JP6621245B2 - Method for producing artificial tissue having three-dimensional structure including controlled microstructure - Google Patents

Description

  The present invention relates to a method for manufacturing an artificial tissue having a three-dimensional structure including a controlled microstructure.

  An artificial tissue having a three-dimensional structure imitating a living tissue or organ (for example, an organ) is expected to be applied to the field of drug discovery and regenerative medicine. Artificial tissue having a three-dimensional structure is usually formed by assembling cells at high density. The biggest problem in producing an artificial tissue having such a three-dimensional structure is to supply oxygen and nutrients necessary for survival to cells inside the artificial tissue and / or functional substances produced by the cells. And the introduction of a microstructure such as a vascular network to efficiently release waste products to the outside of the artificial tissue. The vascular network has a specific density and a specific form of network structure required for its maintenance and functional expression in a living tissue or organ. Examples of the fine structure in a living tissue or organ include a fine structure containing a plurality of types of cells indispensable for the expression of the function of the tissue or organ, such as hepatic lobule in the liver and nephron in the kidney, in addition to the vascular network. it can. If such a fine structure can be designed and introduced into an artificial tissue in an arbitrary shape, a large artificial tissue having a three-dimensional structure can be efficiently produced. A large artificial tissue having a three-dimensional structure including such a fine structure can be used not only as a transplant material having a specific function in the field of regenerative medicine, but also reproduces various pathologies in the field of drug discovery. It can be applied to pharmacological tests as a tissue or organ model.

  Several techniques have been developed as a method for producing an artificial tissue having a three-dimensional structure. For example, Patent Document 1 discloses a substrate, and a stimulus-responsive layer that is disposed on the substrate and has a cell-adhesive property and includes a stimulus-responsive material that can express cell non-adhesiveness by stimulation. A cell adhesion inhibiting layer including a cell adhesion inhibiting material having a cell non-adhesive property, arranged in a pattern on the substrate, and the surface of the cell adhesion inhibiting layer is on the surface of the stimulus-responsive layer The lower limit of the height of the cell adhesion-inhibiting layer is a height that cannot be overcome by the vascular endothelial cells, and the upper limit is the micropatterning formed on the stimulus-responsive layer. Described is a micropatterned vascular endothelial cell transfer substrate characterized in that the height is such that vascular endothelial cells can be collected. This document is also a method for producing an artificial tissue containing at least micropatterned vascular endothelial cells, wherein the micropatterned vascular endothelial cell transfer substrate is prepared, and the stimulus-responsive layer expresses cell adhesion A micropatterned vascular endothelial cell forming step for forming the micropatterned vascular endothelial cell by seeding and culturing the vascular endothelial cell on the stimulus-responsive layer under conditions, and the micropatterned vascular endothelial cell forming step By contacting the micropatterned vascular endothelial cell with a cell recovery layer including a cell adhesion layer having cell adhesion under the condition that the subsequent stimulus-responsive layer expresses cell non-adhesiveness, A vascular endothelial cell transfer step of transferring a patterned vascular endothelial cell to the cell recovery layer. Law describes.

  Patent Document 2 discloses at least one container for holding a solution containing a material for forming a cell tissue, a substrate to be patterned, a voltage applying unit that applies a voltage to the container, a container, The voltage application using a pattern generation device for patterning a cell tissue on a substrate using an electrostatic ink jet method provided between the substrate and a grounded flat plate electrode provided with a hole When a voltage of 0.5 kv to 5 kv is applied between the container and the plate electrode by the unit, an electric field is generated between the container and the plate electrode, and the solution is removed from the container by electrostatic ink jet phenomenon. Is discharged, and the discharged solution passes through the holes formed in the flat plate electrode and adheres to the substrate, thereby patterning the cell tissue on the substrate and allowing the cells to survive A method for producing a cellular tissue having a three-dimensional structure, comprising a step of discharging a scaffold and cells on a substrate, and attaching and patterning the scaffold and the cells so that the scaffold and the cells are in contact with each other. A method for producing a cellular tissue having a three-dimensional structure in a state in which the cells are alive and are adhered and the three-dimensional structure is maintained by scaffolding will be described.

  Non-Patent Document 1 describes a fibroblast produced using a temperature-responsive culture dish coated with a temperature-responsive polymer capable of changing from cell adhesion to cell non-adhesion in response to temperature changes. A cell sheet-like fibroblast layer and a vascular endothelial cell layer containing a vascular endothelial cell arranged in a specific pattern shape are laminated in any number of layers and in an order, thereby forming a vascular network-like fine layer. The result of producing an artificial tissue having a three-dimensional structure including the structure is described.

  Non-Patent Document 2 describes a three-dimensional structure in which three or five human skeletal muscle myoblast cell layers are laminated using an automated cell sheet laminating apparatus using a temperature-responsive culture dish coated with a temperature-responsive polymer. The result of producing an artificial tissue having a structure will be described.

JP 2011-223944 Japanese Patent No. 5540304

M. Muraoka et al., Biomaterials, Volume 34, 2013, p. 696-703 T. Kikuchi et al., Biomaterials, Volume 35, 2014, p. 2428-2435

  As described above, a method for producing an artificial tissue having a three-dimensional structure is known. However, there are points to be improved in the prior art methods. For example, in the prior art, it has been difficult to stably introduce a fine structure such as a vascular network to a desired position in the three-dimensional structure of the artificial tissue. In the case of the method described in Non-Patent Document 1, vascular endothelial cells arranged so as to form a pattern-shaped fine structure inside the artificial tissue may actively migrate by interacting with other cells. It has been reported. As a result, in the artificial tissue produced by the method described in the document, it was difficult to maintain the introduced microstructure for a long period of several days or more. Such a phenomenon may occur even when cells other than vascular endothelial cells are used in the method described in Non-Patent Document 1.

  Therefore, an object of the present invention is to provide a means for producing an artificial tissue having a three-dimensional structure including a fine structure that can maintain a predetermined fine structure for a certain period of time.

  As a result of various studies on means for solving the above problems, the present inventors have found that a parenchymal tissue having a cell structure containing a parenchymal cell that adheres to and extends on the surface of a cell culture support, and a predetermined fine structure. After laminating a non-parenchymal cell tissue having a cell structure including a vascular endothelial cell that adheres and extends to the surface of a cell culture support having a template corresponding to the shape of the structure, and then contracts. It was found that the artificial tissue having a three-dimensional structure including the microstructure obtained by the above method can maintain the microstructure of the non-parenchymal cell tissue for several days. Based on the above findings, the present inventors have completed the present invention.

That is, the gist of the present invention is as follows.
(1) A method for producing an artificial tissue having a three-dimensional structure including a fine structure,
Preparing a plurality of extended parenchymal cell tissues having a cell structure containing parenchymal cells;
A non-parenchymal cell tissue preparation step of preparing one or a plurality of extended non-parenchymal cell tissues having a cell structure including the non-parenchymal cells having the fine structure;
Laminating the plurality of stretched parenchymal tissue and one or more stretched non-parenchymal tissue having the microstructure so that each of the non-parenchymal tissue is sandwiched between the stretched parenchymal tissue A cell tissue stacking step;
A contraction step of contracting a laminate of cell tissues having a three-dimensional structure including a fine structure obtained in the cell tissue stacking step;
Said method.
(2) The parenchymal cells differentiate into cancer cells, hepatocytes, epithelial cells, fibroblasts, mural cells, cardiomyocytes, myoblasts, bone cells, chondrocytes, fat cells and glial cells, and these cells. The method according to (1) above, wherein the cell is one or more cells selected from the group consisting of pluripotent stem cells having the ability to
(3) The non-parenchymal cell is one or more selected from the group consisting of endothelial cells, neurons, photoreceptors, glandular epithelial cells and pancreatic islet cells, and pluripotent stem cells capable of differentiating into these cells. The method according to (1) or (2) above, which is a cell.
(4) The method according to any one of (1) to (3), wherein in the shrinking step, the laminate is shrunk at a shrinkage rate of 30% or less.
(5) The microstructure comprises a vascular network, lymphatic network, hepatic lobule, nephron, cranial nerve circuit, neuromuscular junction, bone tissue, cartilage tissue, endocrine gland, retina, and microstructure for stem cell culture The method according to any one of (1) to (4), wherein the method is one or more fine structures selected from the above.

