JP5884218B2 - Method for producing tumor tissue model - Google Patents

Description

  The present invention relates to a method for producing a tumor tissue model.

  In the research and development of pharmaceuticals, it is important to evaluate the penetrability of a drug into a disease site and the therapeutic effect obtained by the drug. On the other hand, solid tumors such as pancreatic cancer and Skills gastric cancer have extremely fibrotic tumor stroma and few blood vessels, so that chemotherapy using drugs is extremely difficult.

  So far, various methods for performing drug evaluation on tumors have been proposed. As an in vivo evaluation method, for example, there is a method in which a tumor is formed in an experimental animal such as a mouse and a drug is administered to the experimental animal for evaluation. However, the properties differ greatly depending on the size of the tumor and the place where the tumor is formed, and it has been difficult to evaluate the drug in detail due to the diffusion and adsorption of the drug to other tissues.

  As an in vitro test method, a method for evaluating drug or substance permeability using Caco-2 cells (a cell line derived from human colon cancer) cultured in a monolayer on a culture insert (for example, Non-Patent Document 1), etc. Has been proposed. However, each method has a problem that it is difficult to evaluate the permeability of the drug because the structure is greatly different from the actual tumor tissue.

  Therefore, from the viewpoint of drug evaluation, there is a demand for the construction of a three-dimensional model tissue that reproduces the structure and properties of a disease site such as a solid tumor.

Z. Wang et al., Journal of Mass Spectrometry, 2000, 35:71

  Therefore, the present invention provides a method for producing a new three-dimensional tissue model suitable for drug evaluation for solid tumors.

  The present invention is a method for producing a tumor tissue model, wherein a cell-containing liquid containing cells derived from a solid tumor is disposed to form a cell layer, and the first liquid and the second liquid are alternately disposed, Two or more cell layers are stacked by forming an extracellular matrix on the cell layer, and arranging a cell-containing solution containing cells derived from a solid tumor on the extracellular matrix to form a cell layer. And the combination of the content of the first liquid and the content of the second liquid interacts with the protein or polymer having an RGD sequence and the protein or polymer having the RGD sequence. Or a method for producing a tumor tissue model, which is a combination of a positively charged protein or polymer and a negatively charged protein or polymer. To.

  According to the present invention, a tumor tissue model suitable for drug evaluation for a solid tumor can be produced.

FIG. 1 is a schematic diagram illustrating an example of a method for evaluating a test substance. FIG. 2 is a graph showing an example of the result of a substance permeability test performed using a tumor tissue model. FIG. 3 is a graph showing another example of the result of a substance permeability test performed using a tumor tissue model.

  The present invention provides an environment closer to a tumor tissue than conventional methods by laminating cells derived from a solid tumor while alternately arranging the first liquid and the second liquid described above to form an extracellular matrix. Based on the knowledge that drug permeability can be evaluated.

  According to the present invention, for example, it is possible to produce a tumor tissue model capable of reproducing an environment close to a solid tumor as compared with a conventional method, and preferably to produce a tumor tissue model capable of reproducing a more precise tissue structure. it can. For this reason, by using the tumor tissue model obtained by the production method of the present invention, for example, drug evaluation can be performed in an environment closer to the tumor tissue in the living body than in the conventional method. The tumor tissue model obtained by the production method of the present invention has the effect that it can be a very useful tool in, for example, the evaluation of drug kinetics of various molecular weights in the creation (screening) of new drugs, in particular, the kinetic evaluation of drugs with large molecular weights Preferably it plays.

That is, the present invention
[1] A method for producing a tumor tissue model, wherein a cell-containing liquid containing cells derived from a solid tumor is disposed to form a cell layer, and the first liquid and the second liquid are alternately disposed to form the cell Forming an extracellular matrix on the layer, and laminating two or more cell layers by forming a cell layer by placing a cell-containing solution containing cells derived from a solid tumor on the extracellular matrix A combination of the content of the first liquid and the content of the second liquid is a protein or polymer having an RGD sequence and a protein or polymer interacting with the protein or polymer having the RGD sequence Or a method for producing a tumor tissue model, which is a combination of a positively charged protein or polymer and a negatively charged protein or polymer;
[2] The method for producing a tumor tissue model according to [1], wherein the cell layers are laminated on a membrane filter;
[3] The method for producing a tumor tissue model according to [1] or [2], wherein the cells derived from a solid tumor are fibroblasts derived from a solid tumor;
[4] A tumor tissue model produced by the production method according to any one of [1] to [3];
[5] The tumor tissue model according to [4], comprising a cell layer containing cells derived from at least two layers of solid tumors and an extracellular matrix, wherein the extracellular matrix is formed at least between the cell layers. ;
[6] A method for evaluating a test substance, comprising evaluating the permeability of the test substance to the tumor tissue using the tumor tissue model according to [4] or [5];
[7] An evaluation kit used for evaluating the permeability of a test substance, the evaluation kit having the tumor tissue model according to [4] or [5];
About.

