Pulsating three dimensional structure and method for producing the same

Abstract

To provide a dynamically pulsating three dimensional structure and a method for manufacturing the same.SOLUTION: The three dimensional structure contains crosslinked collagen and cultured cells. The three dimensional structure beats as a whole in the culture medium. The method for producing a three dimensional structure includes a step of allowing a molded article made of crosslinked collagen to absorb a coating liquid containing an extracellular matrix, and a step of pressing the molded article after the liquid absorption to allow the coating liquid to flow out from the molded article, and then bringing a cell suspension into contact with the molded article to seed and culture cells in the molded article.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a three-dimensional structure, and more particularly to a three-dimensional structure obtained by cell culture.

[0002] In recent years, various techniques have been developed to culture cells on a culture substrate to form structures that are the same as or similar to biological tissues. In particular, the development of three-dimensional structures that pulsate in the same way as in vivo by cell culture has attracted attention.

[0003] Conventionally, a method of stacking thin cell sheets to construct a three-dimensional structure of cultured cells has been known, but the stacked sheets do not all pulsate. JP 2008-99565 A (Patent Document 1) discloses a technique for culturing cardiomyocytes using a sponge-like sheet made of collagen as a culture substrate to obtain myocardial tissue with pulsating ability.

[0004] Japanese Patent Application Laid-Open Publication No. 2019-34126 (Patent Document 2) discloses a self-moving myocardial tissue-collagen membrane capable of self-repetitive flapping-like movement, which comprises a membranous collagen and a myocardial tissue-like structure consisting of cultured cells formed on the upper and / or lower surfaces of the membranous collagen. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2008-99565 [Patent Document 2] Japanese Patent Application Publication No. 2019-34126 Summary of the Invention [Problem to be solved by the invention]

[0006] According to Example 1 of Patent Document 1, a sponge-like sheet is produced by placing an atelocollagen solution in a tray to a depth of 1 cm, treating it with ammonia, and then freeze-drying it. Therefore, it is presumed that a thick sponge-like sheet equivalent to 1 cm in thickness can be obtained. When such a thick sponge-like sheet is used, even if the cultured cardiomyocytes or myocardium-like tissue itself pulsates, it is difficult to induce a bending movement of the entire structure including the sponge-like sheet.

[0007] The self-moving myocardial tissue-collagen membrane disclosed in Patent Document 2 is capable of self-repetitive flapping-like movement as a whole membrane, but it has a macroscopic two-dimensional shape and is not a three-dimensional structure that mimics biological tissue.

[0008] An object of the present invention is to provide a three-dimensional structure that can be given a three-dimensional shape that mimics biological tissue and that pulsates dynamically as a whole, and a method for manufacturing the same. [Means for solving the problem]

[0009] The three-dimensional structure according to the present invention comprises cross-linked collagen and cultured cells, and the three-dimensional structure as a whole beats in a culture medium.

[0010] The method for manufacturing this three-dimensional structure includes: A step of allowing a coating liquid containing an extracellular matrix to be absorbed into a molded body made of crosslinked collagen; a step of pressing the liquid-absorbed molded body to cause the coating liquid to flow out of the molded body, and then bringing the molded body into contact with a cell suspension, thereby seeding and culturing cells on the molded body; It has the following characteristics. [Effects of the Invention]

[0011] According to the present invention, a three-dimensional structure that exhibits dynamic pulsating motion as a whole, which has not been seen before, is provided. This three-dimensional structure can have a three-dimensional shape that mimics biological tissue. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a photograph showing the appearance of the three-dimensional structure of Example 1 immersed in a culture medium. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will be described in detail below based on preferred embodiments, with reference to the accompanying drawings. Note that the present invention is not limited to the following embodiments, and various modifications are possible within the scope of the claims.

[0014] (three-dimensional structure) As a result of extensive research, the present inventors discovered that a three-dimensional structure that pulsates as a whole can be obtained by sufficiently absorbing a cell suspension into a molded body made of crosslinked collagen and culturing the cell suspension, thereby completing the present invention. Specifically, the three-dimensional structure of the present invention comprises crosslinked collagen and cultured cells, and pulsates as a whole in a culture medium. Specifically, the three-dimensional structure comprises crosslinked collagen as a scaffold for cell culture and cultured cells cultured on the crosslinked collagen as a scaffold. In this three-dimensional structure, the cultured cells may be adhered or attached to the crosslinked collagen. Here, "pulsation" of the three-dimensional structure of the present invention refers to the repeated periodic contraction and expansion of the entire structure when observed with the naked eye. This pulsation may be autonomous, like that of a heart, or may be induced by an external stimulus such as electrical stimulation. Furthermore, "pulsation as a whole" refers to the pulsation of the entire three-dimensional structure as a whole, synchronously, as a unit. The "beating" caused by the three-dimensional structure according to the present invention is essentially different from the autonomous beating caused by cardiac muscle cells or aggregates thereof that can be observed under a microscope.

