a lectin, and a cell adhesion molecule. The method for producing a spheroid according to any one of claims 9 to 11, wherein the cell aggregating agent is a lectin. The method for producing a spheroid according to claim 13, wherein the lectin is a galectin or an annexin. Use of the spheroid according to any one of claims 1 to 7 for drug screening. Use of the spheroid according to any one of claims 1 to 7 for evaluation of drug toxicity. Use of the spheroid according to any one of claims 1 to 7 for producing a disease state model animal. A spheroid cell culture kit comprising the spheroid according to any one of claims 1 to 7 and a cell culture container. Use of antibodies for spheroid formation. Use of lectins for spheroid formation. Use of cell adhesion molecules for spheroid formation.

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