  As used herein, a “solid tumor” is a tumor mass that is nourished by tumor blood vessels and can be formed by tumor cells and tumor stroma such as fibroblasts and extracellular matrix, unlike blood tumors such as leukemia. That means. Examples of solid tumors include pancreatic cancer, breast cancer, stomach cancer, lung cancer, colon cancer, prostate cancer, renal cell cancer, malignant mesothelioma, malignant lymphoma, osteosarcoma, Ewing sarcoma, chondrosarcoma, leiomyosarcoma and the like. Solid tumors include not only primary cancers (primary tumors) but also tumor masses (metastatic cancers) formed by their invasion and metastasis.

  In the present specification, the “tumor tissue” refers to a tissue composed of an assembly of cells including at least one of tumor cells and tumor stroma and an extracellular matrix. Examples of the tumor tissue include a tumor stroma (stromal cell) constituting a stroma with a tumor cell in a solid tumor, a tissue composed of a tumor cell and an extracellular matrix, a stroma cell and an extracellular matrix. Tissue, tumor cells, and tissues composed of extracellular matrix. Tumor stroma (stromal cells) are stromal cells in a solid tumor, such as fibroblasts, vascular endothelial cells, vascular wall cells and vascular smooth muscle cells, lymphatic endothelial cells, macrophages, lymph Examples thereof include immune cells such as spheres and dendritic cells.

  As used herein, “solid tumor-derived cell” refers to a cell present in a solid tumor, and may include, for example, a cell collected from a solid tumor, a cell subcultured from the collected cell, and the like. Examples of the solid tumor-derived cells include tumor cells and stromal cells in solid tumors.

  As used herein, a “tumor tissue model” is a reproduction or mimic of the environment inside and / or surrounding a tumor tissue in a solid tumor and may contain tumor cells. Includes those that reproduce or mimic the surrounding environment of a tissue, those that reproduce or mimic the surrounding environment of a tissue containing tumor cells and stromal cells in a solid tumor, those that reproduce or mimic the surrounding environment of a tumor cell in a solid tumor, etc. . In addition, the tumor tissue model preferably includes an experimental or test tool that can be used for drug evaluation on a solid tumor, and more preferably includes a tumor tissue model manufactured by the manufacturing method of the present invention.

[Method for producing tumor tissue model]
The present invention is a method for producing a tumor tissue model, wherein a cell-containing liquid containing cells derived from a solid tumor is disposed to form a cell layer, and the first liquid and the second liquid are alternately disposed, Forming an extracellular matrix on the cell layer, and laminating two or more cell layers by forming a cell layer by placing a cell-containing solution containing cells derived from a solid tumor on the extracellular matrix. A combination of the content of the first liquid and the content of the second liquid is a protein or polymer having an RGD sequence and a protein or polymer interacting with the protein or polymer having the RGD sequence. The present invention relates to a method for producing a tumor tissue model, which is a combination or a combination of a positively charged protein or polymer and a negatively charged protein or polymer. . In the method for producing a tumor tissue model of the present invention, the order of cell layer formation and extracellular matrix formation is not particularly limited, and the cell layer may be formed first, or the extracellular matrix may be formed first. May be.

[Formation of extracellular matrix]
In the present specification, the term “extracellular matrix” refers to a living body that fills a space outside a cell in a living body and performs functions such as a skeletal role, a role of providing a scaffold, and / or a role of holding a biological factor. It refers to a substance containing a substance in the body, and may further contain a substance capable of performing functions such as a skeletal role, a role of providing a scaffold and / or a role of retaining a biological factor in in vitro cell culture. Unless otherwise specified in the present specification, the “extracellular matrix” means one formed by alternately arranging the first liquid and the second liquid on the cell layer and / or the substrate. Formation of such an extracellular matrix can be performed with reference to, for example, Japanese Patent Application Laid-Open No. 2007-279015, US publication No. 2007-0207540, and the like.

  The combination of the content of the first liquid and the content of the second liquid is a protein having an RGD sequence, from the viewpoint of ease of forming operation, ease of thickness adjustment, and efficiency of three-dimensional cell culture. A combination of a polymer (hereinafter also referred to as “first substance having RGD sequence”) and a protein or polymer that interacts with the first substance having RGD sequence (hereinafter also referred to as “second substance that interacts”). Or a protein or polymer having a positive charge (hereinafter also referred to as “first substance having a positive charge”) and a protein or polymer having a negative charge (hereinafter referred to as “a second substance having a negative charge”). ")"). In this specification, “interact” means, for example, electrostatic interaction, hydrophobic interaction, hydrogen bond, charge transfer interaction, covalent bond formation, specific interaction between proteins, van der Waals force, etc. Thus, it is preferable to mean that the first substance and the second substance are close enough to be bonded, adhered, adsorbed, or exchanged of electrons chemically and / or physically.

