JP2011155945A - Incubator for tissue culture, and method for manufacturing three-dimensional tissue culture - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an incubator for tissue culture capable of conveniently increasing the dynamic strength of a three-dimensional tissue culture having orientation and a method of manufacturing the three-dimensional tissue culture using the incubator for tissue culture. <P>SOLUTION: The incubator for tissue culture is configured to manufacture a three-dimensional tissue culture from cells wherein grooves the width of which is larger in upper parts than in bottom parts are continuously or intermittently formed in a prescribed direction on a face to which the cells are adhered so as to be cultured, and a plurality of grooves formed in the prescribed direction are arrayed in a direction crossing the prescribed direction with prescribed intervals. A method of manufacturing the three-dimensional tissue culture includes: a sowing process for sowing the cells in the incubator for tissue culture; an adhesion and culture process for culturing the sowed cells to adhere the cells to the face for culture, and for producing an extracellular matrix from the cells to form a three-dimensional culture in culture solution; and a separation process for separating the culture from the face for culture after a prescribed period. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a tissue culture incubator and a method for producing a three-dimensional tissue culture.

In recent years, when part or all of biological tissue is damaged due to severe organ failure or intractable disease, or severe sports or traffic accidents, make full use of genetic engineering, cell tissue engineering, regenerative medicine, etc. Attention has been focused on regenerative medical treatment to compensate for this defect.
When cell therapy aiming at tissue repair and regeneration has been performed so far, the majority of researches have been conducted to maintain cell accumulation, stabilize cell proliferation, stabilize differentiation function, and protect against mechanical stress applied to the treatment site. Then, organic polymer compounds and biological scaffolds (Scaffold) have been used (for example, Patent Document 1). However, many of the scaffolds contain biological (animal) materials, biopolymer materials, etc., and the safety of using these materials on the living body has been a problem that cannot be predicted for a long time.

  As a cell transplantation method not using a scaffold such as a scaffold, a cell sheet engineering technique using a temperature-sensitive culture dish is known (for example, Non-Patent Document 1). However, when this cell sheet technology is used, a single sheet is often fragile, and in order to obtain a strength that can withstand surgical operations such as transplantation, it has been necessary to devise operations such as overlapping sheets. .

  In order to solve these problems, a cultured tissue containing no scaffold is known (Patent Document 2 and Non-Patent Document 2). Such scaffold-free self-organizing three-dimensional artificial tissue (3DBT: scaffold-free Three-Dimensional Bioengineered Tissue) cultivates stem cells derived from synovial membrane in a cell culture medium containing an extracellular matrix production promoting factor. It is described that it can be obtained. In addition, the production of extracellular matrix is promoted by adding an extracellular matrix production promoting factor. As a result, the obtained cultured tissue or complex is hardened and easily peeled, and contracted when the culture is peeled off. However, it is described that three-dimensionalization, multi-layering, etc. are promoted. Such a cultured tissue and its complex can solve all the problems caused by contamination of the scaffold itself, and enables excellent treatment in the course after transplantation despite the absence of the scaffold. It is described that it can.

However, because the scaffold-free self-organizing three-dimensional artificial tissue has not yet developed a structure such as a fibrous tissue, it should be handled with care so as not to be damaged by an operation in a surgical procedure even if the self-supporting property can be maintained to some extent. Necessary. In the conventional manufacturing method, there was a limit to improving the tensile strength beyond this.
The scaffold-free self-organizing three-dimensional artificial tissue is an isotropic material in which cells and collagen fibers do not have orientation, but soft tissue having mechanical and morphological anisotropy such as ligaments and tendons. As an alternative tissue, it is desirable to have anisotropy that approximates the tissue in a living body.
Thus, there has been a need for a method of simply increasing the strength of a cultured tissue without using a polymer material or the like, and imparting an orientation that mimics the tissue in a living body to the cultured tissue.

In relation to this, as an incubator for seeding and culturing cells, for example, a square groove having a depth of about several μm to several tens of μm is provided on the cell seeding surface, and the cells are seeded on the surface. Incubators for culturing are described (for example, Non-Patent Documents 3 to 5). It is known that a cultured tissue cultured with such an incubator has an orientation along the direction of the groove in the tissue and cells and collagen fibers contained in the tissue.
However, when a tissue cultured in such an incubator is peeled off from the culture surface of the incubator, a phenomenon such that the cultured tissue is easily broken and the strength cannot be increased is caused. ing. Further, since the depth of the groove is shallow, sufficient structural / mechanical anisotropy cannot be imparted to the generated tissue.
Also from such a point, a method for simply increasing the strength of the cultured tissue while imparting orientation to the cultured tissue has been required.

International Publication No. 2002/010349 Pamphlet Special table 2007-528756 gazette

J Biomed. Mater. Res., Vol. 45, pp. 355-362 (1999) Biomaterials, Vol. 38 (36), pp.5462-5470 (2007) Journal of Biomechanics, Vol. 36, pp.97-102 (2003) Biomaterials, Vol. 29, pp.2565-2572 (2008) Biomaterials, Vol. 30, pp.5417-5426 (2009)

  Therefore, the object of the present invention is a heterogeneous tissue in which the cell density and the substrate density of the groove portion are high and the density is low in the portion other than the groove, and there is no tissue in the living body or conventional biomaterials. It is to provide a tissue culture incubator capable of generating a tissue imparted with a novel property, and a method for producing a three-dimensional tissue culture using the same.

The present invention is as follows.
[1] A tissue culture incubator for producing a three-dimensional tissue culture from cells, wherein a groove having an upper width larger than a bottom width is formed in a predetermined direction on a surface on which the cells are attached and cultured. The tissue culture incubator is provided continuously or intermittently toward the predetermined direction, and a plurality of grooves provided in the predetermined direction are arranged at predetermined intervals in a direction intersecting the predetermined direction.

[2] The tissue culture incubator according to [1], wherein a side surface of the groove is formed of a curved wall surface.
[3] The tissue culture incubator according to [1] or [2], wherein the material is an inorganic material.
[4] The tissue culture incubator according to any one of [1] to [3], wherein the depth of the groove is 5 μm to 40 μm.

[5] A production method for producing a three-dimensional tissue culture containing cells and extracellular matrix in a culture solution, wherein the cells are placed in the tissue culture incubator according to any one of [1] to [4] A seeding process for seeding cells, and a cell culture for seeding and adhering to the surface to be cultured, and producing an extracellular matrix from the cells to form a three-dimensional culture in the culture solution; And a separation step of separating the culture from the surface to be cultured after a predetermined period of time.