[0015] Here, the term "three-dimensional structure" refers to an object having any three-dimensional shape, and may be one whose external shape or size changes in response to pulsation. Furthermore, as long as the effects of the present invention are obtained, the three-dimensional structure may be a combination of structures of the same shape or different shapes, or may be a combination of structures of the same size or different sizes.

[0016] The shape of the three-dimensional structure is not particularly limited as long as it is three-dimensional, and examples include a sphere, an ellipsoid, a cube, a rectangular parallelepiped, a polygonal three-dimensional shape, a three-dimensional shape with irregularities, and the shape of an organ such as a heart. The size of the three-dimensional structure is also not particularly limited, and can be appropriately selected within a range that achieves the effects of the present invention. The shape and size of the three-dimensional structure are determined by the shape and size of the molded product made of crosslinked collagen (hereinafter, sometimes simply referred to as "molded product") described below in the production method.

[0017] The three-dimensional structure of the present disclosure may include a porous structure. This porous structure may be derived from a molded article made of crosslinked collagen used as a culture substrate. When the three-dimensional structure has a porous structure, it can be confirmed by microscopic observation.

[0018] "Cultured cells" generally refer to cells cultured outside the body (in vitro). The "cultured cells" contained in the three-dimensional structure of the present invention refer to cells cultured using a shaped body made of crosslinked collagen as a scaffold and / or cells that are being cultured, and are a concept that includes an aggregate of multiple cells. In the three-dimensional structure of the present invention, the cultured cells are contained in an inseparable state with the shaped body made of crosslinked collagen used as a scaffold for cell culture. The density of the cultured cells contained in the three-dimensional structure affects the pulsating state of the three-dimensional structure. The optimal cell density is selected depending on the shape and size of the three-dimensional structure.

[0019] The type of cultured cells may be any that can be cultured using a molded body made of crosslinked collagen as a scaffold, and are typically adherent cells that are cultured in a state where they are attached to the surface of a substrate. From the viewpoint of achieving significant effects of the present invention, the preferred cultured cells are cardiomyocytes. They may be of human origin, may be of non-human origin, or may be prepared from artificial stem cells (iPS cells).

[0020] The cross-linked collagen contained in the three-dimensional structure according to the present invention is derived from the molded article used as a scaffold for cell culture. Details of the cross-linked collagen will be described later in the section on the production method.

[0021] As long as the effects of the present invention are achieved, the three-dimensional structure may contain other components in addition to the cross-linked collagen and cultured cells, such as fibrin, thrombin, gelatin, hyaluronic acid, chondroitin sulfate, and alginic acid.

[0022] From the viewpoint of exerting a greater pulsatile motion, the three-dimensional structure preferably comprises a shell containing crosslinked collagen and cultured cells and a hollow space surrounded by this shell. In other words, this three-dimensional structure is a hollow body. Here, "hollow space" means a space that is not substantially filled with the components of the shell, but the space forming this hollow space may contain a culture solution or the like. Furthermore, the shell containing crosslinked collagen and cultured cells may include a porous structure derived from the molded body made of crosslinked collagen used as a culture substrate.

[0023] The shape of the shell is not particularly limited, and examples thereof include a sphere, an ellipsoid, a cube, a rectangular parallelepiped, a polygonal three-dimensional shape, a three-dimensional shape with irregularities, the shape of an organ such as a heart, etc. For example, the cultured cells are cardiomyocytes, and the outer shape of the shell mimics the outer shape of a human heart.

[0024] The shell may have a single opening or multiple openings as long as the effects of the present invention are obtained. The size of the shell is not particularly limited and can be appropriately selected within the range in which the effects of the present invention are obtained.

[0025] The shape and size of the hollow portion may be selected according to the shape and size of the shell. By appropriately setting the thickness of the shell and the size of the hollow portion, more significant pulsating motion can be achieved. In addition, the density of cultured cells contained in the shell also affects the pulsating state.