  According to the present invention, it is possible to provide a means for producing an artificial tissue having a three-dimensional structure including a fine structure that can maintain a predetermined fine structure for a certain period.

FIG. 1 is a view showing a cell culture support having a microstructured template used for producing the artificial tissues of Examples 1 to 3. FIG. A: Top view of a cell culture support having a microstructured template; B: Schematic diagram of a plurality of stripe-like temperature-responsive regions that are microstructured templates. FIG. 2 is a schematic diagram showing the procedure of the cell tissue laminating step and the shrinking step by the gelatin stamp method. FIG. 3 is a superimposed image of the fluorescence image (red) immediately after lamination and contraction of the three layers of the artificial tissue at the same location in Examples 1 and 2, and the fluorescence image (green) on the fifth day after the lamination and contraction. . A: Image of Example 1; B: Image of Example 2. The scale bar represents 200 μm. FIG. 4 shows the first-layer fibroblast tissue (red), the second-layered vascular endothelial cell tissue (green), and the third-layer fibroblast tissue in the artificial tissue of Example 1. It is a superimposed image of (red) fluorescent images. A: Fluorescence image immediately after lamination and shrinkage; B: Fluorescence image 12 hours after lamination and shrinkage. The scale bar represents 200 μm. FIG. 5 is a fluorescence image of the vascular endothelial cell tissue having the stripe-like microstructure of the second layer of Examples 1 to 3 from immediately after lamination and contraction to the fifth day at the same location. A: fluorescent image of Example 1; B: fluorescent image of Example 2; C: fluorescent image of Example 3. The scale bar represents 200 μm. FIG. 6 shows a fluorescence image from immediately after lamination and contraction of the vascular endothelial cell tissue having the second layer stripe-like microstructure of Example 1 to the fifth day, and a binarized image of the fluorescence image. It is. A: fluorescent image; B: binarized image. FIG. 6 is a diagram showing a change with time of a correlation coefficient by image correlation analysis of an image obtained by time-lapse observation in the artificial tissue of Example 1. 3 is a three-dimensional fluorescence image on the fifth day after the artificial tissue laminating step and the shrinking step of Example 1. FIG.

  In the present specification, features of the present invention will be described with reference to the drawings as appropriate. In the drawings, the size and shape of each part are exaggerated for clarity, and the actual size and shape are not accurately depicted. Therefore, the technical scope of the present invention is not limited to the size and shape of each part shown in these drawings.

<1: Production method of artificial tissue>
The present invention relates to a method for manufacturing an artificial tissue having a three-dimensional structure including a fine structure. In the present invention, “microstructure” means a structure having a specific pattern shape required for maintenance and function expression in a living tissue or organ (for example, an organ). Examples of the microstructure include, for example, a vascular network, a lymphatic network, a hepatic lobule, a nephron, a cranial nerve circuit, a neuromuscular junction, a bone tissue, a cartilage tissue, various endocrine glands, a retina, and a microstructure for stem cell culture. Can be mentioned. In an artificial tissue having a three-dimensional structure including a microstructure produced by a prior art method, it was difficult to maintain the microstructure for a long period of time of several days or more (M. Muraoka et al., Biomaterials 34, 2013, p. 696-703). The present inventors have found that an artificial tissue having a three-dimensional structure including a microstructure produced by the method of the present invention described below can maintain the microstructure for several days. Therefore, according to the method of the present invention, an artificial tissue capable of maintaining a fine structure over a longer period than that of the prior art method can be produced.

  In the artificial tissue produced by the method of the present invention, the microstructure may be a mature state having a structure and function substantially equivalent to those in a living tissue or organ, and in the future, an equivalent structure and It may be an immature state having the ability to express a function. The shape of the microstructure in the mature state is, for example, a luminal shape. In this case, the shape of the microstructure in the immature state is a shape that can form a lumen in the future, for example, a striped sheet shape. It is. For example, when the microstructure is a vascular network, the mature microstructure is preferably a blood vessel-like luminal shape composed of vascular endothelial cells, and the immature microstructure is composed of vascular endothelial cells. It preferably has a striped sheet shape. When the microstructure is a lymphatic network, the mature microstructure preferably has a lymphatic-like luminal shape composed of lymphatic endothelial cells, and the immature microstructure is lymphatic endothelial cells It preferably has a striped sheet shape made of

  The method of the present invention includes a parenchymal cell tissue preparation step, a non-parenchymal cell tissue preparation step, a cell tissue lamination step, and a contraction step. The method of the present invention also optionally further comprises a parenchymal cell tissue forming step and a non-parenchymal cell tissue forming step. Hereafter, each process of the method of this invention is demonstrated in detail.

[1-1: Parenchymal cell tissue preparation process]
The method of the present invention needs to include a parenchymal tissue preparation step of preparing a plurality of stretched parenchymal tissues having cell structures containing parenchymal cells.

  In the present invention, “parenchymal cell” means a cell that constitutes a substrate that occupies the majority in a tissue or organ. Examples of parenchymal cells used in this step include various mesenchymal cells and glial cells, and pluripotent stem cells having the ability to differentiate into these cells. Parenchymal cells have the ability to differentiate into various cancer cells, hepatocytes, epithelial cells, fibroblasts, mural cells, cardiomyocytes, myoblasts, bone cells, chondrocytes, adipocytes and glial cells, and these cells. It is preferably one or more types of cells selected from the group consisting of pluripotent stem cells, and more preferably fibroblasts. The parenchymal cell used in this step may be a cell derived from a primary cell line directly collected from a living tissue or organ, or a cell derived from a subculture cell line in which the primary cell has been passaged several times. May be. The animal from which the cells are derived is not particularly limited, and for example, cells derived from animals such as humans, non-human primates, mice, rats, rabbits, dogs, cats, cows, pigs, horses, goats or sheep are used. Can do. When the parenchymal cell used in this step is a fibroblast, and the non-parenchymal cell used in the non-parenchymal tissue preparation step described below is a vascular endothelial cell, the method of the present invention allows the blood vessel as a fine structure. An artificial tissue having a net can be produced.

  In the present invention, the “cell structure” means an aggregate of cells composed of a plurality of cells, which are produced when cells form an intercellular adhesion via an extracellular matrix. The cell structure constituting the parenchymal cell tissue or non-parenchymal cell tissue prepared in this step or the non-parenchymal cell tissue preparation step described below is a two-dimensional structure formed by arranging a plurality of cells in a plane. It may have a three-dimensional structure formed by arranging a plurality of cells above and / or below a plurality of cells arranged in a plane. In the method of the present invention, examples of the cell structure include a sheet-like form having a two-dimensional structure (hereinafter also referred to as “cell sheet”). When the cell structures constituting the parenchymal cell tissue and the non-parenchymal cell tissue are both in the form of a cell sheet, the method of the present invention allows a non-parenchymal cell tissue composed of a cell sheet having a fine structure to Thus, an artificial tissue having a three-dimensional structure laminated so as to be sandwiched between the parenchymal cell tissues can be produced.

  In the present invention, “stretched” or “stretched” refers to a cell structure including a plurality of cells and a cell tissue having the cell structure, wherein a plurality of cells arranged in a plane maintain cell-cell adhesion. However, the cytoskeleton is stretched in a certain direction by a method such as bonding to a cell culture support to form adhesion spots, so that it is arranged in a deformed form from a spherical shape when floating to an asymmetric shape such as a spindle system. It means the state to be done. As described below, the extended cell structure and tissue can be obtained by seeding cells on the surface of a cell culture support and culturing the parenchymal cells in a suitable medium until they become confluent. it can. The extended cell structure and cell tissue obtained by such a method can maintain the extended state by adhering to the surface of the cell culture support or the surface of another support.