[6] The method for producing a three-dimensional tissue culture according to [5], wherein the cells are mesenchymal stem cells.
[7] The method for producing a three-dimensional tissue culture according to [5] or [6], wherein the cells are cultured in the presence of an extracellular matrix production promoting factor.
[8] The three-dimensional tissue culture according to [7], wherein the extracellular matrix production promoting factor is at least one selected from the group consisting of ascorbic acid, ascorbic acid derivatives, TGF-β1 and TGF-β3. It is a manufacturing method.
[9] The three-dimensional tissue culture according to any one of [5] to [8], wherein the extracellular matrix is at least one selected from the group consisting of collagen I, collagen III, vitronectin and fibronectin. It is a manufacturing method.

  According to the present invention, the cell density and the substrate density of the groove portion are high, and the non-uniform tissue is low density in a portion other than the groove, which is a novel tissue that has not been found in in vivo tissues or biomaterials so far. It is possible to provide a tissue culture incubator capable of generating a tissue imparted with properties, and a method for producing a three-dimensional tissue culture using the same.

It is a schematic perspective view which shows an example of the incubator for three-dimensional tissue culture of this invention. It is a schematic sectional drawing (equivalent to AA sectional drawing of FIG. 1) which shows an example of the bottom part of the incubator for three-dimensional tissue culture of this invention. It is a schematic sectional drawing (equivalent to AA sectional drawing of FIG. 1) which shows another example of the bottom part of the incubator for three-dimensional tissue cultures of this invention. It is a schematic sectional drawing (equivalent to AA sectional drawing of FIG. 1) which shows another example of the bottom part of the incubator for three-dimensional tissue cultures of this invention. It is a schematic perspective view which shows another example of the incubator for three-dimensional tissue culture of this invention. It is a schematic perspective view which shows another example of the incubator for three-dimensional tissue culture of this invention. It is a schematic perspective view which shows another example of the incubator for three-dimensional tissue culture of this invention. It is process drawing which shows an example of the manufacturing method of the incubator for three-dimensional tissue culture of this invention. It is process drawing which shows an example of the manufacturing method of the incubator for three-dimensional tissue culture of this invention.

It is the figure which showed typically preparation of the sample concerning the Example of this invention. It is a differential interference microscope image of the sample obtained in the Example of this invention. It is a stress-strain curve of the sample obtained in the Example of this invention. It is a stress-strain curve of the sample obtained in the Example of this invention. It is a stress-strain curve of the sample obtained in the Example of this invention. It is a stress-strain curve of the sample obtained in the Example of this invention. It is a stress-strain curve of the sample obtained in the Example of this invention.

The tissue culture incubator according to the present invention is a tissue culture incubator for producing a three-dimensional tissue culture from cells, and attaches and cultures the cells (hereinafter referred to as culture surface), Grooves having an upper width larger than the width of the bottom are provided continuously or intermittently in a predetermined direction, and a plurality of grooves provided in the predetermined direction are arranged at predetermined intervals in a direction intersecting the predetermined direction. It is an arrayed tissue culture incubator (hereinafter referred to as an incubator).
The culture surface is generally the bottom surface of the incubator, but may be a side surface.

  The method for producing a three-dimensional tissue culture of the present invention is a method for producing a three-dimensional tissue culture containing cells and an extracellular matrix in a culture solution, and seeds the cells in the incubator of the present invention. A seeding step, and an adherent culture step of culturing the cells after seeding and attaching them to the culture surface of the incubator and producing an extracellular matrix from the cells to form a three-dimensional culture in the culture solution; And a separation step of separating the culture from the surface to be cultured after a predetermined period of time.

According to the method for producing a three-dimensional tissue culture using the incubator of the present invention, the cell density and the substrate density of the groove portion are high, and the non-uniform tissue is low in the portion other than the groove, It is possible to generate a tissue to which a new property that has not been found in in vivo tissues or biomaterials so far is given.
Moreover, according to the method for producing a three-dimensional tissue culture using the incubator of the present invention, it is possible to impart orientation to the three-dimensional tissue culture. Then, by imparting orientation, mechanical strength can be imparted to the three-dimensional tissue culture, and the mechanical strength can be easily increased. This makes it possible to produce a culture that is mechanically useful as a substitute for tissue in a living body. In addition, a culture excellent in handling ability of a surgical technique can be produced.

In the culture surface of the incubator of the present invention, the grooves are provided continuously or intermittently in a predetermined direction, and extracellular matrices such as cells and collagen are aligned along the direction, and a three-dimensional tissue is formed. It is believed that orientation occurs in the culture.
In addition, since the groove formed on the culture surface has a larger width at the top than the width of the bottom, when separating the culture cultured to be embedded in the groove from the culture surface, the groove in the culture It is considered that the portion embedded in the layer is easily removed from the groove and is less likely to be damaged. For this reason, even if the depth of the groove is increased, it is considered that a decrease in strength of the culture due to the damage is suppressed, and as a result, a three-dimensional tissue culture having high strength (tensile strength) can be obtained.

  In the present invention, the term “orientation” is used in the meaning normally used in this technical field. The orientation of the three-dimensional tissue culture can be confirmed morphologically by observation with the naked eye and observation using a magnifier, optical microscope, electron microscope, etc. At this time, tissue staining such as HE staining or immunostaining is performed. You can go and observe. The orientation of the three-dimensional tissue culture can be confirmed mechanically by measuring tensile strength, rigidity, Young's modulus, etc. described later in a plurality of directions.

First, the incubator of the present invention will be described.
For example, as shown in FIGS. 1 and 2, the incubator of the present invention is configured by a circular and transparent glass dish 10, and this bottom surface is used as a culture surface 10 </ b> A.
A plurality of grooves 12 are formed in stripes on the culture surface 10A. Specifically, one groove 12 is continuously formed linearly in a predetermined direction, and a predetermined interval (predetermined) in a direction intersecting (for example, orthogonal to) the predetermined direction (extending direction of the groove 12). Pitch).

The groove 12 has an upper width (width at the opening edge of the groove 12) larger than the width of the bottom. Specifically, for example, the groove 12 has a surface parallel to the culture surface 10A as the bottom surface, and the top from the bottom. The cross-sectional shape gradually increases in width toward. Specifically, the groove | channel 12 is comprised, for example with the wall surface where the bottom face was comprised by the surface parallel to the culture surface 10A, and the side surface was curved.
In particular, the side surface of the groove 12 is preferably composed of a curved wall surface (specifically, both end portions in the width direction at the bottom of the groove 12 are preferably composed of curved wall surfaces). Since the culture to be cultivated does not enter at both ends in the width direction at the bottom of 12 and the culture is separated from the culture surface 10A without applying excessive force, the damage is unlikely to occur, resulting in the strength of the culture. Is increased.
Here, the cross-sectional shape of the groove 12 means that the incubator in which the groove 12 is formed along a predetermined direction is cut along a direction orthogonal to the predetermined direction (extending direction of the groove 12). It means a cross-sectional shape. Moreover, the upper part of the groove | channel 12 means the direction orthogonal to the culture surface 10A and facing the outer side of the culture surface 10A.