(First substance having RGD sequence)
The RGD sequence in the first substance having the RGD sequence, that is, the protein or polymer having the RGD sequence refers to a generally known “Arg-Gly-Asp” sequence. In the present specification, “having an RGD sequence” may originally have an RGD sequence, or may be an RGD sequence chemically bound thereto. The first substance having an RGD sequence is preferably biodegradable and preferably water-soluble. Examples of the protein having an RGD sequence include conventionally known adhesive proteins, and specifically include fibronectin, vitronectin, laminin, cadherin, collagen, and the like. In addition, the protein having an RGD sequence may be, for example, collagen, gelatin, albumin, globulin, proteoglycan, enzyme, antibody or the like to which the RGD sequence is bound. Examples of the polymer having an RGD sequence include naturally-derived polymers and synthetic polymers. Examples of naturally derived polymers having an RGD sequence include water-soluble polypeptides, low molecular peptides, polyamino acids such as polylysine, sugars such as chitin and chitosan, polyesters, polyurethanes, polycarbonates, polyamides, and copolymers thereof. Is mentioned. Examples of the synthetic polymer having an RGD sequence include polymers or copolymers having an RGD sequence such as linear, graft, comb, dendritic, and star. Examples of the polymer or copolymer include poly (N-isopropylacrylamide-co-polyacrylic acid), polyamidoamine dendrimer, polyethylene oxide, polyε-caprolactam, polyacrylamide, poly (methyl methacrylate-γ-polymethacrylic acid). Acid oxyethylene) and the like.

(Interactive second substance)
The interacting second substance is preferably biodegradable and preferably water-soluble. Among the interacting second substances, examples of the protein that interacts with the first substance having an RGD sequence include collagen, gelatin, proteoglycan, integrin, enzyme, and antibody. In addition, examples of the polymer that interacts with the first substance having the RGD sequence include naturally-derived polymers and synthetic polymers. Examples of naturally-derived polymers that interact with the first substance having an RGD sequence include, for example, water-soluble polypeptides, low-molecular peptides, elastin, polyamino acids, polyesters, sugars such as heparin, heparan sulfate, and dextran sulfate, polyurethane, polyamide , Polycarbonate, and copolymers thereof. Examples of the synthetic polymer that interacts with the first substance having the RGD sequence include polymers or copolymers having an RGD sequence such as a linear type, a graft type, a comb type, a dendritic type, and a star type. Examples of the polymer or copolymer include polyacrylic acid, polymethacrylic acid, polyethylene glycol-graft-polyacrylic acid, poly (N-isopropylacrylamide-co-polyacrylic acid), polyamidoamine dendrimer, polyethylene oxide, poly ε-caprolactam, polyacrylamide, poly (methyl methacrylate-γ-polyoxyethylene methacrylate) and the like can be mentioned.

  The combination of the first substance having the RGD sequence and the second substance that interacts is not particularly limited, and may be a combination of different substances that interact. For example, a combination of fibronectin and gelatin, laminin and gelatin, fibronectin and dextran sulfate, polylysine and elastin, fibronectin and collagen, laminin and collagen, vitronectin and collagen, RGD-bound collagen or RGD-bound gelatin and collagen or gelatin and the like can be mentioned. Among these, a combination of fibronectin and gelatin, or a combination of laminin and gelatin is preferable, and a combination of fibronectin and gelatin is more preferable. In addition, the 1st substance which has RGD arrangement | sequence, and the 2nd substance to interact may be one each, respectively, and may use 2 or more types together in the range which shows interaction.

(First substance having a positive charge)
Of the first substances having a positive charge, the protein having a positive charge is preferably a water-soluble protein, for example. Examples of the water-soluble protein include basic collagen, basic gelatin, lysozyme, cytochrome c, peroxidase, myoglobin, and the like. Among the first substances having a positive charge, examples of the polymer having a positive charge include naturally-derived polymers and synthetic polymers. Examples of naturally derived polymers include water-soluble polypeptides, low molecular peptides, polyamino acids, polyesters, sugars such as chitin and chitosan, polyurethanes, polyamides, polycarbonates, and copolymers thereof. Examples of the polyamino acid include polylysine such as poly (α-lysine) and poly (ε-lysine), polyarginine, polyhistidine and the like. Examples of the synthetic polymer include polymers or copolymers of linear type, graft type, comb type, dendritic type, star type and the like. Examples of the polymer or copolymer include polydiallyldimethylammonium chloride, polyallylamine hydrochloride, polyethyleneimine, polyvinylamine, and polyamideamine dendrimer.

(Second substance having negative charge)
Of the second substance having a negative charge, for example, a water-soluble protein is preferable as the protein having a negative charge. Examples of the water-soluble protein include acidic collagen, acidic gelatin, albumin, globulin, catalase, β-lactoglobulin, thyroglobulin, α-lactalbumin, ovalbumin and the like. Among the second substances having a negative charge, examples of the polymer having a negative charge include naturally-derived polymers and synthetic polymers. Examples of the naturally-derived polymer include water-soluble polypeptides, low-molecular peptides, polyamino acids such as poly (β lysine), dextran sulfate, polyester, polyurethane, polyamide, polycarbonate, and copolymers thereof. Examples of the synthetic polymer include polymers or copolymers of linear type, graft type, comb type, dendritic type, star type and the like. Examples of the polymer or copolymer include polyester, polyacrylic acid, polymethacrylic acid, polystyrene sulfonic acid, polyacrylamide methylpropane sulfonic acid, and terminal carboxylated polyethylene glycol.