  The stretched parenchymal tissue prepared in this step has a stretched cell structure. The stretched parenchymal tissue may be composed of a single stretched cell structure or a plurality of stretched cell structures. Either case is included in the embodiment of this step.

  The extended cell structure constituting the extended parenchymal tissue is preferably in the form of a cell sheet. In this case, the shape of the surface of the cell sheet is not particularly limited. Based on the shape of the artificial tissue produced by the method of the present invention, a group consisting of a circle (for example, a perfect circle or an ellipse), and a polygon such as a triangle, a rectangle (for example, a striped rectangle or a square), a pentagon, or a hexagon. The shape is appropriately selected from the more selected shapes. In the case where the extended parenchymal tissue is composed of an extended cell structure in the form of a cell sheet, the cell tissue lamination step described below can be easily performed.

  In this step, the means for preparing the extended parenchymal cell tissue is not particularly limited. For example, a parenchymal tissue may be prepared by performing a parenchymal cell tissue formation step described below, or a previously prepared parenchymal tissue may be purchased and prepared. Either case is included in the embodiment of this step.

[1-2: Nonparenchymal cell tissue preparation process]
The method of the present invention needs to include a non-parenchymal cell tissue preparation step of preparing one or a plurality of extended non-parenchymal cell tissues having a cell structure having a fine structure and containing non-parenchymal cells.

  In the present invention, “non-parenchymal cell” means a cell that constitutes a minimum functional unit of a tissue or organ among cells constituting a tissue or organ of a living body and is not included in the parenchymal cell. Examples of non-parenchymal cells used in this step include various endothelial cells, nerve cells, visual cells, epithelial cells, islet cells, and the like, and pluripotent stem cells having the ability to differentiate into these cells. . Non-parenchymal cells are one or more cells selected from the group consisting of endothelial cells, neurons, photoreceptors, glandular epithelial cells and islet cells, and pluripotent stem cells capable of differentiating into these cells. Are preferable, and vascular endothelial cells are more preferable. The non-parenchymal cells used in this step may be cells derived from primary cell lines collected directly from living tissues or organs, or cells derived from subculture cell lines obtained by subculturing the primary cells for several generations. There may be. The animal from which the cells are derived is not particularly limited, and for example, cells derived from animals such as humans, non-human primates, mice, rats, rabbits, dogs, cats, cows, pigs, horses, goats or sheep are used. Can do. When the non-parenchymal cells used in this step are vascular endothelial cells and the parenchymal cells used in the parenchymal tissue preparation step are fibroblasts, an artificial tissue having a vascular network as a fine structure according to the method of the present invention. Can be produced.

  The non-parenchymal cell tissue prepared in this step has a microstructure contained in the artificial tissue produced by the method of the present invention. Examples of the microstructure include, for example, a vascular network, a lymphatic network, a hepatic lobule, a nephron, a cranial nerve circuit, a neuromuscular junction, a bone tissue, a cartilage tissue, various endocrine glands, a retina, and a microstructure for stem cell culture. Can be mentioned. The microstructure may be a mature state having a structure and function substantially equivalent to those in a living tissue or organ, and an immature state having the ability to express an equivalent structure and function in the future. May be. The non-parenchymal tissue prepared in this step is a mature microstructure having a luminal shape, or a shape that can form a lumen in the future, for example, an immature state having a striped sheet shape. It is preferable to have a fine structure. For example, when the microstructure is a vascular network, the mature microstructure is preferably a blood vessel-like luminal shape composed of vascular endothelial cells, and the immature microstructure is composed of vascular endothelial cells. It preferably has a striped sheet shape. When the microstructure is in an immature state having a striped sheet shape, the specific shape of the stripe is not particularly limited. The immature microstructure can have, for example, a striped sheet shape which is a linear shape, a curved shape, a branched shape, a mesh shape, or a combination thereof. In addition, the immature microstructure may have a shape in which a plurality of striped sheets having the above-described shape are arranged in parallel or intersecting with each other in whole or in part. The immature microstructure is, for example, a shape in which a plurality of linear striped sheets are arranged in parallel with each other. When the microstructure contained in the non-parenchymal tissue is an immature microstructure having the above shape, in the artificial tissue produced by the method of the present invention, the immature microstructure is a luminal shape. Can develop into a mature microstructure with

  The extended parenchymal tissue prepared in this step has an extended cell structure having a fine structure. The stretched non-parenchymal cell tissue may consist of one stretched cell structure having a fine structure, or a combination of a plurality of stretched cell structures having a fine structure. Either case is included in the embodiment of this step. For example, when the immature microstructure has a plurality of straight stripe-like sheet shapes, the extended non-parenchymal cell tissue having the microstructure has a plurality of extended straight stripe-like sheet shapes. Having a combination of cell structures.

  The extended cell structure constituting the extended non-parenchymal tissue is preferably in the form of a cell sheet. In this case, the shape of the surface of the cell sheet is based on the shape of the microstructure, and is a circle (for example, a perfect circle or an ellipse) and a polygon such as a triangle, a rectangle (for example, a striped rectangle or a square), a pentagon, or a hexagon. A shape selected from the group consisting of: When the extended non-parenchymal cell tissue has an extended cell structure in the form of a cell sheet, the cell tissue lamination step described below can be easily performed.

  In this step, means for preparing a non-parenchymal cell tissue having an extended microstructure is not particularly limited. For example, a non-parenchymal tissue may be prepared by carrying out a non-parenchymal tissue forming process described below, or a previously prepared non-parenchymal tissue may be purchased and prepared. Either case is included in the embodiment of this step.

[1-3: Parenchymal cell tissue formation process]
The method of the present invention optionally includes a parenchymal cell tissue forming step of culturing parenchymal cells on the surface of a cell culture support to form an extended parenchymal cell tissue having a cell structure comprising the parenchymal cells. it can.

  This step can be carried out by applying a method for producing a cell structure that is a form of a cell sheet that is usually used in the art. This step includes, for example, a sub-step of placing a cell culture support on the culture surface of a cell culture container, a sub-step of seeding a parenchymal cell on the surface of the cell culture support, culturing the parenchymal cell, A sub-step of forming an extended cell structure including a sub-process, and a sub-step of removing the extended cell structure including a parenchymal cell from a cell culture support to obtain an extended parenchymal tissue having a cell structure including a parenchymal cell. Can be implemented. In this case, the parenchymal cells are preferably cultured until they become confluent. By culturing until the parenchymal cells become confluent, an extended parenchymal tissue having a cell structure adhered to the surface of the cell culture support and stretched can be obtained.

In this step, the conditions for seeding the parenchymal cells, and the culture conditions such as the medium for culturing the parenchymal cells and the external environment (eg, temperature, humidity, photoperiod or CO ₂ concentration) are based on the type of parenchymal cells used. Inoculation conditions and culture conditions that are usually used in the art can be applied as appropriate.

  In this step, various cell culture containers that are usually used in the art and have a shape such as a culture dish or a culture flask can be used. Since the resulting extended parenchymal cell tissue can be easily collected, it is preferable to use a cell culture vessel having the shape of a culture dish. The material forming the cell culture vessel can be appropriately selected from various materials usually used in the production of cell structures in the form of cell sheets in the art. Examples of the material for forming the cell culture container include polystyrene resin, polyester resin, polyethylene resin, polyethylene terephthalate resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluorine resin, polycarbonate resin, polyurethane resin, methylpentene resin, and phenol. Examples thereof include resin materials such as resin, melamine resin, epoxy resin and vinyl chloride resin, and inorganic materials such as glass and quartz. If desired, the resin material may be used in combination with one or more additional resin materials. In addition, the resin material may be subjected to a post-treatment such as a hydrophilic treatment on the surface, if desired. The material forming the cell culture vessel used in this step is preferably at least one resin material selected from the above group, and more preferably a polystyrene resin or a polyethylene terephthalate resin.