  The depth R1 of the groove 12 is, for example, 5 to 40 μm, preferably 10 to 40 μm, more preferably 15 to 35 μm, still more preferably 20 to 30 μm, and particularly preferably 25 to 30 μm. By setting the depth R1 of the groove 12 in the above range, the orientation of the obtained culture can be easily obtained, and when the obtained culture is separated from the culture surface 10A of the incubator, Damage is suppressed.

  The width R2 of the groove 12 (width at the opening edge of the groove 12: maximum width) is, for example, preferably 40 to 150 μm, preferably 50 to 120 μm, and more preferably 60 to 100 μm. By setting the width R2 of the groove 12 in the above range, it is easy to obtain the orientation of the obtained culture, and when the obtained culture is separated from the culture surface 10A of the incubator, the culture is damaged. Is suppressed.

  The pitch R3 (arrangement interval) of the grooves 12 is, for example, preferably 100 to 220 μm, preferably 120 to 200 μm, more preferably 140 to 180 μm, and still more preferably 140 to 160 μm. By setting the pitch R3 of the groove 12 in the above range, the orientation of the obtained culture can be easily obtained, and the culture is damaged when the obtained culture is separated from the culture surface 10A of the incubator. Is suppressed.

  The ratio (R1: R2) between the depth R1 and the width R2 of the groove 12 is, for example, 1:30 to 1: 1, and preferably 1: 5 to 1: 2. By setting the ratio of the depth R1 and the width R2 of the groove 12 to the above range, the orientation of the obtained culture can be easily obtained, and when the obtained culture is separated from the culture surface 10A of the incubator. In addition, damage to the culture is suppressed.

  In addition to the cross-sectional shape described above, for example, 1) as shown in FIG. A U-shaped cross-sectional shape having a surface parallel to the culture surface 10A, gradually increasing in width from the bottom toward the top, and constant in width, 2) As shown in FIG. 4, these cross-sectional shapes In addition, a cross-sectional shape in which the opening edge of the groove 12 is chamfered (for example, the cross-sectional shape shown in FIG. 2 in FIG. 4 shows a cross-sectional shape chamfered in a curved shape) and the like.

  Moreover, the groove | channel 12 is not restricted to the form in which the one groove | channel 12 was continuously provided in linear form, For example, as shown in FIG. 5, the form provided intermittently may be sufficient. Even if the groove 12 is provided intermittently, orientation can be imparted to the culture.

  Further, the groove 12 is not limited to a form in which a plurality of grooves 12 are arranged at a predetermined interval (pitch) and formed in a stripe shape. As shown in FIG. 6, one groove 12 is formed on the culture surface 10A in a predetermined direction. The first groove group is formed continuously in a straight line toward the surface and arranged at a predetermined interval (predetermined pitch) in a direction intersecting the predetermined direction (extending direction of the groove 12). A configuration in which a second groove group similar to the first groove group is disposed in a direction (for example, an orthogonal direction) intersecting the predetermined direction with respect to the first groove group formed in the predetermined direction ( In other words, the groove 12 may be provided in a lattice shape. Thereby, a culture having orientation in two directions, that is, a culture in which strength (tensile strength) is given to the two directions is obtained.

  Further, the incubator of the present invention is not limited to the form in which the bottom surface of the circular glass dish 10 is the culture surface 10A and the grooves are provided in the culture surface. For example, as shown in FIG. The surface may be a culture surface, and grooves 12 may be formed in the culture surface. For example, the plate-like body 11 provided with the grooves 12 is used by being arranged on the bottom surface of a circular glass dish 10 as shown in FIG.

Next, the manufacturing method of the incubator of the present invention will be described.
In the method for producing an incubator of the present invention, for example, an incubator is prepared, and an etching process (for example, a dry etching process or a wet etching process) is performed on the culture surface, whereby a groove is provided on the culture surface. And a method for obtaining an incubator. Of course, the method for producing the incubator of the present invention is not particularly limited, and a well-known method can be adopted if a groove can be formed on the culture surface.

  Examples of the method for producing the incubator of the present invention include the methods shown in FIGS. This method for producing an incubator is a method in which a wet etching process is performed on a glass circular dish to form grooves on the culture surface.

Specifically, first, a circular glass dish 10 or a circular plate body 11 (for example, Tempax glass: Chiba science glass) is prepared. These members are not limited to a circular shape.
Hereinafter, the case where the circular glass dish 10 is used will be described as an example, but the same applies to the case where the circular plate body 11 is used.
As shown in FIG. 8 (A), the bottom surface that becomes the culture surface 10A of the circular glass dish 10 was ultrasonically cleaned in the order of isopropanol alcohol (Taisei Chemical Co., Ltd.) and pure water for 15 minutes, respectively. The dirt adhering to the bottom surface of 10 is removed. After the cleaning, for example, water attached to the bottom surface of the glass dish 10 is blown off by an air gun, for example, heated on a hot plate set at 120 ° C. for 30 minutes, and the water is completely blown off.

  Next, as shown in FIG. 8B, when the glass dish 10 is cooled, a metal mask layer 20 (for example, a Cr film) is deposited on the bottom surface of the glass dish 10 by, for example, a three-way sputtering apparatus. At this time, it is confirmed that the surface (bottom surface) on which the metal mask layer 20 is deposited is a clean surface without scratches.

Next, as shown in FIG. 8C, a photosensitive resin (for example, OFPR: Tokyo Ohka Kogyo Co., Ltd.) is uniformly applied to the bottom surface of the glass dish 10 on the metal mask layer 20 by, for example, a spin coater. Forming a layer. In this spin coater rotation program, for example, the initial speed of 300 rpm is set to 5 s, the main speed of 5000 rpm is set to 15 s, and the final speed of 6000 rpm is set to 0.2 s.
Then, for example, after spin coating, pre-baking is performed at 110 ° C. for 5 minutes to cure the liquid photosensitive resin (for example, OFPR: Tokyo Ohka Kogyo) to form the photosensitive resin layer 22.

  Next, as shown in FIG. 8D, the photosensitive resin layer 22 is irradiated with a pattern of ultraviolet rays for 20 s by a single-side mask aligner, and then immersed in a developer (for example, NMD-3: Tokyo Ohka Kogyo Co., Ltd.). Development processing is performed to form an opening pattern 22 </ b> A in the photosensitive resin layer 22. Thereafter, for example, in order to completely remove the developer, the surface is washed with pure water three times, water on the surface is removed by an air gun, and the photosensitive resin layer 22 is brought into close contact with the lower layer (metal mask layer 20). Post bake at 5 ° C. for 5 minutes.