  Examples of the combination of the first substance having a positive charge and the second substance having a negative charge include, for example, a combination of chitosan and dextran sulfate, a combination of polyallylamine hydrochloride and polystyrene sulfonic acid, and polydiallyldimethylammonium chloride. And a combination of polystyrene sulfonic acid and the like. Note that the first substance having a positive charge and the second substance having a negative charge may each be one kind, or two or more kinds may be used in combination within a range showing an interaction.

  Arrangement | positioning of a 1st liquid and a 2nd liquid can be performed by making a 1st liquid and a 2nd liquid contact a cell layer, for example. The contact between the first liquid and the second liquid can be performed by, for example, coating, dipping, dropping, spraying, or the like.

  The first liquid can be prepared, for example, by dissolving or dispersing the first substance having the above RGD arrangement or the first substance having a positive charge (hereinafter also referred to as “first substance”) in a solvent or a dispersion medium. . The content of the first substance in the first liquid is, for example, preferably 0.0001 to 1% by mass, more preferably 0.01 to 0.5% by mass, and still more preferably 0.02 to 0.1% by mass. is there.

  The second liquid can be prepared by, for example, dissolving or dispersing the second substance having the above RGD sequence or the second substance having a negative charge (hereinafter also referred to as “second substance”) in a solvent or a dispersion medium. . The content of the second substance in the second liquid is, for example, preferably 0.0001 to 1% by mass, more preferably 0.01 to 0.5% by mass, and still more preferably 0.02 to 0.1% by mass. is there.

  The solvent or dispersion medium (hereinafter also simply referred to as “solvent”) in the first liquid and the second liquid is not particularly limited, and for example, an aqueous solvent such as water, phosphate buffered saline (PBS), and buffer solution may be used. Can be mentioned. Examples of the buffer include Tris buffer such as Tris-HCl buffer, phosphate buffer, HEPES buffer, citrate-phosphate buffer, glycylglycine-sodium hydroxide buffer, Britton-Robinson buffer , GTA buffer and the like. Although the pH in particular of a solvent is not restrict | limited, For example, it is 3-11, Preferably it is 6-8, More preferably, it is 7.2-7.4.

  Although the order in which the first liquid and the second liquid are arranged is not particularly limited, it is preferable to arrange the second liquid containing the second substance after arranging the first liquid containing the first substance, and in this order. More preferably, the first liquid and the second liquid are alternately arranged.

  The thickness of the extracellular matrix membrane formed by arranging the first liquid and the second liquid once is usually about 1 to 20 nm, and the desired thickness can be obtained by repeatedly arranging the first liquid and the second liquid. A thick extracellular matrix layer can be formed. Moreover, you may adjust the thickness of the extracellular matrix layer formed according to content of the 1st substance and the 2nd substance which are contained in the 1st liquid and the 2nd liquid, respectively. By these methods, for example, an extracellular matrix layer having a thickness of 1 to 1000 nm, preferably 1 to 300 nm, more preferably 5 to 100 nm can be formed.

  In addition, it is known that extracellular matrix such as collagen and fibronectin existing in solid tumors are greatly related to tumor cell invasion / metastasis and drug delivery / permeation. For this reason, from the point of manufacturing a tumor tissue model closer to the environment in the living body, the amount of the first liquid and the second liquid, the thickness of the extracellular matrix layer, and the like are appropriately adjusted according to the tumor tissue model to be produced. It is preferable to do.

The first liquid and the second liquid further contain salts such as sodium chloride, calcium chloride, sodium hydrogen carbonate, sodium acetate, sodium citrate, potassium chloride, sodium hydrogen phosphate, magnesium sulfate, sodium succinate and the like. Also good. One kind of salt may be contained, or two or more kinds of salts may be contained. Both the first liquid and the second liquid may contain a salt, or either one may contain a salt. The salt concentration is not particularly limited, but is, for example, 1 × 10 ^{−6 to} 2M, preferably 1 × 10 ^{−4 to} 1M, more preferably 1 × 10 ^{−4 to} 0.05M.

  The first liquid and the second liquid are pharmaceuticals such as cell growth factor, cytokine, chemokine, hormone, bioactive peptide, disease therapeutic agent, preventive agent, inhibitor, antibacterial agent, anti-inflammatory agent, etc. You may contain a composition etc.

[Formation of cell layer]
The cell layer is formed by arranging a cell-containing solution containing cells derived from a solid tumor. The arrangement of the cell-containing liquid is preferably performed, for example, by arranging the cell-containing solution on a predetermined region on the substrate and / or an extracellular matrix formed in the predetermined region. In addition, arrangement | positioning of a cell containing solution can be performed similarly to arrangement | positioning of a 1st liquid and a 2nd liquid. In addition, the cell layer may be formed directly on a membrane filter to be described later or on an extracellular matrix formed on the membrane filter.