  The sub-step of placing the cell culture support on the culture surface of the cell culture container can be performed by using a cell culture support that is usually used in the art. This sub-step may be carried out, for example, by placing a cell culture support prepared as a separate member on at least a part of the culture surface of the cell culture container, and at least one of the culture surfaces of the cell culture container. Part may be used as a cell culture support. In the case of the latter embodiment, for example, at least a part of the culture surface of the cell culture container is formed by the material of the cell culture support, or at least a part of the culture surface of the cell culture container is coated with the material of the cell culture support By doing so, a cell culture vessel capable of using at least a part of the culture surface as a cell culture support can be prepared.

  The material for forming the cell culture support can be appropriately selected from various materials usually used in the production of a cell structure in the form of a cell sheet in the technical field. Examples of the material forming the cell culture support include metals, glass, ceramics, resins and elastomers, and composite materials thereof. Examples of the resin material include polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetyl cellulose), polyimide (PI), nylon (Ny), low density polyethylene (LDPE), medium density polyethylene ( MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyether sulfone, polyethylene naphthalate, polypropylene and acrylic. The resin material may be a biocompatible polymer such as polylactic acid, polyvinyl alcohol, polyglycolic acid or polycaprolactan, or a copolymer thereof. When the material forming the cell culture support is the biocompatible polymer, an artificial tissue produced using the parenchymal tissue obtained in this step can be used as a highly safe transplant material.

  In the sub-step of obtaining an extended parenchymal tissue having a cell structure containing parenchymal cells by peeling the stretched cell structure containing parenchymal cells from the cell culture support, the stretched cell structures containing parenchymal cells are treated as cells. The means for peeling from the culture support is not particularly limited. For example, a means of treating a cell structure that adheres and extends to the surface of a cell culture support with one or more proteolytic enzymes such as trypsin, chymotrypsin, pepsin, papain, dispase, elastase or hyaluronidase. it can. By the means described above, adhesion by the extracellular matrix can be cut, and the cell structure can be detached from the surface of the cell culture support to obtain an extended parenchymal tissue having a cell structure containing parenchymal cells. In the case of this embodiment, the conditions for treatment with one or more types of proteolytic enzymes can be appropriately set according to the combination of the parenchymal cells and the enzymes used.

  Alternatively, means for using a cell culture support having a region containing a stimulus-responsive polymer capable of changing from cell-adhesive to non-cell-adhesive in response to a predetermined stimulus on at least a part of its surface. Can be mentioned. By the above means, a predetermined stimulus can be applied to peel the cell structure from the surface of the cell culture support to obtain an extended parenchymal cell tissue having a cell structure containing parenchymal cells. The cell culture support used in this step is a region containing a stimulus-responsive polymer capable of changing from cell-adhesive to non-cell-adhesive in response to a predetermined stimulus on at least a part of its surface. It is preferable to have. In the present invention, the “stimulus responsive polymer” is a state in which cell adhesion is exhibited under conditions for culturing cells (for example, temperature, pH, ionic strength, or light intensity). It means a polymer that changes to a state showing adhesiveness. Examples of the stimulus-responsive polymer used in this step include a temperature-responsive polymer, a pH-responsive polymer, an ion-responsive polymer, and a photo-responsive polymer. It is preferable to use a cell culture support having a region containing a temperature-responsive polymer because stimulation can be easily applied. By carrying out this step using a cell culture support having a region containing a stimulus-responsive polymer on at least a part of its surface, a predetermined stimulus is applied and the expanded parenchymal tissue is applied to the cell culture support. Can be easily peeled off.

  In the cell culture support used in this step, the stimulus-responsive polymer may be disposed so as to cover substantially the entire surface of the cell culture support, and is disposed so as to cover at least a part of the surface. May be. Either case is included in the embodiment of the present invention.

  In the present invention, “cell adhesion” and “cell non-adhesion” mean the relative strength of the degree of cell adhesion between one surface region and the other surface region of the cell culture support. “Cell adhesion” means a surface property to which cells are likely to adhere. Cell adhesion is usually determined based on whether or not cell adhesion and / or spreading is likely to occur due to the chemical and / or physical properties of the surface of the cell culture support. As an index for determining the cell adhesion of the cell culture support, for example, the cell adhesion extension rate when cells are actually cultured on the surface of the cell culture support can be used. In the present invention, when the region containing the stimulus-responsive polymer is in a state of showing cell adhesion, the surface of the region containing the stimulus-responsive polymer preferably has a cell adhesion extension rate of 60% or more. The rate is more preferably 80% or more. When the cell adhesion extension rate in the region containing the stimulus-responsive polymer is high, the cells can be cultured efficiently. In addition, when the region containing the stimulus-responsive polymer is non-cell-adhesive, the surface of the region containing the stimulus-responsive polymer preferably has a cell adhesion extension rate of less than 60%, less than 40%. More preferably, it is more preferably 5% or less, and particularly preferably 2% or less.

In addition, the cell adhesion extension rate used as an index of cell adhesiveness can be determined by the following method, for example. Cells to be cultured are seeded on the surface to be measured at an initial seeding density in the range of 4000 to 30000 cells / cm ² and then cultured under conditions of 37 ° C. and 5% CO ₂ concentration. Three hours after the start of the culture, the medium is changed to remove non-adhered cells, and then several places where the cells adhere are observed. The cell adhesion spreading rate is calculated based on the following formula: ({(number of adherent cells) / (number of seeded cells)} × 100 (%)).

Examples of the temperature-responsive polymer include acrylic polymers and methacrylic polymers, such as poly-N-isopropylacrylamide (PNIPAAm) (lower critical solution temperature, T _c = 32 ° C.), poly-Nn-propyl. Acrylamide (T _c = 21 ° C), Poly-Nn-propylmethacrylamide (T _c = 32 ° C), Poly-N-ethoxyethylacrylamide (Tc = about 35 ° C), Poly-N-tetrahydrofurfurylacrylamide (T _c = -28 ° C.), poly-N-tetrahydrofurfuryl methacrylamide (T _c = about 35 ° C.) and poly-N, N-diethylacrylamide (T _c = 32 ° C.). The temperature-responsive polymer may be a copolymer formed by copolymerizing two or more monomers capable of forming the polymer. The temperature-responsive polymer is preferably PNIPAAm, poly-Nn-propyl methacrylamide or poly-N, N-diethylacrylamide, more preferably PNIPAAm. The temperature-responsive polymer has a _Tc in the preferred range described above. Therefore, when the temperature-responsive polymer is used, cell damage can be reduced as much as possible when the cells are cultured in this step.

  As the monomer for forming the temperature-responsive polymer, it is preferable to use a monomer that can be polymerized by radiation irradiation. Examples of the monomer include (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives, (meth) acrylamide derivatives having a cyclic group, and vinyl ether derivatives. When polymerizing using the monomer alone, the resulting temperature-responsive polymer is in the form of a homopolymer, and when copolymerizing using two or more of the monomers, the resulting temperature-responsive polymer is a copolymer. (Copolymer or heteropolymer). Any form of temperature-responsive polymer can be used in this step.

The temperature-responsive polymer may be a copolymer formed by copolymerizing one or more kinds of monomers described above and a further monomer other than those described above. Alternatively, the temperature-responsive polymer may be obtained by graft polymerization or blocking a polymer or copolymer formed by polymerizing or copolymerizing one or more kinds of monomers described above and a further polymer or copolymer. It may be a graft copolymer or a block copolymer formed by polymerization. Alternatively, the temperature-responsive polymer may be in the form of a mixture of one or more polymers or copolymers described above. In these forms, _Tc can be adjusted to a desired range by appropriately selecting the material to be used.