[0026] FIG. 1 is a photograph showing an example of a three-dimensional structure according to the present invention. As shown in the figure, in this embodiment, the external shape of the three-dimensional structure is formed to resemble a human heart and is immersed in a culture solution. As will be described later in the Examples, this three-dimensional structure is hollow and contains cardiomyocytes as cultured cells. In this three-dimensional structure, the pulsation of the entire structure can be observed with the naked eye in a culture environment.

[0027] (Method of manufacturing three-dimensional structures) As described above, the three-dimensional structure according to the present invention can be obtained by culturing cells using a molded body made of cross-linked collagen as a scaffold. In detail, the method for producing the three-dimensional structure according to the present invention includes the following steps: a step of allowing a coating liquid containing an extracellular matrix to be absorbed into a molded body made of crosslinked collagen; and a step of pressing the liquid-absorbed molded body to cause the coating liquid to flow out of the molded body, and then bringing a cell suspension into contact with the molded body, thereby seeding and culturing cells on the molded body; It has the following characteristics.

[0028] This manufacturing method is characterized by having a scaffold molded body absorb a coating liquid to attach an extracellular matrix, and then pressing the molded body containing the coating liquid. This pressing causes the coating liquid in the molded body to flow out, and then a cell suspension is added, allowing a large number of cells to be efficiently seeded into the molded body, resulting in a three-dimensional structure containing cultured cells at a high density. This manufacturing method uses a molded body made of crosslinked collagen that has the strength and flexibility to withstand this pressing when wet.

[0029] In one embodiment, the shaped article made of crosslinked collagen may be a hollow body composed of a shell made of crosslinked collagen and a hollow portion surrounded by this shell, and in another embodiment, the shaped article made of crosslinked collagen may be a porous body.

[0030] A method for producing a molded article made of crosslinked collagen will be described below.

[0031] (First manufacturing method) A preferred embodiment of the method for producing a molded article made of crosslinked collagen includes a first step of forming a fibrous collagen gel produced by fibrillating a solubilized collagen solution to obtain a gel molded article, and a second step of performing a crosslinking treatment. Hereinafter, this embodiment of the method will be referred to as the first production method.

[0032] A solubilized collagen solution is an aqueous solution in which collagen is dissolved. The collagen is preferably water-soluble collagen having a triple helix structure. Although the solubilized collagen solution may contain peptides, amino acids, gelatin, etc., it is preferable to exclude these as much as possible.

[0033] Water-soluble collagen with a triple helix structure can be obtained by known methods from collagen-containing tissues of biological sources such as mammals, seafood, birds, and reptiles. Examples include [1] acid-solubilized collagen obtained by extraction with dilute acid, [2] enzyme-solubilized collagen obtained by enzymatic solubilization, and [3] alkali-solubilized collagen obtained by alkali solubilization. Acid-solubilized collagen and enzyme-solubilized collagen are soluble under acidic conditions, while alkali-solubilized collagen is soluble under alkaline conditions. However, both collagens are known to undergo fibrillation when the ionic strength and pH of the solubilized collagen solution are set within appropriate ranges. Particularly when intended for in vivo use, it is preferable to use atelocollagen, in which telopeptides, the antigenic determinants of collagen, have been removed. The type of collagen is not particularly limited, but type I collagen, which is abundant in living organisms, is preferred.

[0034] Collagen derived from fish that do not share viruses with humans is particularly suitable, and from the viewpoint of applicability to various uses, collagen with a relatively high denaturation temperature is preferred, and a good example is collagen derived from the Oreochromis genus. Of the Oreochromis genus, tilapia is particularly preferred because it is farmed primarily for food from China to Southeast Asia and is easily available.

[0035] Here, we will explain the method for obtaining the enzyme-solubilized collagen described in [2] above. The method for obtaining the collagen is not particularly limited and may be any conventional method. Examples include the methods described in Japanese Patent No. 4863433 or Japanese Patent No. 5692770. To briefly explain one embodiment of the method using scales as an example, scales are decalcified with acid and treated with a protease such as pepsin to ateloidize the collagen, followed by a purification process as necessary to obtain enzyme-solubilized collagen. Examples of purification methods that can be used include the salting-out method and the method using activated carbon at a pH of 7 or less described in Japanese Patent No. 5522857.