  Examples of the solid tumor-derived cells include solid tumor-derived stromal cells and tumor cells. Examples of solid tumor-derived stromal cells include fibroblasts derived from pancreatic cancer, gastric cancer, renal cancer, prostate cancer, pharyngeal cancer, colon cancer, and the like. Although it does not restrict | limit especially as a fibroblast, For example, a mouse | mouth pancreatic intraepithelial carcinoma origin fibroblast (K643f cell), a prostate cancer origin fibroblast (FB-D-5, FB-D-6), nasopharyngeal cancer Derived fibroblasts (NPC-fib), other fibroblasts established directly from human surgical resection material, and the like. Stromal cells may be those collected from human or non-human mammals (mouse, rat, rabbit, pig, etc.), may be cells derived from primary cancer, or are derived from metastatic cancer. It may be a cell.

  Examples of tumor cells include tumor cells derived from pancreatic cancer, stomach cancer, kidney cancer, prostate cancer, pharyngeal cancer, colon cancer and the like. Tumor cells may be those collected from human or non-human mammals (mouse, rat, rabbit, pig, etc.), may be cells derived from primary cancer, or may be derived from metastatic cancer. It may be a cell.

  In the formation of the cell layer, the cells to be used can be appropriately determined according to the purpose of the tumor tissue model to be produced, and one type of cell or a plurality of types of cells may be used. Examples of the combination of cells include a combination of a stromal cell derived from a solid tumor and a tumor cell, a combination of a stromal cell, a tumor cell and an immune cell, and a combination of a normal stromal cell and a tumor cell. .

  When a plurality of types of cells are used, a plurality of types of cells may be arranged in one cell layer, or cell layers having different cell types may be stacked. When a plurality of types of cells are arranged in one cell layer, the cells may be arranged using a cell-containing solution in which the cells to be arranged are mixed in advance, or each cell using a cell-containing solution containing each cell. A cell-containing solution may be placed.

  In cell layer formation, it is preferable to incubate for a certain period of time after placing the cell-containing solution. This incubation makes it easier for the arranged cells to grow two-dimensionally (in the plane direction) to form a monolayer cell. Incubation conditions are not particularly limited and can be appropriately determined depending on the cells. As general conditions, the temperature is, for example, 4 to 60 ° C., preferably 20 to 40 ° C., more preferably 30 to 37 ° C., and the time is, for example, 1 to 168 hours, preferably 3 to 24 hours. More preferably, it is 3 to 12 hours. Moreover, the culture medium used for cell culture is not particularly limited, and can be appropriately determined according to the cells. For example, Eagle's MEM medium, Dulbecco's Modified Eagle medium (DMEM), Modified Eagle medium (MEM), Minimum Essential medium, RDMI, GlutaMax medium, serum-free medium and the like can be used.

The cell concentration in the cell-containing solution is preferably (1.0) × 10 ⁴ to (1.0) × 10 ⁹ cells / mL, more preferably (1.0) × from the viewpoint of the efficiency of cell layer formation. 10 ⁵ to (1.0) × 10 ⁸ cells / mL, more preferably (1.0) × 10 ⁶ to (1.0) × 10 ⁷ cells / mL.

  As the medium for the cell-containing solution, the above-mentioned medium and / or Tris buffer, phosphate buffer, HEPES, PBS and the like can be used.

  A step of washing the cell layer after forming the cell layer may be included. The washing can be performed using, for example, the above aqueous solvent or the like, and is preferably performed using the solvent of the first liquid and / or the second liquid.

  In the method for producing a tumor tissue model of the present invention, the cell layer is preferably laminated on a membrane filter from the viewpoint of easy drug evaluation. From the viewpoint of handling, the cell layer is preferably laminated on a culture plate having a membrane filter, more preferably a culture plate having a housing part and a base part, and the base part being a membrane filter. That is. The housing part is preferably transparent. A commercial item may be used for this culture plate. Examples of commercially available products include Transwell (registered trademark), Cell Culture Insert (trade name), and the like.

  The pore size of the membrane filter is not particularly limited as long as the cultured cells can be retained on the membrane filter, and is, for example, 0.1 to 2 μm, and preferably 0.4 to 1.0 μm. Examples of the membrane material include polyethylene terephthalate (PET), polycarbonate, and polytetrafluoroethylene (PTFE).

[Tumor tissue model]
As another aspect, the present invention relates to a tumor tissue model produced by the production method of the present invention. According to the tumor tissue model of the present invention, for example, an effect can be obtained that drug evaluation can be performed in an environment close to an actual tumor tissue as compared with a conventional method. In addition, the tumor tissue model of the present invention can be an extremely useful tool, for example, in the kinetic evaluation of drugs with various molecular weights in the creation (screening) of new drugs, especially in the kinetic evaluation of drugs with large molecular weights.