  In this step, the means for recovering the stretched parenchymal cell tissue having a cell structure containing parenchymal cells detached from the cell culture support is not particularly limited. For example, an extended parenchymal tissue having a cell structure containing parenchymal cells can be collected from the surface of the cell culture support using tweezers, an aspirator, a pipette, or the like. Alternatively, a known method for recovering cell structures such as cells or cell sheets using gelatin gel as a support (hereinafter also referred to as “gelatin stamp method”) (Y. Tsuda et al., Biomaterials Vol. 28, (2007) p. 4939-4946) can be applied to recover extended parenchymal tissue with cell structures containing parenchymal cells from the surface of the cell culture support. Since the cell tissue laminating step described below can be carried out continuously, it is preferable to recover an extended parenchymal tissue having a cell structure containing parenchymal cells using a gelatin stamp method.

[1-4: Nonparenchymal cell tissue formation process]
The method of the present invention optionally cultivates non-parenchymal cells on the surface of a cell culture support to form a stretched non-parenchymal tissue having a cellular structure comprising non-parenchymal cells having a fine structure. A parenchymal cell tissue formation step can be included.

  This step can be carried out by applying a method for producing a cell structure that is a form of a cell sheet that is usually used in the art. In this step, for example, a sub-step of placing a cell culture support having a microstructured template on the culture surface of a cell culture vessel, and seeding non-parenchymal cells on the surface of the cell culture support having the microstructured template A sub-step, a sub-step of culturing the non-parenchymal cell to form an extended cell structure including a non-parenchymal cell having a fine structure, and an extended cell structure including a non-parenchymal cell having a fine structure. It can be carried out by a sub-process of exfoliating from the cell culture support to obtain an extended non-parenchymal cell tissue having a cell structure containing non-parenchymal cells.

  Each sub-step in this step can be carried out by applying the same material and method as in the parenchymal cell tissue forming step described above. The cell culture vessel and cell culture support used in this step preferably have the same characteristics as those used in the parenchymal cell tissue formation step described above.

  In the present invention, the “microstructured template” means a template placed on the surface of the cell culture support to form a microstructure having the shape described above. The microstructure mold preferably has the same size and / or shape as the microstructure surface of the extended non-parenchymal cell tissue described above.

  The sub-step of placing the cell culture support having a microstructured template on the culture surface of the cell culture vessel is by using the cell culture support in the form of a microstructured template usually used in the art. Can be implemented. This sub-step may be carried out, for example, by placing a cell culture support in the form of a microstructured template prepared as a separate member on at least a part of the culture surface of the cell culture vessel. You may implement by using at least one part of the culture surface of a culture container as a cell culture support body which is the shape of the mold of a microstructure. In the case of the latter embodiment, a cell culture support having a microstructured template can be prepared, for example, by the following means. Prepare a cell culture support with a cell-adhesive surface and a mask corresponding to the shape of the microstructured template, and use photolithography to chemistry the areas other than the microstructured template on the surface of the cell culture support It is modified to change to cell non-adhesiveness. Alternatively, a cell culture support having a non-cell-adhesive surface and a mask corresponding to the shape of the microstructure template are prepared, and the microstructure template region on the surface of the cell culture support is prepared using a photolithographic technique. Can be chemically modified to modify cell adhesion. For example, when using a cell culture support that has a region containing PNIPAAm, a temperature-responsive polymer, polymerize water-soluble camphorquinone on the surface of the cell culture support using a photolithographic technique, except for the microstructure template. By chemically modifying polyacrylamide (PAAm) as an initiator, it can be modified to be non-cell-adhesive.

  In this step, the means for recovering the stretched non-parenchymal cell tissue having a fine structure and a cell structure containing non-parenchymal cells is not particularly limited. For example, an extended non-parenchymal cell tissue having a cell structure including a non-parenchymal cell having a fine structure can be recovered from the surface of the cell culture support using tweezers, an aspirator, a pipette, or the like. Alternatively, by applying a known method such as the gelatin stamp method described above, an extended non-parenchymal cell tissue having a cell structure including a non-parenchymal cell having a fine structure is removed from the surface of the cell culture support. It can be recovered. Since the cell tissue laminating step described below can be carried out continuously, a stretched non-parenchymal tissue having a cell structure containing a non-parenchymal cell having a fine structure is used as a cell culture support by using a gelatin stamp method. It is preferable to recover from the surface.

[1-5: Cell tissue lamination process]
The method of the present invention needs to include a cell tissue lamination step of laminating the plurality of extended parenchymal cell tissues and one or more extended non-parenchymal cell tissues having the microstructure. By this step, it is possible to obtain a stretched cell tissue laminate composed of a plurality of stretched parenchymal cell tissues and one or a plurality of stretched non-parenchymal cell tissues having a fine structure.

  This step is performed such that each of the non-parenchymal cell tissues having one or more extended microstructures is sandwiched between the plurality of extended parenchymal tissue. Therefore, the cell tissue laminate obtained by this process has a structure in which each of the non-parenchymal tissue having one or more stretched microstructures is sandwiched between the plurality of stretched parenchymal tissues. Have. By having such a structure, in the cell tissue laminate obtained by this step, the parenchymal cell tissue can be used as a scaffold for non-parenchymal cells contained in the non-parenchymal cell tissue having a fine structure.

  In this step, the means for laminating a plurality of stretched parenchymal tissue and one or a plurality of stretched non-parenchymal tissue having a fine structure is not particularly limited. For example, the stretched parenchymal tissue and the stretched non-parenchymal tissue having a fine structure can be grasped and stacked using tweezers or the like. Alternatively, by applying a known method such as the gelatin stamp method described above, a plurality of extended parenchymal cell tissues and one or more extended non-parenchymal cell tissues having a fine structure are laminated. Can do. Since a plurality of extended parenchymal tissues and one or more extended non-parenchymal cell tissues having a fine structure can be successively laminated in a desired order, the cell tissues are laminated using a gelatin stamp method. It is preferable to do.

[1-6: Shrinkage process]
The method of the present invention needs to include a contraction step of contracting a laminate of cell tissues having a three-dimensional structure including the fine structure obtained in the cell tissue stacking step.

  The present inventors provide a stretched cell tissue laminate comprising a plurality of stretched parenchymal cell tissues obtained in the cell tissue laminating step and one or a plurality of stretched non-parenchymal cell tissues having a fine structure. It has been found that when contracted, the fine structure contained in the non-parenchymal cell tissue is stably maintained for several days. The reason why the method of the present invention has such an effect can be explained as follows. Note that the present invention is not limited to the following actions and principles. In this step, the state of the parenchymal cell tissue used as a scaffold for non-parenchymal cells contained in the non-parenchymal cell tissue having a fine structure is obtained by contracting the cell tissue laminate obtained in the cell tissue laminating step. Change. Thereby, it is considered that migration of non-parenchymal cells is remarkably suppressed and a predetermined fine structure is stably maintained.