  Next, as shown in FIG. 9E, for example, the metal mask layer 20 is etched with a metal etchant (for example, ETCH-1: Sanyo Kasei) to form an opening pattern 20A. Thereafter, for example, the metal etching solution is completely removed by washing three times with pure water, and moisture is blown off with an air gun.

  Next, as shown in FIG. 9F, after confirming whether the metal mask layer 20 is etched with the desired opening pattern 20A, for example, with hydrofluoric acid until the depth of the predetermined groove 12 is reached. Etching is performed on the bottom surface of the glass dish 10 to form grooves 12. Then, for example, it is immediately washed three times with pure water, hydrofluoric acid is completely removed, and moisture is blown off with an air gun.

Next, as shown in FIG. 9G, the photosensitive resin layer 22 is removed using, for example, a stripping solution warmed to 70 ° C. (for example, stripping solution 105: Tokyo Ohka Kogyo Co., Ltd.). Thereafter, for example, after the stripping solution is replaced with isopropanol alcohol, it is washed twice with pure water.
Then, the metal mask layer 20 is removed with a metal etching solution (for example, ETCH-1: Sanyo Chemical). Similarly, it is purely washed three times to completely remove the metal etching solution and blow off moisture with an air gun.

An incubator is obtained through the above steps.
In the above manufacturing method, for example, the width and depth of the groove 12 to be formed depending on the shape of the opening pattern formed in the metal mask layer 20 and the photosensitive resin layer 22, the etching time with respect to the bottom surface of the glass dish 10, and the like. The arrangement interval (pitch) and the like are controlled.
Further, when the above wet etching process is employed, the width of the upper portion is larger than the width of the bottom portion, and the groove 12 constituted by a curved wall surface is easily obtained.

  Further, in the incubator of the present invention, the material using glass as the material has been described, but the material is not limited to this, and a resin such as plastic, a polymer such as PDMS (polydimethylsiloxane), etc. It may be adopted. Incubators made of inorganic materials such as glass, ceramic, and metal (for example, silicon) have little deformation and can be used repeatedly if sterilized. In addition, by employing a transparent glass incubator, it becomes possible to observe cells in real time.

Next, the manufacturing method of the three-dimensional tissue culture of this invention is demonstrated.
The method for producing a three-dimensional tissue culture of the present invention is a method for producing a three-dimensional tissue culture containing cells and an extracellular matrix in a culture solution using the incubator of the present invention. Specifically, the seeding step of seeding the cells in the incubator of the present invention, the cells after seeding are attached to the surface to be cultured and cultured, and the extracellular matrix is produced from the cells to be three-dimensional in the culture solution. An adherent culture step for forming the culture, and a separation step for separating the culture from the surface to be cultured after a predetermined period.

The cells to be seeded in the incubator are not particularly limited, but stem cells are preferable from the viewpoint of proliferation ability and differentiation ability.
In the present invention, the stem cell refers to a cell having self-renewal ability and differentiation ability. Examples of the stem cells in the present invention include embryonic stem (ES) cells and somatic stem cells, and artificially prepared pluripotent stem cells may also be used as long as they have the above-mentioned ability. Corresponds to stem cells in Embryonic stem cells refer to pluripotent stem cells derived from early embryos, and somatic stem cells are cells whose direction of differentiation is more limited than embryonic stem cells. As somatic stem cells, there are stem cells derived from ectoderm, stem cells derived from mesoderm, stem cells derived from endoderm. For example, stem cells derived from ectoderm are derived from skin system, nervous system, mesoderm. Stem cells are classified into the myeloid system, and stem cells derived from the endoderm are classified into the digestive system. Examples of cutaneous somatic stem cells include epidermal stem cells and hair follicle stem cells. Examples of digestive somatic stem cells include pancreatic (common) stem cells and hepatic stem cells. Examples of myeloid somatic stem cells include hematopoietic stem cells, mesenchymal stem cells (for example, fat-derived, bone marrow-derived), and the like. Examples of neural somatic stem cells include neural stem cells and retinal stem cells. In the present invention, it is preferably used for mesenchymal stem cells.

  Mesenchymal stem cells have the ability to proliferate and differentiate into bone cells, chondrocytes, muscle cells, stromal cells, tendon cells, adipocytes, and the like. In the present invention, stem cells capable of differentiating into tendon cells, bone cells, chondrocytes and the like can be preferably used.

  These mesenchymal stem cells can be obtained from a tissue in which these stem cells can exist. Tissues preferably used for obtaining stem cells in the present invention include bone, cartilage, tendon, ligament, meniscus, intervertebral disc, periosteum, blood vessel, blood vessel-like tissue, heart, heart valve, pericardium, dura mater, etc. Can be mentioned. In particular, when stem cells that can differentiate into tendon cells are used, tissues such as synovium can be used as a supply source, and it is preferable to use synovial stromal cells. When stem cells that can be differentiated into bone cells or chondrocytes are used, tissues such as fat can be used as the supply source, and it is preferable to use adipose tissue-derived cells.

Moreover, as a cell seed | inoculated to a culture device, the cell with high productivity of an extracellular matrix is preferable. Examples of cells with high productivity of extracellular matrix include mesenchymal stem cells, fibroblasts, bone cells, chondrocytes, stromal cells, tendon cells, synovial cells, adipocytes, and the like.
The extracellular matrix is not particularly limited, but at least one of collagen I, collagen III, vitronectin and fibronectin is preferable.

  The origin of the cell is not particularly limited, but when a three-dimensional tissue culture is used as a graft for humans, it is appropriately selected from humans and other mammals in consideration of species compatibility. It is preferable. Examples of other mammals include, for example, rodents, carnivores, carnivores, long noses, odd hoofs, even hoofs, rodents, scales, sea cattle, cetaceans, primates, rodents Specific examples include animals such as monkeys, cows, pigs, horses, cats, and dogs. From the viewpoint of tissue compatibility, autotransplantation is preferable.

  These cells may be so-called primary cultures collected from a living body or those that have been passaged, but from the viewpoint of improving cell density and improving extracellular matrix production ability, they are more than 4 passages and 7 passages. It is preferable to use cultured cells of the following generation. In the present invention, “passage” is used in the meaning normally used in the art, and the cells in the present invention are represented by the number of passages since the primary culture collected from a living body. If the cell in the present invention is a cultured cell having 4 or more passages, a sufficiently strong culture can be obtained, and if the cell has 7 or less passages, the cell growth ability is sufficiently high, which is preferable. Moreover, if it is a cultured cell of 5 to 7 passages, the balance between the production of extracellular matrix and the cell density when used as a three-dimensional tissue culture can be improved at a high level. preferable.