[0036] A conventional method for producing a fibrous collagen gel by fibrillating a solubilized collagen solution may be used. For example, a common method involves adding an appropriate aqueous solution to the solubilized collagen solution to achieve an appropriate ionic strength and pH. The pH of the aqueous solution is preferably set appropriately within the range of 3 to 10 depending on the type of collagen (acid-solubilized collagen, enzyme-solubilized collagen, alkali-solubilized collagen, etc.). For enzyme-solubilized collagen, for example, a buffer solution, buffered saline, neutral salt aqueous solution, etc., with a pH range of 6 to 8 is preferably used. Preferred examples include phosphate buffer, Tris buffer, HEPES buffer, acetate buffer, carbonate buffer, citrate buffer, PBS, D-PBS, Tris-buffered saline, HEPES-buffered saline, etc. Furthermore, an aqueous solution of an alkali metal bicarbonate may be used as the aqueous solution. Preferred alkali metal bicarbonate salts are sodium bicarbonate and potassium bicarbonate. The aqueous solution of an alkali metal bicarbonate may contain alkali metal carbonates, provided that the effects of the present invention are not impaired.

[0037] The generation of fibrous collagen can be confirmed by the clouding of the mixture of the solubilized collagen solution and the aqueous solution. As the clouding progresses, the gelation of the entire mixture also progresses. The gelled mixture is called a "fibrous collagen gel."

[0038] A suitable method for molding a fibrous collagen gel to obtain a gel molded body is to mold the fibrous collagen gel using a mold for producing a molded body of the desired shape. Alternatively, a gel molded body consisting of a shell made of fibrous collagen gel and a hollow portion can be obtained by joining two separate pieces of a gel molded body with a shell structure or by using a core according to a conventional method. The size and shape of the gel molded body are not particularly limited and can be selected appropriately depending on the size and shape of the desired three-dimensional structure, its application, etc.

[0039] In the second step, the gel molded body is crosslinked. Examples of crosslinking treatments include (1) physical crosslinking treatments using gamma ray irradiation, electron beam irradiation, plasma irradiation, UV irradiation, or thermal dehydration, and (2) chemical crosslinking treatments using a water-soluble chemical crosslinking agent or a chemical crosslinking agent with vaporization capability. Either (1) or (2) may be used alone, or (1) and (2) may be used in combination. Of course, multiple crosslinking treatments may be used for each of (1) and (2), for example, gamma ray irradiation may be performed after UV irradiation. The degree of crosslinking may be appropriately set depending on the purpose.

[0040] Among the physical crosslinking methods (1), crosslinking treatments using gamma-ray irradiation, electron beam irradiation, plasma irradiation, and UV irradiation can simultaneously perform sterilization by appropriately setting the irradiation conditions. Therefore, if the packaging is appropriately selected to maintain a sealed state during and after crosslinking, the product can be distributed to the market as a sterilized product. Of the above irradiation methods, gamma-ray irradiation is particularly preferred because of its high penetration power and ability to achieve uniform crosslinking. With gamma-ray irradiation, a fixed dose rate radiation source is used, and the desired irradiation dose can be easily obtained by appropriately setting conditions such as irradiation time. For example, when a cobalt-60 radiation source is used, crosslinking treatment can be performed with an absorbed dose of 5 to 75 kGy, with 5 to 50 kGy being preferred, 10 to 50 kGy being more preferred, and 15 to 30 kGy being even more preferred.

[0041] The crosslinking treatment using irradiation may also be performed in the presence of a liquid. Here, "in the presence of a liquid" refers to a state in which the entire surface of the gel molded body is covered with the liquid during the crosslinking treatment. For example, the gel molded body may be in a wet state, but preferably the entire gel molded body is immersed in the liquid. Therefore, the volume of the liquid is not limited as long as the entire surface of the gel molded body is covered with the liquid. However, the volume of the liquid is preferably 2 to 100 times, and more preferably 10 to 50 times, the volume of the gel molded body. The liquid is not limited as long as it contains water, and examples thereof include water, buffer solutions, and aqueous solutions of alkali metal bicarbonates. Specific examples of buffer solutions include phosphate buffer, Tris buffer, HEPES buffer, acetate buffer, carbonate buffer, and citrate buffer. Physiological saline solutions such as PBS, D-PBS, Tris-buffered saline, and HEPES-buffered saline may also be used.

[0042] As the water-soluble chemical crosslinking agent or the chemical crosslinking agent having vaporization ability, known agents may be used, such as glutaraldehyde, polyepoxy compounds (ethylene glycol diglycidyl ether, glycerol polyglycidyl ether, etc.), carbodiimide compounds (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, etc.), and reducing sugars (ribose, etc.), and crosslinking treatment may be carried out according to a conventional method.