  In this step, the contraction rate for contracting the stretched cell tissue laminate is determined by the non-parenchymal tissue having the microstructure after the contraction step relative to the surface dimension of the stretched non-parenchymal tissue having the microstructure before the contraction step. Defined as a percentage of the surface dimensions. For example, the shrinkage rate can be defined as a percentage of the microstructure size on the surface of the non-parenchymal tissue after the contraction step relative to the size of the microstructure on the surface of the stretched non-parenchymal cell tissue before the contraction step. For example, when the microstructure has a shape in which a plurality of linear stripe sheet-like cell structures are arranged in parallel with each other, the shrinkage rate is the non-stretched stretch before the shrinking process. Two adjacent striped cell structures on the surface of the non-parenchymal tissue after the contraction process relative to the distance between the longitudinal centerlines of the two adjacent striped cell structures on the surface of the parenchymal cell tissue It can be defined as the percentage of the distance between the longitudinal centerlines of the body. In this case, the distance between the center lines in the longitudinal direction of two adjacent striped cell structures on the surface of the non-parenchymal cell tissue is, for example, previously labeled non-parenchymal cells, confocal laser microscope, etc. It is possible to measure by observing the shape of the striped cell structure in the artificial tissue using the label confirmation means. The shrinkage rate is preferably 30% or less, and more preferably in the range of 20 to 30%. When the contraction rate exceeds the upper limit, migration of non-parenchymal cells cannot be sufficiently suppressed, and as a result, there is a possibility that the fine structure contained in the non-parenchymal tissue cannot be maintained for a long time. When the contraction rate is less than the lower limit, oxygen and nutrients cannot be sufficiently supplied to non-parenchymal cells and parenchymal cells, causing oxygen deficiency or nutrient deficiency, or excessive pressure is applied to non-parenchymal cells and parenchymal cells. May cause cell rupture. Therefore, an artificial tissue capable of stably maintaining the fine structure for several days can be obtained by contracting the laminated body of cell tissues extended at a contraction rate in the above range.

  In this step, the means for contracting the stretched cell tissue laminate is not particularly limited. For example, in the laminating process, when using tweezers or the like to hold and laminate the extended parenchymal tissue and the extended non-parenchymal cell tissue having a fine structure, the extended cell tissue laminate is peeled from the cell culture support. Has been. In this case, the stretched cellular tissue laminate can shrink to the shrinkage rate in the above range, usually immediately after lamination, typically within a few minutes, for example within 1 to 10 minutes. Alternatively, in the laminating step, when the stretched parenchymal tissue and the stretched non-parenchymal tissue having a fine structure are laminated using the gelatin stamp method described above, the stretched cell tissue laminate is formed of the gelatin stamp. The stretched form is maintained by adsorbing to the gelatin gel. For this reason, the laminated body of the extended cell tissue can be contracted by heating and dissolving the gelatin gel. In this case, the gelatin gel adsorbing the stretched tissue stack is usually dissolved at 25 to 40 ° C, typically 25 to 37 ° C, usually 30 minutes to 1 hour. Thus, the extended laminate can be contracted at a contraction rate in the above range.

In this step, culture conditions such as a culture medium and an external environment (for example, temperature, humidity, photoperiod or CO ₂ concentration) for culturing the stretched cell tissue laminate and the contracted cell tissue laminate are used. Based on the types of parenchymal cells and non-parenchymal cells, culture conditions usually used in the art can be applied as appropriate. As will be described below, in the method of the present invention, migration of non-parenchymal cells is remarkably suppressed, and therefore it is preferable to culture the stretched laminate and the contracted laminate in the presence of a growth factor. For example, when the non-parenchymal cells are vascular endothelial cells, the growth factors are fetal bovine serum (FBS), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) Preferably, ascorbic acid, heparin, hydrocortisone, or insulin-like growth factor. An artificial tissue having a three-dimensional structure including a fine structure developed into a luminal mature state is obtained by culturing a stretch of cellular tissue and a laminate of contracted cellular tissue in the presence of the growth factor. be able to.

<2: Artificial tissue having a three-dimensional structure including a fine structure>
According to the method of the present invention, an artificial tissue capable of stably maintaining a fine structure over several days can be obtained. Therefore, the present invention also relates to an artificial tissue having a three-dimensional structure including a fine structure.

  In the artificial tissue of the present invention, the microstructure is stably maintained over a period of several months, usually within several days, particularly within five days after the artificial tissue is produced. When the artificial tissue of the present invention is cultured for more than several days, an appropriate medium exchange and / or selection of a culture substrate for supplying oxygen and nutrients to the cells constituting the artificial tissue is performed, whereby a fine structure is obtained. There is a possibility that it can be stably cultured for a longer period of time.

  In the artificial tissue of the present invention, the fine structure is stably maintained because, for example, the parenchymal cells and the non-parenchymal cells are labeled in advance, and the artificial tissue is labeled using a label confirmation means such as a confocal laser microscope. This can be confirmed by observing the shape of the microstructure in the tissue. Alternatively, it is possible to confirm by fluorescently labeling parenchymal cells and non-parenchymal cells in advance and time-lapse observation of the shape change of the fine structure in the artificial tissue using a time-lapse fluorescence microscope apparatus. In the case of time-lapse observation, after the obtained fluorescence image is binarized and processed, the microstructure is stably maintained by performing image correlation analysis of the processed image and determining the correlation coefficient. Can be quantitatively evaluated.

  Artificial tissues having a three-dimensional structure including a fine structure formed by laminating a parenchymal cell tissue and a non-parenchymal cell tissue having a fine structure are known in the art (for example, M. Muraoka et al., Biomaterials 34, (2013) p. 696-703). However, in known artificial tissues, it has not been known that when a non-parenchymal cell tissue having an immature microstructure is laminated, the microstructure develops to a mature state. In the artificial tissue produced by the method of the present invention, the extended non-parenchymal tissue having an immature microstructure such as a plurality of striped cell structures, and the extended parenchymal cell tissue, It has been found that a plurality of striped cell structures develop into a mature state like a lumen. Therefore, the present invention also relates to an artificial tissue having a three-dimensional structure including a luminal microstructure.

  The artificial tissue having a three-dimensional structure including the luminal microstructure of the present invention is obtained, for example, by using a non-parenchymal cell tissue and a resultant artificial tissue in the presence of a growth factor in the method for producing an artificial tissue of the present invention. It can be obtained by culturing. For example, when the non-parenchymal cells are vascular endothelial cells, the growth factors are fetal bovine serum (FBS), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) Preferably, ascorbic acid, heparin, hydrocortisone, or insulin-like growth factor. By culturing the non-parenchymal cell tissue and the resultant artificial tissue in the presence of the growth factor, an artificial tissue having a three-dimensional structure including a fine structure developed into a luminal mature state can be obtained.

  The artificial tissue of the present invention can have a three-dimensional structure including a luminal microstructure selected from the group consisting of a vascular network, a lymphatic network, liver lobule, and various endocrine glands. The artificial tissue of the present invention preferably has a three-dimensional structure including a luminal microstructure that is a vascular network. By having the above characteristics, the artificial tissue of the present invention can be used as a transplant material having a vascular network.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.

[I: Production of cell culture support having a microstructured template]
A temperature-responsive culture dish (35 mm in diameter, UpCell (trademark), Cellseed Co., Ltd.) having a culture surface coated with a temperature-responsive polymer was prepared. On the culture surface of a temperature-responsive culture dish, the photo-lithography technique is used to partially chemically modify polyacrylamide (PAAm) with water-soluble camphorquinone as a polymerization initiator, thereby applying it to the culture surface of the temperature-responsive culture dish. A cell culture support having a microstructured template was prepared. FIG. 1 shows a cell culture support having a microstructured template prepared by the above treatment. By the treatment, the cell non-adhesive region 11 and the cell non-adhesive region 11 formed inside the cell non-adhesive region 11 on the culture surface of the temperature-responsive culture dish in response to a temperature change from cell adhesive to cell non-adhesive A cell culture support 110 having a microstructure template composed of a plurality of stripe-like temperature-responsive regions 12 that can be changed into a shape was produced (FIG. 1A). The microstructure mold has a stripe-shaped temperature-responsive region 12 having a width (length in the short direction, L) of 500 μm, and is a non-cell-adhesive region between adjacent temperature-responsive regions 12. The width (S) was set to 500, 1000, or 2000 μm (FIG. 1B). A temperature-responsive culture dish containing a cell culture support having a microstructured template produced by the above treatment was sterilized with ethylene oxide gas.