The culture solution used for culturing cells may be any one that is generally used for culturing target cells. For example, DMEM, MEM, F12, DME, RPMI1640, MCDB104, 199, MCDB153, L15, SkBM Basal medium and the like can be used. In addition, various components that can be generally added, such as glucose, FBS (fetal bovine serum) or human serum, and antibiotics (such as penicillin and streptomycin) may be added to these culture solutions. In addition, although the density | concentration at the time of adding serum can be suitably changed according to a culture state, it can usually be 10% (v / v). For cell culture, normal culture conditions, for example, culture in an incubator with a temperature of 37 ° C. and a concentration of 5% CO ₂ are applied.

The seeding of cells can be appropriately adjusted depending on the type of cells, the number of passages of cells, the culture period, etc., but in general, seeding can be performed at a cell density of 5 × 10 ⁴ to 1 × 10 ⁶ cells / cm ² .
The method for seeding cells is not particularly limited, and can be performed, for example, by injecting a culture solution in which cells are suspended into a culture vessel.

When the seeding of the cells is completed, in the adhesion culture step, the cells are cultured to attach the cells to the culture surface, and an extracellular matrix is produced from the cells to form a three-dimensional culture in the culture solution.
Adhesion of the cells to the culture surface is more easily achieved by attaching the cells to the culture surface according to the properties of the cells under normal culture conditions. Depending on the width and depth of the grooves formed on the culture surface and the size and nature of the seeded cells, the cells can also adhere to the walls of the grooves.
Cell attachment does not have to be firmly attached to the culture surface, it is sufficient that the shape of the culture surface is attached with an adhesive force that can be perceived by the cell, and it does not float independently due to the nature of the cell. In addition, the contact with the culture surface of the incubator may be sufficient, and in the separation step to be described later, the adhesion can be easily separated by a predetermined separation means, so that damage to the culture can be suppressed. This is preferable.

Further, by continuing the culture period, extracellular matrix is produced from the cells.
Examples of the extracellular matrix produced from the cells include collagen I, collagen III, collagen V, elastin, vitronectin, fibronectin, laminin, thrombospondin, proteoglycans (eg, decorin, biglycan, fibrojuline, lumican, hyaluron) Acid, aggrecan, and the like), but are not limited to these, and any extracellular matrix that is responsible for cell adhesion may be used. More preferably, the extracellular matrix includes all collagen (type I, type III, etc.), vitronectin and fibronectin. By producing these extracellular matrixes, a three-dimensional culture composed of the cells in which the extracellular matrix network is formed and the extracellular matrix is formed.

In order to promote the production of extracellular matrix from the cells, it is preferable that the culture of the cells is performed in the presence of an extracellular matrix production promoting factor. For this reason, it is preferable that an extracellular matrix production promoting factor is added to the culture solution.
The extracellular matrix production promoting factor may be present in the adhesion culture step, may be added to the culture solution from the beginning of the culture, or may be added to the culture solution after cell seeding.
By culturing cells in the presence of these extracellular matrix production promoting factors, the culture can be more easily separated in the separation step described later.

  The extracellular matrix production promoting factor added to the culture medium is a factor that promotes the secretion of extracellular matrix of the cell. For example, TGF-β1, TGF-β3, ascorbic acid, ascorbic acid diphosphate or Examples thereof include derivatives thereof and salts thereof. From the viewpoint of collagen production, ascorbic acid, ascorbic acid diphosphate or a derivative thereof and a salt thereof (for example, sodium salt, magnesium salt, potassium salt, etc.) can be preferably used. Ascorbic acid in the present invention is preferably L-form, but is not limited thereto.

  In the case of ascorbic acid 2-phosphate, for example, the amount of the extracellular matrix production promoting factor used in the present invention is usually 0.01 mM or more, preferably from the viewpoint of the tensile strength of the three-dimensional tissue culture. It can be 0.05 mM or more, more preferably 0.1 mM or more, more preferably 0.2 mM or more. In the case of ascorbic acid diphosphate, the addition amount may be increased, for example, 5.0 mM or less, but is more preferably 1.0 mM from the viewpoint of the tensile strength improvement efficiency with respect to the addition amount.

  The adhesion culture period for forming a three-dimensional tissue culture varies depending on the number of cells seeded, the type of cells, the type of target tissue, etc., but can generally be at least 7 days. From the viewpoint of expecting an increase in strength of the culture, it is also preferable to extend the adhesion culture period to, for example, about 15 days, more preferably about 21 days, more preferably about 28 days, and particularly preferably about 35 days. . Thereby, a three-dimensional tissue culture having a size and strength suitable for the purpose can be formed.

In the separation step, the culture being cultured is separated from the culture surface.
The three-dimensional tissue culture formed by the culturing process floats in the culture medium by separation from the culture surface. By applying this separation stimulus, self-contraction occurs, resulting in hardening of the culture (an increase in stiffness and strength due to cross-linking of fibrous tissue).

  As a means for separation, any stimulus that causes self-contraction in the culture while not damaging the culture may be used. Examples of such separation means include physical means (for example, pipetting of a medium), chemical means (addition of a substance), and the like. Among these, physical means such as pipetting is preferable from the viewpoint of preventing foreign matters from being mixed into the obtained culture. In addition, exfoliation can be promoted by applying a physical stimulus (for example, applying a physical means such as application of shear stress, pipetting, or deformation of the incubator with a rod at the corner of the incubator). it can. Self-shrinking occurs naturally after such debonding when a physical stimulus is applied. In the case of chemical stimulation, self-contraction and peeling occur in parallel. It is thought that the self-contraction caused by such stimulation promotes biological binding, particularly in the third dimension, so that the culture can maintain its form as a three-dimensional structure. .

In the method for producing a three-dimensional tissue culture of the present invention, a suspension culture step of suspension culture in a state where the culture is separated from the incubator can be provided after the separation step. The three-dimensional culture separated from the culture surface in the separation step naturally becomes a floating state in the culture solution, and the floating culture step is performed by continuing the culture in such a floating state for a predetermined period. The strength of the three-dimensional culture can be improved by the suspension culture process.
The culture period in a floating state is not particularly limited, but it is preferable to continue for at least one day. Sufficient self-shrinkage can be produced by setting it as 3 days or more.

  The three-dimensional tissue culture obtained by the present invention is a so-called scaffold-free tissue culture that does not contain an artifact, and is sufficient to ensure operability without being damaged by an operation in a surgical technique. It has strength. Moreover, it is useful as a substitute for the tissue in the living body to which the orientation imitating the tissue in the living body is imparted mechanically and morphologically.

  Here, as a culture, sufficient strength to ensure operability without being damaged by operation in the surgical technique means that it is grasped with tweezers etc. in the surgical technique in addition to sufficient strength for self-supporting It means the strength not to be destroyed when doing. “Self-supporting” in the present invention refers to a property that the tissue is not substantially destroyed when at least one point of the tissue is fixed in space. The three-dimensional tissue culture in the present invention exhibits a strength greater than a strength sufficient for self-supporting, and can obtain a remarkable tensile strength compared to a tissue obtained by a conventional culture method. is there.