[0043] (Second manufacturing method) A preferred embodiment of the method for producing a porous shaped article made of crosslinked collagen includes a first step of fibrillating a solubilized collagen solution to produce a fibrous collagen gel and molding the resulting gel shaped article, a second step of freeze-drying the fibrous collagen gel, and a third step of crosslinking the fibrous collagen gel. Hereinafter, this type of production method will be referred to as the second production method.

[0044] The second production method differs from the first production method in that a freeze-drying step is carried out after the first step. This freeze-drying allows a porous molded body to be obtained. Furthermore, by the method described above in the first production method, a gel molded body composed of a shell made of fibrous collagen gel and a hollow portion is obtained in the first step, and then freeze-drying can be carried out to obtain a molded body having a porous shell and a hollow portion surrounded by this shell.

[0045] The aqueous solution used to fibrillate the solubilized collagen solution in the first step of the second production method may be one exemplified in the first production method, with a preferred example being an aqueous solution of alkali metal bicarbonate. An aqueous solution of alkali metal bicarbonate can result in the formation of a more porous, open-pore, spongy structure during the freeze-drying step in the second step. The amount of alkali metal bicarbonate applied is not particularly limited as long as it is within a range that can produce a fibrous collagen gel. However, particularly for the stable production of a gel-shaped body composed of fibrous collagen, it is preferable to prepare a fibrous collagen gel that has been sufficiently gelled and whitened. To this end, it is preferable to appropriately set the mixing conditions, particularly the amount of alkali metal bicarbonate applied relative to the amount of collagen in the solubilized collagen solution. For example, if the amount of alkali metal bicarbonate applied relative to the amount of collagen in the solubilized collagen solution is too small, collagen fibrillation will be insufficient. On the other hand, if the amount is too large, the collagen may not be fibrillated (defibrillated) during the freeze-drying step in the second step. A preferred example of the amount of alkali metal bicarbonate to be applied is as follows: when the molecular weight of collagen is 300,000, the molar ratio of alkali metal bicarbonate to collagen in the solubilized collagen solution is 3×10 2 ~3×10 4 It is a quantity that falls within the range of

[0046] In the second step of the second production method, the gel molded body is freeze-dried to obtain a porous molded body. Although freeze-drying is usually performed while the gel molded body is still in the mold, freeze-drying may be performed after removal from the mold if the structure of the gel molded body can be maintained. A known freeze-drying method may be employed. The freeze-drying conditions may be appropriately set so that a porous molded body is obtained by a conventional method. For example, the freezing temperature is preferably in the range of -10 to -60°C, more preferably in the range of -20 to -50°C. The freeze-drying time is preferably 0.5 to 60 hours, more preferably 1 to 48 hours. Because fibrous collagen gel is adhesive and sticky, it is preferable to select a mold made of a material to which the fibrous collagen gel does not easily adhere, so that it can be easily removed after freeze-drying. The surface of the mold may be hydrophobized, and molds coated with a release agent or mold release agent, or molds with properties such as water repellency and moisture permeability may be used as needed.

[0047] The porous molded body is then subjected to a crosslinking treatment (third step) to obtain a molded body made of crosslinked collagen. The crosslinking treatment may be carried out in the same manner as in the second step of the first production method.

[0048] (porous body) Among porous shaped articles made of crosslinked collagen, porous shaped articles (including those having a porous shell and a hollow space surrounded by this shell) (hereinafter referred to as "porous articles") capable of three-dimensional cell culture are not particularly limited as long as they have a spongy structure with continuous pores of a pore size suitable for smooth cell migration, allowing cells to penetrate into the substrate. A preferred embodiment of the pore size is one in which, in a scanning electron microscope image of the porous article, the number of pores observed on the outermost surface within a given compartment is at least 30, and when the maximum number of pores is n, the average pore size calculated by the formula: average pore size = {Σ(maximum width of pore i + minimum width of pore i) / 2} / n (where i = 1 to n) is in the range of 50 to 300 μm. A more preferred range for the average pore size is 70 to 250 μm. Note that the pores referred to herein are complete pores and do not include incomplete pores, which are pores separated by compartment boundaries. The magnification of the scanning electron microscope image for determining the average pore size is preferably about 100 times.

[0049] (An example of a suitable manufacturing method using a porous body) The cell culture method for effectively distributing cells three-dimensionally in a porous body by utilizing the mechanical properties of the porous body is as follows.