  The tumor tissue model of the present invention comprises a laminated cell layer containing cells derived from at least two solid tumors and an extracellular matrix, and the extracellular matrix is formed between cell layers containing at least cells derived from solid tumors. Preferably it is. The solid tumor-derived cells and extracellular matrix are as described above.

  The number of cell layers laminated in the tumor tissue model is not particularly limited, but is preferably 3 layers or more, more preferably 4 layers or more, and still more preferably from the viewpoint of exerting properties and functions equivalent to those of living tissues such as humans. Is 5 layers or more, and more preferably 6 layers or more. The upper limit of the number of cells to be stacked is not particularly limited.

  The tumor tissue model is preferably formed on a substrate from the viewpoint of handling. Examples of the substrate include the membrane filter described above.

[Test substance evaluation method]
As yet another aspect, the present invention relates to a test substance evaluation method including evaluating the permeability of a test substance to a tumor tissue using the tumor tissue model of the present invention. According to the evaluation method of the present invention, for example, it is possible to achieve an effect that drug evaluation can be performed in an environment close to an actual tumor tissue as compared with a conventional method. In addition, the evaluation method of the present invention can be an extremely useful tool, for example, in the kinetic evaluation of drugs with various molecular weights in the creation (screening) of new drugs, especially in the kinetic evaluation of drugs with large molecular weights.

  The evaluation method of the present invention can be performed, for example, by bringing a test substance into contact with the tumor tissue model of the present invention and detecting the test substance that has passed through the tumor tissue model. In addition, the permeability of the test substance can be evaluated by, for example, the concentration of the test substance that has passed through the tumor tissue model. The test substance refers to a substance to be evaluated, and examples thereof include inorganic compounds and organic compounds. Moreover, the permeability test of the test substance in the evaluation method of the present invention can be performed, for example, by the method described in the examples.

  The evaluation method of the present invention is suitable for drug evaluation for solid tumors that tend to have severe stroma fibrosis. Examples of solid tumors that tend to have intense stroma fibrosis include pancreatic cancer and Skills gastric cancer.

  According to the evaluation method of the present invention, a tissue model in which normal fibroblasts are stacked is prepared, a tissue model of normal fibroblasts and a test substance are contacted, and a test in the tissue model of normal fibroblasts is performed. It may include evaluating the permeability of the test substance by evaluating the permeability of the test substance and the permeability of the test substance in the tumor tissue model.

  A method for evaluating the permeability of a test substance will be described with reference to the drawings, taking a form using a plate (cell culture insert) including a membrane filter as an example. FIG. 1 is a schematic diagram showing an example of a test substance permeability evaluation method.

  As shown in FIG. 1, the tumor tissue model 12 is formed on a cell culture insert 11, and the cell culture insert 11 is arranged on a culture dish (plate) 13. A test substance or a solution containing the test substance is dropped or applied from the upper stage of the cell culture insert 11 (arrow A), and the test substance and the tumor tissue model 12 are brought into contact with each other. Next, after culturing for a predetermined time, sampling is performed from the culture dish 13 (also referred to as the lower part of the cell culture insert) (arrow B), and the test substance is detected. Thereby, the permeability | transmittance of the to-be-tested substance with respect to a tumor tissue model can be evaluated.

  The contact with the test substance may be performed once or two or more times. The culture conditions can be appropriately set according to the purpose, and it is preferable to set the culture conditions suitable for the survival of the solid tumor-derived cells contained in the tumor tissue model.

[Evaluation kit]
In still another aspect, the present invention relates to an evaluation kit used for evaluating the permeability of a test substance, which includes the tumor tissue model of the present invention. According to the evaluation kit of the present invention, the method for evaluating a test substance of the present invention can be performed more simply.

  In the evaluation kit of the present invention, the tumor tissue model is preferably formed on a membrane filter, and more preferably is formed on a plate provided with the membrane filter.

  The evaluation kit may further include a product including at least one of a reagent, a material, a tool, and a device used for a predetermined test, and an instruction for the evaluation (an instruction manual).

  The evaluation kit may include a tissue model in which normal fibroblasts are stacked. The tissue model can be produced in the same manner as in the production method except that the cells to be stacked in the production method of the tumor tissue model are normal fibroblasts.

  The present invention will be further described below using examples. However, the present invention is not construed as being limited to the following examples.