  The mechanical properties of the tissue that resists the tension of the cultured tissue, three-dimensional structure, etc. in the present invention can be generally determined by conducting a tensile test and measuring the tensile strength, rigidity, Young's modulus, and the like. .

  Further, the mechanical properties of the cultured tissue, the three-dimensional structure and the like in the present invention can be obtained by measuring stress / strain properties in a mechanical test. Briefly, a load is applied to a sample, for example, 1ch is strain, 2ch is input to each AD converter (eg ELK-5000), stress and strain are measured, tensile strength and tangential coefficient (The hardness of the material) can be obtained.

  Mechanical properties can also be determined by testing creep properties. The creep characteristic indentation test is a test for examining how the material expands with time under a certain load. For the indentation test of a minute material, a thin material, etc., an indenter having a radius of about 0.1 to 1 μm, for example, a triangular pyramid indenter is pushed into the test object. First, an indenter is pushed into the test piece to give a load. Then, when the test piece is pushed into the test piece by about several tens nm to several μm, the indenter is returned and unloaded. The hardness, Young's modulus, and the like can be obtained from the load load obtained from the load unloading curve obtained by such a test method and the behavior of the indentation depth.

  The high strength of the three-dimensional tissue culture of the present invention can be evaluated using a numerical value based on tensile strength as an index. The numerical value based on the tensile strength is preferably at least 0.1 MPa or more from the viewpoint of handling of the surgical technique, and the higher the better. This tensile strength can be confirmed by a tensile test. The intensity is defined as measured under the following conditions.

  In the tensile test, each sample is sandwiched between aluminum alloy (A6061) clamps in PBS maintained at 37 ° C., and both sides are trimmed so that the width is 6 mm. The temperature of the PBS is controlled by a silicon rubber heater (general SR: Three High) arranged on the bottom of the bath and a temperature controller. One of the clamps is connected to an actuator (LAH-46-3002-F-PA-B1-SP, Harmonic Drive Systems), and the other is integrated with the load detector. A load detection part makes it possible to detect a micro load correctly also by sticking a strain gauge (KGF-02-120-C1-23, Kyowa Dengyo) two sheets on each side. In order to measure the strain of each sample in a non-contact manner during the test, the marker was prepared using a micro magnet (mass 0.002 g, dimensions 1 × 1 × 1 mm) and expanded polystyrene beads (φ2 mm). Insert and fix. The cross-sectional area is calculated from the thickness and width measured with a microscope (VHX-100, Keyence). 2 mN is applied as a preload, the state is defined as zero strain, and a tensile load is applied until the sample breaks at a tensile speed of 0.05 mm / s.

  Examples of the present invention will be described below, but the present invention is not limited thereto. Moreover,% in an Example is a mass reference | standard unless there is particular notice.

[Example 1]
<Preparation of three-dimensional tissue culture from human synovial stromal cells>
(1) Maintenance of cultured cells A cell for obtaining a three-dimensional tissue culture was prepared using human synovial stromal cells collected after obtaining consent from the patient.
Synovial tissue pieces were collected from the knee joint, treated with collagenase, and cultured in DMEM culture solution (10% FBS, 1% penicillin / streptomycin) at 5% CO ₂ and 37 ° C. Cultured cells were maintained by replacing about 1/3 of the culture solution (20 ml) every 7 days, and when subcultured, the cells were subcultured every 14 days.

(2) Preparation of Test Specimen (Sample A) A cell group that has been subcultured 7 times by the method of (1) above to increase the number of cells so that the initial cell density is 4.0 × 10 ⁵ cells / cm ² And suspended in DMEM culture solution (10% FBS, 1% penicillin / streptomycin, 0.2 mM ascorbic acid 2-phosphate), and inoculated in an incubator, and the culture was started under conditions of 5% CO ₂ and 37 ° C. . The incubator used here has a groove having a semicircular cross-sectional shape as illustrated in FIG. 2 formed on the culture surface (bottom surface) in a stripe shape as illustrated in FIG. The pitch is 200 μm, the width is 100 μm, the depth is 30 μm, and the bottom material is glass (Tempax glass: Chiba Science glass), diameter 35 mm). The cells attached to the bottom of the incubator soon after seeding.

On the 35th day of culture, the culture was detached from the bottom of the plate using the tip of a micropipette and maintained for 1 hour in a floating state to self-contract. After self-contraction, the culture was taken out of the culture medium and used as sample A (see FIG. 10).
A differential interference microscope image of sample A is shown in the center of FIG. As shown in the center of FIG. 11, Sample A had a texture oriented. The direction of this orientation almost coincided with the direction of the groove on the culture surface (bottom surface), and cells and extracellular matrix were aligned along the direction of the groove.

(3) Preparation of Comparative Test Specimen (Sample B) A cell group that has been subcultured seven times by the method of (1) above is suspended in a culture solution in the same manner as in (2) above, and is suspended in a 6-well plate (BD Falcon). After seeding, the culture was started under conditions of 5% CO ₂ and 37 ° C. The cells attached to the bottom of the plate shortly after seeding.

On the 35th day of culture, the culture was detached from the bottom of the plate using the tip of a micropipette and maintained for 1 hour in a floating state to self-contract. After self-contraction, the culture was removed from the culture medium and used as sample B.
A differential interference microscope image of Sample B is shown on the left of FIG. As shown in the left of FIG. 11, the sample B was not oriented in structure.

(4) Evaluation of test piece The mechanical characteristics of the samples A and B obtained above were evaluated as follows.
(A) Thickness Spread each sheet-shaped sample on a slide glass, add 10 μl of PBS, sandwich it with a cover glass, and use a digital microscope (VHX-100, Keyence) to set the distance between the slide glass and the cover glass at five locations. The average of the measured values was taken as the thickness of each sample. The cross-sectional area of each sample was determined from the thickness and width of each sample. The results are shown in Table 1.

(B) Tensile strength and rigidity The tensile strength and rigidity of each sample were evaluated by conducting the following tensile tests.
Each sample was sandwiched between aluminum alloy clamps, and both sides were cut to a width of 6 mm. A tensile load is applied to the sample at a tensile speed of 0.05 mm / s until the sample breaks (breaking point, BP), and the tensile load at the time the sample breaks is divided by the cross-sectional area of the sample. Strength. Note that a tensile load was applied to the sample A in the direction of tissue orientation.
At the same time, the strain of the sample was measured, the tangential coefficient in the strain range 0% to 5% of the stress-strain diagram was obtained, and this numerical value was taken as the stiffness.
The strain was measured in a non-contact manner by using an image sensor (CV-750, Keyence) for a marker attached to each sample. The marker was manufactured using a micro magnet (weight 0.002 g, dimensions 1 × 1 × 1 mm) and polystyrene foam beads (φ2 mm), and the sample was sandwiched from above and below and fixed to the sample. A preload of 2 mN was applied by a linear actuator (LAH-46-3002-F-PA-V1-SP, Harmonic Drive Systems), and the state was defined as zero strain.
The stress was determined by dividing the load output of the strain gauge by the initial cross-sectional area of each sample. Here, the cross-sectional area was calculated from the thickness and width measured with a microscope (VHX-100, Keyence).
During the tensile test, in order to simulate the test environment in the living body, each sample is immersed in PBS in the bath, and the temperature of the PBS is adjusted using a silicon rubber heater (general SR, three high) and a temperature controller (Kyowa Electronics Industry). Maintained at 37 ° C.