[0050] First, the porous body is dried or dehydrated. If the porous body is already dry or dehydrated, it may be used as is. However, if a porous body is to be used that is wet due to storage in a liquid, for example, it is first dried or dehydrated. When drying or dehydrating, it is preferable to use a technique in which the solvent is replaced with a buffer solution or medium that is isotonic with the cells (preferably the same medium as that used in the subsequent cell culture step), and then drying or dehydration is performed. When drying, it is preferable to dry at a low temperature to prevent collagen denaturation, for example, by low-temperature ventilation. Furthermore, dehydration may be performed by centrifugal dehydration, but if the mechanical strength is sufficient, a simple method of dehydration is to compress it with fingers or tweezers.

[0051] Next, the dried or dehydrated porous body is allowed to absorb a coating solution containing an extracellular matrix. At this time, the coating solution penetrates into the porous body, and the extracellular matrix adheres to the surface of each pore. After that, the porous body is dried or dehydrated in the same manner as described above.

[0052] Next, a cell suspension is absorbed into the dried or dehydrated porous body. During absorption of the cell suspension, the cells enter the porous body together with the medium, allowing the cells to be distributed three-dimensionally within the porous body. In a preferred embodiment, the cells are distributed from the outer surface to the deepest part of the porous body. The cell suspension absorption procedure may be performed multiple times. After this procedure, normal cell culture can be performed. For example, in the case of cardiomyocytes, they can be cultured using Dulbecco's modified Eagle's medium (DMEM) containing a Rho kinase inhibitor and fetal bovine serum (FBS) at a temperature of 37°C and a CO2 concentration of 5%. By continuing to culture in an environment appropriate for the cell type, a three-dimensional structure that beats as a whole can be obtained.

[0053] In the method for producing a three-dimensional structure according to the present invention, the type of cells to be cultured is not particularly limited, as long as a three-dimensional structure that pulsates as a whole in a culture medium can be obtained. For example, cardiomyocytes are preferred from the viewpoint of obtaining an autonomously pulsating three-dimensional structure. Examples of cardiomyocytes include cardiomyocytes isolated from cardiac muscle, ES cell-derived cardiomyocytes, iPS cell-derived cardiomyocytes, cardiomyocytes differentiated from cardiac tissue stem cells, and commercially available cardiomyocytes. Cells capable of differentiating into cardiomyocytes are not particularly limited as long as they can differentiate into cardiomyocytes, and examples include ES cells, iPS cells, and cardiac tissue stem cells. As long as the objective of the present invention is not impaired, cardiomyocytes or cells capable of differentiating into cardiomyocytes may be mixed with other types of cells, such as fibroblasts and vascular cells, and other types of cells may be seeded into a molded body together with cardiomyocytes or cells capable of differentiating into cardiomyocytes.

[0054] The extracellular matrix can be selected depending on the type of cells. For example, when culturing cardiomyocytes, a preferred extracellular matrix may be one or more selected from the group consisting of iMatrix511, iMatrix511-MG, iMatrix511-silk, iMatrix221, and fibronectin. When iMatrix511, iMatrix511-MG, iMatrix511-silk, or iMatrix221 is used as the extracellular matrix, the concentration of the coating solution is preferably 0.5 to 5.0 μg / mL. When fibronectin is used as the extracellular matrix, the concentration of the coating solution is preferably 5.0 to 50 μg / mL.

[0055] The number of cells to be seeded into the molded body can be set depending on the shape and size of the molded body, the type of cells, etc. For example, when culturing using a hollow body with a porous shell, the seeding density can be calculated from the volume of the shell that serves as a scaffold for culture and the number of cells to be seeded. The seeding density is adjusted so that the entire structure is in a pulsating state.

[0056] The method for managing the three-dimensional structure by maintaining a pulsating state as a whole in the culture medium may be under environmental conditions suitable for the survival of the cultured cells. For example, environmental conditions in which the type of culture medium (culture medium), temperature, etc. are appropriately set are preferred. One suitable embodiment is to set the cell culture conditions until pulsation of the three-dimensional structure is observed.

[0057] (Application) Applications of the three-dimensional structure of the present invention include, but are not limited to, drug evaluation methods and regenerative medical applications, since the structure can assume a shape that mimics biological tissue. [Example]

[0058] The effects of the present invention will be clarified below by examples, but the present invention should not be construed as being limited based on the descriptions of these examples.