The width of the stripe-like temperature-responsive region 12 (L), the width of the cell non-adhesive region existing between the adjacent temperature-responsive regions 12 (S), and the two adjacent stripe-like temperature-responsive regions Table 1 shows the relationship with the distance (D _temp ) between the twelve longitudinal centerlines.

[II: Preparation of stretched parenchymal cell tissue and stretched non-parenchymal cell tissue]
By using fibroblasts as parenchymal cells, extended cell structures in the form of cell sheets were produced. 1 × 10 ⁶ cells of normal human skin fibroblasts (NHDF) (Lonza) are used as a cell culture support on the culture surface of a temperature-responsive culture dish (diameter 35 mm, UpCell ™, Cellseed). / Seeded at the concentration of the culture dish. The seeded cells were cultured in a fibroblast growth medium (FGM-2) (Lonza) for 3 days in a 37 ° C., 5% CO ₂ concentration environment until reaching confluence. By the culture, a fibroblast sheet adhered to and extended on the surface of the cell culture support (culture surface of the temperature-responsive culture dish) was obtained. The culture was performed in duplicate, and two extended fibroblast tissues (NHDF cell tissues) composed of extended fibroblast sheets were prepared.

By using vascular endothelial cells as non-parenchymal cells, an extended cell structure having a fine structure in the form of a plurality of extended striped cell sheets was prepared. Green fluorescent protein-expressing normal human umbilical vein endothelial cells (GFP-HUVEC) (Angio-proteomie) on the surface of the cell culture support having the microstructured template of Examples 1 to 3 prepared according to the above procedure I Seeding was performed at a concentration of 0.25 × 10 ⁶ cells / culture dish. Multiple striped temperature responsiveness of seeded cells in vascular endothelial cell growth medium (EGM-2) (Lonza) supplemented with 20% by weight fetal bovine serum (FBS) (Japan Bioseam) The cells were cultured for 3 days in a 37 ° C., 5% CO ₂ concentration environment until the surface of the region reached confluence. By the culture, a plurality of striped vascular endothelial cell sheets adhered to and extended on the surfaces of the plurality of striped temperature-responsive regions were obtained. By the culture, one extended vascular endothelial cell tissue (striped GFP-HUVEC cell tissue) having a plurality of striped microstructures having a plurality of extended striped vascular endothelial cell sheets was prepared.

[III: Lamination of stretched parenchymal tissue and stretched non-parenchymal tissue]
By using a cell sheet laminated manipulator that collects cells using a gelatin gel as a support (hereinafter also referred to as “gelatin stamp method”) (Y. Tsuda et al., Biomaterials Vol. 28, (2007) p. 4939-4946) The extended fibroblast tissue and the extended vascular endothelial cell tissue having a plurality of striped microstructures were laminated. In each of the artificial tissues of Examples 1 to 3, the first layer extended fibroblast tissue, the second layer extended vascular endothelial cell tissue having a plurality of stripe-like microstructures, and the third layer extended The layers were laminated so as to have a three-layer structure in the order of fibroblast tissue. The procedure of the lamination process is shown in FIG.

  The gelatin gel used for the cell sheet laminated manipulator was prepared by the following procedure. After dissolving porcine skin-derived gelatin (G1890, Sigma-Aldrich) in Hank's balanced salt solution (Sigma-Aldrich) in a 60 ° C water bath, the pH is adjusted using 1 M NaOH aqueous solution. A 7.5% by weight gelatin solution was prepared. The prepared gelatin solution was poured into a disk-shaped silicon rubber mold. A plunger was brought into close contact with the gel poured into the mold. The mold was cooled on ice to gelatinize the gelatin poured into the mold. Thus, a gelatin gel (gelatin stamp) used for the cell sheet laminated manipulator was obtained.

  The cell tissue laminating step by the gelatin stamp method was performed according to the following procedure (FIG. 2). The disc-shaped gelatin gel adsorbed on the plunger of the cell sheet laminated manipulator prepared in the above procedure was adhered to the cell culture support on the culture surface of the temperature-responsive culture dish at 20 ° C. It was placed on the surface and brought into close contact. In this state, by maintaining the culture temperature at 20 ° C., the surface of the cell culture support is changed from cell adhesive to cell non-adhesive, and the fibroblast tissue extends from the surface of the cell culture support. Was peeled off. By lifting the plunger part of the cell sheet laminated manipulator, the first-layer extended fibroblast tissue adsorbed on the gelatin gel of the plunger part was collected. The recovered fibroblast tissue was transferred to a gelatin gel by surface tension while maintaining the extended form on the surface of the cell culture support. Next, the gelatin gel in the plunger part of the cell sheet laminated manipulator adsorbed with the first layer of expanded fibroblast tissue was adhered to the surface of a plurality of stripe-like temperature-responsive regions at 20 ° C. And placed on the surface of a vascular endothelial cell tissue having a striped microstructure. The vascular endothelial cell tissue having a plurality of stripe-like microstructures extended by the same procedure as described above is peeled off from the culture surface, and is applied to the surface of the first-layer extended fibroblast tissue adsorbed on the gelatin gel of the plunger part. The extended vascular endothelial cell tissue having a plurality of stripe-like microstructures in the second layer was transferred. Furthermore, the gelatin gel of the plunger part of the cell sheet laminated manipulator adsorbed with the extended fibroblast tissue in the first layer and the extended vascular endothelial cell tissue having a plurality of stripe-like microstructures in the second layer Then, it was placed on and adhered to the surface of the expanded fibroblast tissue adhered to the culture surface of another temperature-responsive culture dish. In the same procedure as described above, the fibroblast tissue expanded in the third layer is detached from the culture surface, the fibroblast tissue expanded in the first layer adsorbed to the gelatin gel of the plunger part, and the plurality of stripes in the second layer The stretched fibroblast tissue of the third layer was transferred to the surface of the stretched vascular endothelial cell tissue having a fine microstructure, and an stretched laminate composed of the stretched cell tissue of three layers was obtained.

[IV: Contraction of the laminated body of stretched cell tissues]
The stretched cell tissue laminate comprising the stretched cell tissue of the three layers obtained by the procedure of III was peeled off from the plunger portion of the cell sheet laminate manipulator together with the gelatin gel. The gelatin gel and the extended laminate were placed in a glass bottom dish (Asahi Glass Co., Ltd.) and incubated at 37 ° C. for 1 hour. By re-dissolving the gelatin gel by the above treatment, the stretched cell tissue laminate was contracted. The layered structure of contracted cell tissues composed of three layers of cell tissues was washed while adhered to the bottom surface of the glass, and the dissolved gelatin was removed. The obtained artificial tissue composed of three layers of cell tissue was used for the following observations in a state where it was adhered to the bottom surface of the glass.

[V: Cultivation and observation of artificial tissue with three-dimensional structure]
Using the microscope stage-mounted incubator (INUG2-KI3) (Tokai Hit Co., Ltd.), the artificial tissue consisting of the contracted three-layered cellular tissue obtained by the above procedure was used as a 50 ng / mL recombinant human vascular endothelial cell growth factor. The cells were cultured in EGM-2 supplemented with (rhVEGF) (R & DSystems) at 37 ° C. in a CO ₂ concentration environment of 5% for 5 days. Using a time-lapse fluorescence microscope apparatus (BZ-9000, Keyence), observation was performed by acquiring time-lapse fluorescence images of the same part of the artificial tissue at the start of culture and every day after the start of culture. After the fluorescence image obtained by the time lapse observation was binarized, noise removal processing and pattern extraction processing were performed. Using ImageJ (National Institutes of Health, NIH), image correlation analysis of the processed image was performed to determine the correlation coefficient.

  The three-dimensional structure of the artificial tissue composed of the three-layered cellular tissue obtained by the above procedure is a confocal laser microscope (FV1200) (Olympus) and an oil immersion objective (UPlanSApo, 30 ×, NA 1.05) (Olympus). Was observed. In the observation, three-dimensionalization by stacking two-dimensional images was performed using observation software (Fluo View) (Olympus).