  Tensile strength and tangential coefficient were tested by t-test, with a significance level of 5%. The results are shown in Table 1. A stress-strain curve is shown in FIG. In FIG. 12, the white square represents sample A and the black circle represents sample B.

As shown in Table 1, the sample A has no significant difference in thickness as compared with the sample B, but the tensile strength is significantly increased (P <0.05) and the rigidity (strain range 0%). The tangential coefficient at ˜5%) also increased significantly (P <0.05).
From this, orientation can be imparted to the culture by the method of culturing using the incubator of the present invention without applying mechanical stimulation or chemical stimulation. It has been found that the strength and rigidity can be increased.

[Example 2]
<Examination of groove arrangement direction>
(1) Preparation of test piece (sample C) In the preparation of sample A of Example 1, a culture was prepared in the same manner as the preparation of sample A except that the culture period was 28 days. . The differential interference microscope image of Sample C was the same as Sample A. In Sample C, the tissue was oriented, and the direction of this orientation almost coincided with the direction of the groove on the culture surface (bottom surface), and cells and extracellular matrix were aligned along the direction of the groove.

(2) Preparation of test piece (sample D) In the preparation of sample A of Example 1, the culture period was 28 days, and the culture was performed in the same manner as the preparation of sample A, except that the incubator used was changed. An object was prepared as Sample D. The incubator used here has grooves with a semicircular cross-sectional shape as illustrated in FIG. 2 formed in a lattice shape as illustrated in FIG. 6 on the culture surface (bottom surface) (first). The groove group and the second groove group have a pitch of 200 μm, a width of 100 μm, a depth of 30 μm, the first groove group and the second groove group are orthogonal, and the bottom material is glass (Tempax glass: Chiba science glass), diameter 35 mm).
A differential interference microscope image of Sample D is shown on the right side of FIG. As shown in the right side of FIG. 11, the sample D had the tissue oriented in two directions. The direction of this orientation almost coincided with the direction of the groove on the culture surface (bottom surface), and cells and extracellular matrix were aligned in two directions along the direction of the groove.

(3) Evaluation of Specimen Samples C and D were measured for thickness, tensile strength, and tangential coefficient in the same manner as in Example 1. However, for the tangent coefficient, the tangential coefficient in the strain range of 0% to 5% and the tangential coefficient in BP-5% to BP were obtained. For sample C, the thickness was also measured in a direction orthogonal to the orientation direction of the tissue (sometimes referred to as “vertical direction”), and a tensile test and a tangential coefficient were measured. For sample D, a tensile load was applied along one of the two orientations when the tensile test was performed.
The results are shown in Table 2. A stress-strain curve is shown in FIG. In FIG. 13, the white square represents the orientation direction of the sample C, the white circle represents the vertical direction of the sample C, and the black diamond represents the sample D.

As shown in Table 2, there was no significant difference in thickness between the orientation direction of sample C, the vertical direction of sample C, and sample D.
The orientation direction of sample C significantly increased the tensile strength as compared to the vertical direction of sample C (P <0.05). In addition, there was no difference in the stiffness in the low strain range (tangential coefficient in the strain range of 0% to 5%), but the stiffness in the high strain range (BP-5% to tangential coefficient in the BP) was significantly increased. (P <0.05).
The orientation direction of sample C significantly increased the tensile strength compared to sample D (P <0.05). In addition, no significant difference was observed in the stiffness in the low strain region (tangential coefficient in the strain range of 0% to 5%), but the stiffness in the high strain range (BP-5% to tangential coefficient in the BP) was significant. (P <0.05).
Although sample D was not significantly different from the vertical direction of sample C, the tensile strength and the rigidity in the high strain region (BP-5% to tangential coefficient in BP) increased.
From this, it was found that mechanical anisotropy can be imparted to the culture by the method of culturing using the incubator of the present invention.

[Example 3]
<Examination of groove spacing>
(1) Preparation of Specimen (Sample E) In the preparation of Sample A of Example 1, the sample had the initial cell density of 1.0 × 10 ⁵ cells / cm ² and the culture period was 2 months. A culture was prepared in the same manner as in preparation of A, and used as sample E.

(2) Preparation of test piece (sample F) In the preparation of sample A of Example 1, the initial cell density was 1.0 × 10 ⁵ cells / cm ² , the culture period was 2 months, and the culture used A culture was prepared in the same manner as the preparation of sample A except that the vessel was changed, and was used as sample F. The incubator used here has a groove having a semicircular cross-sectional shape as illustrated in FIG. 2 formed on the culture surface (bottom surface) in a stripe shape as illustrated in FIG. The pitch is 160 μm, the width is 100 μm, the depth is 30 μm, and the bottom material is glass (Tempax glass: Chiba science glass), diameter 35 mm).

(3) Evaluation of test piece For Samples E and F, the thickness, tensile strength, and tangential coefficient were measured in the same manner as in Example 1. However, for the tangent coefficient, the tangential coefficient in a strain region of 10% to 40% was obtained.
The results are shown in Table 3. A stress-strain curve is shown in FIG. In FIG. 14, the black triangle represents the sample E, and the white square represents the sample F.

As shown in Table 3, sample F (pitch 160 μm) showed significant differences in thickness and tensile strength compared to sample E (pitch 200 μm) (P <0.05).
Regarding the rigidity (tangential coefficient in the strain range of 10% to 40%), no significant difference was observed, but the value of Sample F was larger than that of Sample E.
From this, it was found that the thickness, strength and rigidity of the culture can be increased by narrowing the interval (pitch) at which the grooves on the culture surface are arranged.

[Example 4]
<Examination of groove depth>
(1) Preparation of test piece (sample G) In the preparation of sample A of Example 1, the initial cell density was 6.0 × 10 ⁵ cells / cm ² , the culture period was 21 days, and the incubator used A culture was prepared in the same manner as in the preparation of Sample A, except that the sample was changed to Sample G. The incubator used here is a culture surface (bottom surface) in which grooves having a semicircular cross-sectional shape as illustrated in FIG. 2 are formed in stripes as illustrated in FIG. The depth is 10 μm (groove pitch 200 μm, width 100 μm. The material of the bottom surface is glass (Tempax glass: Chiba Science glass), diameter 35 mm).
The differential interference microscope image of Sample G was the same as Sample A. In Sample G, the tissue was oriented, and the direction of this orientation almost coincided with the direction of the groove on the culture surface (bottom surface), and cells and extracellular matrix were aligned along the direction of the groove.