In the description of the present specification, the following abbreviations are used.
FN: Fibronectin FITC: Fluoresceinisothiocyanato
FBS: fetal bovine serum DMEM medium: Dulbecco's modified Eagle medium PBS: phosphate buffered saline

Example 1
[Preparation of a layered culture (tumor tissue model) of pancreatic carcinoma in situ derived from fibroblasts]
A cell culture insert (trade name, manufactured by BD; pore size: 0.4 μm (HD)) placed in a 12-well culture plate was mixed with a first solution for forming an extracellular matrix (0.2 mg / mL fibronectin (Sigma), 10 mM phosphorus). Acid buffered saline (pH 7.4)) and a second solution for forming an extracellular matrix (0.2 mg / mL gelatin, 10 mM phosphate buffered saline (pH 7.4)) were alternately added. Subsequently, mouse pancreatic in situ carcinoma fibroblasts (K643f cells) dispersed in a small amount of medium were added to cell culture inserts coated with FN / gelatin (1.44 × 10 ⁵ cells / well). Thirty minutes after the addition of the cells, the medium was added to each of the upper and lower inserts (culture plate) of the insert, and incubated in a cell culture incubator (37 ° C., 5% CO ₂ ) for 12 to 24 hours. A cell layer was formed. Subsequently, PBS was added to the cell culture insert to wash the cell layer. By alternately adding the first liquid for forming an extracellular matrix and the second liquid for forming an extracellular matrix to the cell culture insert after washing, a FN / gelatin thin film (extracellular matrix) is formed on the surface of the K643f cell layer. ) Was formed. Furthermore, formation of the cell layer, washing, and formation of the FN / gelatin thin film were repeated twice. As the medium, RPMI1640 medium (Gibco) containing 20% FBS and 1% P / S was used.

  By the above method, a culture body (K643f cell tissue model) in which three K643f cell layers were laminated on the cell culture insert could be produced.

(Example 2)
The dextran permeability test was performed under the following conditions using the tumor tissue model in which three K643f cell layers prepared in Example 1 were laminated.

[Dextran permeability test]
The cell culture insert on which the tumor tissue model of Example 1 in which three K643f cell layers were laminated was washed with the above medium. 0.1 mg / mL FITC-dextran (dextran molecular weight: 250 kDa, hydrodynamic diameter: 12 nm, manufactured by Sigma) was added to the upper stage (11 in FIG. 1) of the cell culture insert and allowed to stand. After 1 hour, 4 hours, 8 hours, and 24 hours, the solutions were sampled from the top of the cell culture insert (11 in FIG. 1) and the bottom of the cell culture insert (culture plate, 13 in FIG. 1), respectively, and NanoDrop The fluorescence intensity of FITC-dextran in each was measured using a 3300 fluorescence spectrometer (trade name, manufactured by Thermo). The obtained fluorescence intensity (fluorescence intensity in the solution collected from the upper stage and fluorescence intensity in the solution collected from the lower stage) was converted to FITC-dextran concentration, and the ratio of dextran that permeated the tumor tissue model was measured from the following formula. The result is shown in FIG. In FIG. 2, the vertical axis represents the ratio of dextran that has passed through the tumor tissue model, and the horizontal axis represents the elapsed time after the addition of dextran.
Permeable dextran ratio (%) = [(Lower concentration) x (Lower volume) / (Initial upper density) x (Upper volume)] x 100

(Reference example)
As a reference example, mouse fetal skin-derived fibroblasts (NIH3T3 cells, 0.72 × 10 ⁵ cells / well) were used instead of K643f cells (1.44 × 10 ⁵ cells / well), and the medium was 10%. A culture in which three layers of NIH3T3 cells were laminated in the same manner as in Example 1 except that DMEM medium (Gibco) containing FBS (fetal bovine serum) and 1% P / S was used, and a dextran permeability test was performed. Went. The result is shown in FIG.

  As shown in FIG. 2, it was confirmed that the permeability of dextran was different between the tumor tissue model of Example 1 in which three K643f cell layers were laminated and the culture in the reference example in which three NIH3T3 cell layers were laminated. . Moreover, it was confirmed that the tumor tissue model of Example 1 has low dextran permeability compared to the culture of the reference example.

  In addition, cancer stromal tissue is considered to be a dense tissue compared to normal stromal tissue. As described above, the tumor tissue model of Example 1 in which three K643f cell layers were laminated had lower dextran permeability than the culture in which three NIH3T3 cell layers were laminated. The laminated structure of the tumor tissue model of Example 1 is considered to be a denser structure than the culture of the reference example in which three NIH3T3 cell layers are laminated. Therefore, it was suggested that the tumor tissue model of Example 1 in which three K643f cell layers were laminated could reproduce the cancer stromal tissue in three dimensions. Therefore, it is considered that the tumor tissue model of the present invention can be an extremely useful technique for evaluating the permeability of drugs such as anticancer agents.

(Example 3)
A tumor tissue model in which two K643f cell layers were stacked was prepared by forming an extracellular matrix and stacking the cell layers in the same manner as in Example 1 except that the cell layers were stacked in two layers.

Example 4
A tumor tissue model in which five K643f cell layers were stacked was prepared by forming an extracellular matrix and stacking the cell layers in the same manner as in Example 1 except that the cell layers were stacked in five layers.

(Example 5)
Using the tumor tissue model prepared in Example 3 (two layers of K643f cell layer) and the tumor tissue model prepared in Example 4 (layered five layers of K643f cell layer), dextran permeability was performed in the same manner as in Example 2. A test was conducted. As a result, the tumor tissue model of Example 4 in which five layers of the K643f cell layer were laminated had lower dextran permeability than the tumor tissue model of Example 3 in which two layers of the K643f cell layer were laminated. It was confirmed (date not shown).