  Images acquired using a time-lapse fluorescence microscope apparatus are shown in FIGS. FIG. 3 is a superimposed image of the fluorescence image (red) immediately after lamination and contraction of the three-layer artificial tissue at the same location in Examples 1 and 2, and the fluorescence image (green) on the fifth day after lamination and contraction. . In FIG. 3, A is an image of Example 1, and B is an image of Example 2. FIG. 4 shows the first-layer fibroblast tissue (red), the second-layered vascular endothelial cell tissue (green), and the third-layer fibroblast tissue in the artificial tissue of Example 1. It is a superimposed image of (red) fluorescent images. In FIG. 4, A is a fluorescence image immediately after lamination and shrinkage, and B is a fluorescence image 12 hours after lamination and shrinkage. FIG. 5 is a fluorescence image of the vascular endothelial cell tissue having the stripe-like microstructure of the second layer of Examples 1 to 3 from immediately after lamination and contraction to the fifth day at the same location. In FIG. 5, A is the fluorescence image of Example 1, B is the fluorescence image of Example 2, and C is the fluorescence image of Example 3. FIG. 6 shows a fluorescence image from immediately after lamination and contraction of the vascular endothelial cell tissue having the second layer stripe-like microstructure of Example 1 to the fifth day, and a binarized image of the fluorescence image. It is. In FIG. 6, A is a fluorescence image, and B is a binarized image. By the time-lapse observation, in the artificial tissue after lamination and contraction, the localization of vascular endothelial cells in the three-dimensional structure is controlled by a plurality of stripe-like microstructures in the second-layer vascular endothelial cell tissue, It was also revealed that this localization was maintained for 5 days after lamination and shrinkage.

  FIG. 7 shows the change over time in the correlation coefficient by image correlation analysis of the image obtained by the time-lapse observation in the artificial tissue of Example 1. FIG. The correlation coefficient by image correlation analysis of the image obtained by the time lapse observation decreased until the second day after lamination and shrinkage, but remained at a substantially constant value from the second day to the fifth day after lamination and shrinkage. (Figure 7). From this result, in the artificial tissue composed of the three-layered cell tissue, the vascular endothelial cells constituting the vascular endothelial cell tissue having a plurality of stripe-like microstructures in the second layer are substantially omitted by the second day after lamination and contraction. It became clear that this localized position was maintained after that.

In Examples 1 to 3, the distance (D _dish ) between the longitudinal center lines of the two stripe-like temperature-responsive regions 12 in the temperature-responsive culture dish used, and the fifth day after lamination and shrinkage Table 2 shows the distance between the center lines in the longitudinal direction of two adjacent striped vascular endothelial cell sheets (D _tissue ) and the ratio between D _dish and D _tissue .

In Examples 1 to 3, the distance between the longitudinal center lines of two adjacent striped vascular endothelial cell sheets before the shrinking process, that is, when laminated, is the same as the two in the temperature-responsive culture dish used. This corresponds to the distance (D _dish ) between the longitudinal center lines of the stripe-like temperature-responsive regions 12. Therefore, the ratio of D _dish to D _tissue is the shrinkage relative to the distance between the longitudinal centerlines of two adjacent striped cell structures on the surface of the stretched non-parenchymal tissue before the shrinking process. This corresponds to the ratio of the distance between the center lines in the longitudinal direction of two adjacent striped cell structures on the surface of the non-parenchymal cell tissue after the process, that is, the contraction rate of the artificial tissue of the example. As shown in Table 2, the artificial tissues of Examples 1 to 3 contracted at a contraction rate in the range of 22.8 to 28.1% by the contraction process.

  In an artificial tissue made by a prior art method that does not include a step of contracting the stretched cell tissue after laminating, the distance between the longitudinal center lines of adjacent striped vascular endothelial cell sheets is about 500 μm In this case, it was confirmed that vascular endothelial cells forming a vascular endothelial cell tissue composed of striped vascular endothelial cell sheets migrated randomly on the first day after lamination (M. Muraoka et al., Biomaterials 34, (2013) p. 696-703). In the prior art method, culturing was carried out under conditions substantially free of factors that promote migration and / or proliferation of vascular endothelial cells such as VEGF and FBS. For this reason, the method of the prior art is an advantageous condition in that the localization of the vascular endothelial cells that form the fine structure in the artificial tissue is maintained. On the other hand, the artificial tissue composed of the three-layered cell tissue prepared according to the example of the present invention was cultured under conditions to which factors that promote migration and / or proliferation of vascular endothelial cells such as VEGF and FBS were added. Nevertheless, the localization of vascular endothelial cells within the three-dimensional structure was maintained. The artificial tissue composed of the three-layered cell tissue prepared according to the example of the present invention was able to maintain the localization of the vascular endothelial cells in the three-dimensional structure for several days after the stretched cell tissue was laminated. It is presumed that the contraction caused a change in the state of the fibroblast tissue serving as a scaffold for vascular endothelial cells.

  FIG. 8 shows a three-dimensional fluorescent image on the fifth day after the artificial tissue laminating step and the shrinking step in Example 1. Surprisingly, in the artificial tissue of Example 1 on the fifth day after the lamination step and the contraction step, the cell structure of vascular endothelial cells forming a plurality of stripe-like microstructures in the vascular endothelial cell tissue of the second layer is surprisingly provided. It was confirmed that a lumen was formed along the stripe shape (FIG. 8). The above results indicate that in the artificial tissue produced according to the embodiment of the present invention, the immature microstructure designed in two dimensions forms a three-dimensional structure, which is substantially the same as that in a living tissue or organ. It is suggested that it can develop into a mature microstructure having a structure and function equivalent to The artificial tissue prepared according to the embodiment of the present invention was able to form a three-dimensional structure having a mature microstructure having a specific function because the state of the fibroblast tissue that becomes a scaffold for vascular endothelial cells was changed. In addition to this, it is presumed that it was caused by culturing under conditions to which vascular endothelial cell growth factors such as VEGF and FBS were added. Therefore, by the method of the present invention, there is a possibility that an artificial tissue having a three-dimensional structure including a fine structure having a specific function such as a vascular network may be produced.

110 ... Cell culture support with microstructured template
11 ... Cell non-adhesive region
12… Striped temperature responsive region

Claims (4)

A method for producing an artificial tissue having a three-dimensional structure including a fine structure,
Preparing a plurality of extended parenchymal cell tissues having a cell structure containing parenchymal cells;
A non-parenchymal cell tissue preparation step of preparing one or a plurality of extended non-parenchymal cell tissues having a cell structure including the non-parenchymal cells having the fine structure;
Laminating the plurality of stretched parenchymal tissue and one or more stretched non-parenchymal tissue having the microstructure so that each of the non-parenchymal tissue is sandwiched between the stretched parenchymal tissue A cell tissue stacking step;
A shrinking step of shrinking a laminate of cell tissues having a three-dimensional structure including a fine structure obtained in the cell tissue laminating step at a shrinkage rate of 30% or less;
Only including,
The parenchymal cells are fibroblasts;
The non-parenchymal cells are vascular endothelial cells;
The method, wherein the microstructure is a vascular network . The method according to claim 1, wherein in the shrinking step, the laminate is shrunk at a shrinkage rate in the range of 20 to 30%. In the cell tissue laminating step, using a gelatin gel as a support, laminating the plurality of stretched parenchymal tissue and one or a plurality of stretched non-parenchymal tissue having the microstructure,
3. The method according to claim 1, wherein in the shrinking step, the stretched cell tissue laminate is shrunk by heating and dissolving the gelatin gel. Prior Symbol shrinking step, a laminate, fetal bovine serum (FBS), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), ascorbic acid, heparin, The method according to any one of claims 1 to 3 , wherein the culture is performed in the presence of one or more growth factors selected from the group consisting of hydrocortisone and insulin-like growth factor.

Images