[0059] [Example 1] (Solubilized collagen solution) The solubilized collagen solution used was a colorless, transparent solution (hereinafter referred to as "collagen solution A") prepared by dissolving "Cellcampus FD-08G" sponge manufactured by Taki Chemical Co., Ltd., which is made from tilapia scales, in an HCl solution of pH 3 to adjust the collagen concentration to 2.2% by mass.

[0060] (Molded body) A fibrous collagen gel was obtained by mixing 9 parts by volume of collagen solution A with 1 part by volume of 0.5 M aqueous sodium bicarbonate solution. Using inner and outer molds modeled after the human heart, a gel molded article consisting of a fibrous collagen gel composed of a shell and a hollow portion was produced so that the shell thickness was as uniform as possible. As a guide to the size of the mold, the distance in a straight line connecting the apex of the heart and the point furthest from the apex to the right atrium was 22.0 mm for the external mold and 18.3 mm for the internal mold. Furthermore, the width of the ventricular portion, measured on a line passing approximately midway between the apex and the left atrioventricular orifice and approximately perpendicular to the ventricular septum, was 14.6 mm for the external mold and 12.2 mm for the internal mold. The gel-shaped body was freeze-dried at -35°C for 3 hours, and then immersed in a 0.05M aqueous solution of sodium bicarbonate and irradiated with 25 kGy of gamma rays to obtain a heart-shaped body made of cross-linked collagen with a porous shell (pore diameter 150-200 μm). The thickness of the shell was measured at multiple points in the left and right ventricles (excluding the area near the apex and the areas near the left and right atrium), and was found to be approximately 0.7-1.4 mm.

[0061] (cell culture) <Day (-1)> 1. 3-5 mL of PBS(-) (Phosphate Buffered Saline without Calcium and Magnesium) was added to each well of a 12-well plate. 2. The heart-shaped molded body was immersed in the PBS(-) prepared in 1 to allow it to absorb the liquid. 3. Using a micropipette (Gilson's "Pipetman P1000"), the PBS (-) was removed from the well. At this time, the heart-shaped molded body was crushed to remove as much of the PBS (-) absorbed by the heart-shaped molded body as possible. 4. Using a micropipette (the aforementioned Pipetman P1000), 3 to 5 mL of coating solution containing ECM (extracellular matrix) was added to the heart-shaped molded body so that it soaked into the body. As ECM, iMatrix511, iMatrix511-MG, iMatrix511-silk, iMatrix221, and fibronectin can be used alone or in combination. The concentrations used are as follows: iMatrix511, iMatrix511-MG, iMatrix511-silk, iMatrix221: 0.5~5.0 μg / mL Fibronectin: 5.0 to 50 μg / mL 5. The 12-well plate containing the heart-shaped molded body to which the coating solution had been added was left to stand for at least 1 hour in an incubator set at 37°C and a CO2 concentration of 5%.

[0062] <day0> 1. A medium (20% FBS / DMEM+Y) was prepared by adding the Rho kinase inhibitor Y-27632 to 20% FBS / DMEM to a final concentration of 10 μM. FBS and DMEM are abbreviations for fetal bovine serum and Dulbecco's modified Eagle's medium, respectively. 2. Cryopreserved cardiomyocytes were thawed and added to a medium (20% FBS / DMEM+Y) to prepare a cell suspension. 3. The 12-well plate was removed from the incubator, and the coating solution was removed from the wells using a micropipette (the aforementioned Pipetman P1000). At this time, the heart-shaped molded bodies were crushed to remove as much of the coating solution as possible that had been absorbed into the heart-shaped molded bodies. 3. Using a micropipette (the aforementioned Pipetman P1000), 1 to 5 mL of the cell suspension was added to the heart-shaped molded bodies so that the cell count was 5.0E+06 to 5.0E+07 cells per heart-shaped molded body. 4. The 12-well plate containing the heart-shaped molded bodies with the cell suspension added was placed in an incubator set at 37°C and 5% CO2 for 1-3 hours, after which the medium was removed so that 1-2 mL remained in each well. An equal volume of fresh medium (20% FBS / DMEM+Y) was then added. 5. The cells were cultured in an incubator set at 37°C and 5% CO2.

[0063] <day1> 1. After 24 hours or more had passed since the cardiomyocytes were seeded, the medium was removed so that 1-2 mL remained in each well, and then an equal volume of fresh medium (20% FBS / DMEM) was added. 2. The cells were cultured in an incubator set at 37°C and 5% CO2.