(Example 6)
A dextran permeability test was conducted in the same manner as in Example 5 except that FITC-dextran (molecular weight of dextran: 2000 kDa, hydrodynamic diameter: 31 nm, manufactured by Sigma) was used instead of FITC-dextran (250 kDa, manufactured by Sigma). It was. As a result, the tumor tissue model of Example 4 in which five layers of the K643f cell layer were laminated had lower dextran permeability than the tumor tissue model of Example 3 in which two layers of the K643f cell layer were laminated. It was confirmed (date not shown). Further, in Example 6 using 2,000 kDa dextran, the permeability of dextran was improved in both the two-layer tumor tissue model and the five-layer tumor tissue model as compared with Example 5 using 250 kDa dextran. It was confirmed that it was low (date not shown).

  As described above, in Examples 5 and 6, the greater the number of cell layers of K643f cells, the lower the permeability of dextran, and the greater the molecular weight (hydrodynamic diameter) of dextran, the lower the permeability of dextran. It could be confirmed. Therefore, it was suggested that the tumor tissue model produced in Examples 1, 3, and 4 in which the K643f cells were stacked could reproduce the cancer stromal tissue in three dimensions. Therefore, it is considered that the tumor tissue model of the present invention can be a very useful technique for evaluating the permeability of drugs such as anticancer agents.

(Example 7)
An extracellular matrix is formed and a cell layer is laminated in the same manner as in Example 1 except that instead of K643f cells, cells in which cancer cells (K399 cells) and K643f cells are mixed at a ratio of 1: 2 are used. Thus, a tumor tissue model in which three cell layers including K643f cells and K399 cells were laminated was prepared.

(Example 8)
Except for using K399 cells instead of K643f cells, three layers of cell layers containing K643f cells and K399 cells were laminated by forming an extracellular matrix and laminating cell layers in the same manner as in Example 1. A tumor tissue model was prepared.

Example 9
Using the tumor tissue model prepared in Example 7 (three layers of cell layers containing K643f cells and K399 cells) and the tumor tissue model prepared in Example 8 (three layers of K399 cell layers) were used. The dextran permeability test was conducted in the same manner as in 2. The results are shown in FIG. 3 together with the results of Example 2 (results of the tumor tissue model (three layers of K643f cell layers prepared in Example 1) prepared in Example 1). In FIG. 3, the vertical axis represents the ratio of dextran that has passed through the tumor tissue model, and the horizontal axis represents the elapsed time after the addition of dextran.

  As shown in FIG. 3, the tumor tissue model of Example 7 in which three cell layers containing K643f cells and K399 cells were laminated, and the tumor tissue model in Example 8 in which three K399 cell layers were laminated, Compared with the tumor tissue model of Example 1 in which three K643f cell layers were laminated, it was confirmed that the permeability of dextran was low. From these results, the structure of the tumor tissue model of Example 7 in which three cell layers containing K643f cells and K399 cells were laminated and the structure of the tumor tissue model in Example 8 in which three K399 cell layers were laminated were NIH3T3. It is considered that the structure is denser than the culture of the reference example in which three cell layers are laminated. Therefore, it was suggested that the tumor tissue models of Examples 7 and 8 may reproduce the surrounding environment of the solid tumor in three dimensions.

  Further, as described above, it was confirmed that the tumor tissue models of Examples 7 and 8 had lower dextran permeability than the tumor tissue model of Example 1. For this reason, the structure of the tumor tissue model of Examples 7 and 8 is considered to be a finer structure than the tumor tissue model of Example 1 in which three K643f cell layers are stacked. Therefore, it was suggested that the surrounding environment of the solid tumor in the living body may be reproduced or imitated in three dimensions by using the tumor cell (cancer cell) and the stromal cell derived from the solid tumor. Therefore, it is considered that the tumor tissue model of the present invention can be a very useful technique for evaluating the permeability of drugs such as anticancer agents.

  The present invention is useful, for example, in the fields of medicine, pharmaceuticals, cosmetics, foods and the like.

11 Cell culture insert 12 Tumor tissue model 13 Culture dish

Claims (2)

A method for producing a tumor tissue model, comprising:
Arranging a cell-containing liquid containing cells derived from a solid tumor to form a cell layer;
Alternately arranging the first liquid and the second liquid to form an extracellular matrix on the cell layer; and
Laminating two or more cell layers on the extracellular matrix by disposing a cell-containing solution containing cells derived from a solid tumor to form a cell layer;
The combination of the content of the first liquid and the content of the second liquid is a combination of fibronectin and gelatin or a combination of laminin and collagen,
The method for producing a tumor tissue model, wherein the solid tumor-derived cells include at least one of solid tumor-derived fibroblasts and tumor cells. The method for producing a tumor tissue model according to claim 1, wherein the cell layers are laminated on a membrane filter.

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