(2) Preparation of test piece (sample H) Sample A was prepared except that the initial cell density was 6.0 × 10 ⁵ cells / cm ² and the culture period was 21 days in the preparation of sample A of Example 1. A culture was prepared in the same manner as in the preparation of Sample H. That is, the incubator used here is a culture surface (bottom surface) having a semicircular groove having a cross-sectional shape as illustrated in FIG. 2 formed in a stripe shape as illustrated in FIG. The depth of the groove is 30 μm (the groove pitch is 200 μm, the width is 100 μm. The material of the bottom surface is glass (Tempax Glass: Chiba Science Glass), diameter 35 mm).
The differential interference microscope image of Sample H was the same as Sample A. In Sample H, the tissue was oriented, and the direction of this orientation almost coincided with the direction of the groove on the culture surface (bottom surface), and cells and extracellular matrix were aligned along the direction of the groove.

(3) Evaluation of test piece In the same manner as in Example 1, the thickness, tensile strength, and tangent coefficient of Samples G and H were measured. The results are shown in Table 4. A stress-strain curve is shown in FIG. In FIG. 15, white circles represent the sample G, and black squares represent the sample H.

As shown in Table 4, there was a significant difference in tensile strength between sample G and sample H (P <0.05), and sample H had a larger value.
On the other hand, there was no significant difference in thickness between sample G and sample H. No significant difference was observed in the rigidity (tangential coefficient in the strain range of 5% to 10%), but the tangential coefficient of sample H was larger.
Further, from the results of the observation of the structures of Sample G and Sample H, the unevenness of the culture surface was clearly seen in Sample H.
From these facts, it was found that the degree of mechanical anisotropy of the culture can be changed by changing the depth of the groove on the culture surface. It was found that the strength of the culture can be increased when the groove depth on the culture surface is 30 μm rather than 10 μm.

[Example 5]
<Preparation of three-dimensional tissue culture from human skin fibroblasts>
(1) Maintenance of cultured cells Human skin fibroblasts (Cell Systems, 2F0-C75) are cultured in DMEM culture medium (10% FBS, 1% penicillin / streptomycin) at 5% CO ₂ and 37 ° C. Went. Cultured cells were maintained by replacing about 1/3 of the culture solution (20 ml) every 7 days, and when subcultured, the cells were subcultured every 14 days.

(2) Preparation of test piece (sample I) Using a cell group in which human skin fibroblasts were subcultured 4 times to increase the number of cells, the initial cell density was 6.0 × 10 ⁵ cells / cm ² , A culture was prepared as Sample I in the same manner as in the preparation of Sample A of Example 1, except that the culture period was 15 days.
The differential interference microscope image of Sample I was the same as Sample A. In Sample I, the tissue was oriented, and the direction of this orientation almost coincided with the direction of the groove on the culture surface (bottom surface), and cells and extracellular matrix were aligned along the direction of the groove.

(3) Preparation of Comparative Specimen (Sample J) Using a cell group in which human dermal fibroblasts were subcultured 4 times to increase the number of cells, the initial cell density was 6.0 × 10 ⁵ cells / cm ^2. A culture was prepared as Sample J in the same manner as in the preparation of Sample B of Example 1, except that the culture period was 15 days.
The differential interference microscope image of Sample J was the same as Sample B. In Sample J, the structure was not oriented.

(4) Evaluation of Specimen Samples I and J were measured for thickness, tensile strength, and tangent coefficient in the same manner as in Example 1. The results are shown in Table 5. A stress-strain curve is shown in FIG. In FIG. 16, the white square represents Sample I, and the black square represents Sample J.

As shown in Table 5, the tensile strength was significantly increased in Sample I compared to Sample J, although there was no significant difference in thickness and stiffness (tangential coefficient in the strain range of 5% to 10%). (P <0.05).
From this, orientation can be imparted to the culture by the method of culturing using the incubator of the present invention without applying mechanical stimulation or chemical stimulation. It was found that the strength could be increased.

During the culture for preparing Sample J, a phenomenon was observed in which part of the tissue was detached from the culture surface. On the other hand, in the preparation of Sample I, no tissue detachment from the culture surface was observed.
From this, it was found that the culture can be easily obtained by culturing using the incubator of the present invention without peeling the culture from the culture surface during the culture.

  According to the present invention, the cell density and the substrate density of the groove portion are high, and the non-uniform tissue is low density in a portion other than the groove, which is a novel tissue that has not been found in in vivo tissues or biomaterials so far. A tissue imparted with properties can be generated.

DESCRIPTION OF SYMBOLS 10 ... Glass dish 10A ... Culture surface 12 ... Groove 20 ... Metal mask layer 22 ... Photosensitive resin layer

Claims (9)

A tissue culture incubator for producing a three-dimensional tissue culture from cells,
On the surface to which the cells are attached and cultured, a groove having a width larger than the width of the bottom is continuously or intermittently provided in a predetermined direction, and the groove provided in the predetermined direction includes the groove A tissue culture incubator arranged in a plurality at predetermined intervals in a direction crossing a predetermined direction.   The incubator for tissue culture according to claim 1, wherein a side surface of the groove is formed of a curved wall surface.   The tissue culture incubator according to claim 1 or 2, wherein the material is an inorganic material.   The incubator for tissue culture according to any one of claims 1 to 3, wherein the groove has a depth of 5 to 40 µm. A production method for producing a three-dimensional tissue culture containing cells and extracellular matrix in a culture solution,
A seeding step of seeding cells in the tissue culture incubator according to any one of claims 1 to 4;
An adherent culture step of culturing cells after seeding and attaching them to the surface to be cultured, and producing an extracellular matrix from the cells to form a three-dimensional culture in the culture solution;
A separation step of separating the culture from the surface to be cultured after a predetermined period;
A method for producing a three-dimensional tissue culture comprising:   The method for producing a three-dimensional tissue culture according to claim 5, wherein the cells are mesenchymal stem cells.   The method for producing a three-dimensional tissue culture according to claim 5 or 6, wherein the cells are cultured in the presence of an extracellular matrix production promoting factor.   The method for producing a three-dimensional tissue culture according to claim 7, wherein the extracellular matrix production promoting factor is at least one selected from the group consisting of ascorbic acid, ascorbic acid derivatives, TGF-β1 and TGF-β3.   The method for producing a three-dimensional tissue culture according to any one of claims 5 to 8, wherein the extracellular matrix is at least one selected from the group consisting of collagen I, collagen III, vitronectin and fibronectin. .