[0064] <day3> 1. 6-10 mL of fresh medium (20% FBS / DMEM) was added to each well of a 6-well plate. 2. The heart-shaped bodies being cultured in the 12-well plate were transferred to a 6-well plate using tweezers, and the culture was continued in an incubator set at 37°C and a CO2 concentration of 5%.

[0065] <Day4~> After the fourth day of culture, when the medium turned yellow due to phenol red, a pH indicator contained in the medium, the medium was removed so that 1 to 2 mL remained in each well, and then an equal volume of fresh medium (20% FBS / DMEM) was added to continue culturing, and this process was repeated to obtain the three-dimensional structure of Example 1. This three-dimensional structure had a three-dimensional shape mimicking the external shape of the human heart, and was a composite of a porous shell made of cross-linked collagen and an aggregate of cardiomyocytes (myocardium-like tissue) cultured using this shell as a scaffold.

[0066] (Operation confirmation) The three-dimensional structure of Example 1 was maintained in the above culture environment, and its behavior was observed. In this three-dimensional structure, autonomous beating of myocardial-like tissue was observed from approximately four days after seeding, and beating movement of the entire three-dimensional structure was observed from approximately six days after seeding. Figure 1 shows a photograph of the appearance of the three-dimensional structure in culture medium on day 10 after seeding. When the size of the beating three-dimensional structure is indicated by the linear distance connecting the upper and lower ends (double arrow in Figure 1), it was 17 mm at its maximum contraction and 18 mm at its maximum expansion.

[0067] [Disclosure items] Each of the following sections discloses a preferred embodiment.

[0068] [Item 1] A three-dimensional structure containing cross-linked collagen and cultured cells that pulsates as a whole in a culture medium.

[0069] [Item 2] Item 2. The three-dimensional structure according to item 1, which is composed of a shell containing the crosslinked collagen and the cultured cells, and a hollow portion surrounded by this shell.

[0070] [Item 3] 3. The three-dimensional structure according to item 1 or 2, wherein the cultured cells are cardiomyocytes.

[0071] [Item 4] Item 4. The three-dimensional structure according to item 2 or 3, wherein the outer shape of the shell mimics the outer shape of a human heart.

[0072] [Item 5] a step of allowing a coating liquid containing an extracellular matrix to be absorbed into a molded body made of crosslinked collagen; and a step of pressing the liquid-absorbed molded body to cause the coating liquid to flow out of the molded body, and then bringing a cell suspension into contact with the molded body, thereby seeding and culturing cells on the molded body; Item 2. The method for producing a three-dimensional structure according to Item 1, comprising:

[0073] [Item 6] Item 6. The method according to item 5, wherein the molded body comprises a shell made of crosslinked collagen and a hollow portion surrounded by the shell.

[0074] [Item 7] Item 7. The method according to item 6, wherein the shell is porous.

[0075] [Item 8] The shell has an outer shape that resembles the outer shape of a human heart, 8. The method according to item 6 or 7, wherein the cells are cardiomyocytes. [Industrial Applicability]

[0076] The method for manufacturing a three-dimensional structure described above can be applied to cells other than cardiomyocytes. Furthermore, this three-dimensional structure can be shaped to resemble various biological tissues, and can be used in drug evaluation, regenerative medicine, etc.

Claims

1. A three-dimensional structure containing cross-linked collagen and cultured cells that pulsates as a whole in a culture medium.

2. 2. The three-dimensional structure according to claim 1, comprising a shell containing the crosslinked collagen and the cultured cells, and a hollow portion surrounded by the shell.

3. The three-dimensional structure according to claim 1, wherein the cultured cells are cardiomyocytes.

4. 3. The three-dimensional structure of claim 2, wherein the shell has a contour that mimics the contour of a human heart.

5. a step of allowing a coating liquid containing an extracellular matrix to be absorbed into a molded body made of crosslinked collagen; and a step of pressing the liquid-absorbed molded body to cause the coating liquid to flow out of the molded body, and then bringing a cell suspension into contact with the molded body, thereby seeding and culturing cells on the molded body; The method for producing a three-dimensional structure according to claim 1 , comprising:

6. 6. The method according to claim 5, wherein the molded article comprises a shell made of cross-linked collagen and a hollow portion surrounded by the shell.

7. The method of claim 6 wherein the shell is porous.

8. The shell has an outer shape that resembles the outer shape of a human heart, The method of claim 6, wherein the cells are cardiomyocytes.

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