Tissue structure, tissue structure forming device, and method for forming tissue structure

Abstract

Provided are tissue constructs that facilitate the administration of pluripotent stem cells to disease sites, and methods for forming the tissue constructs. [Solution] A tissue structure formed by a partition wall 12 placed in an environment containing biological tissue, the partition wall 12 having an inner surface 14 surrounding a hollow portion 11 and a plurality of through holes 13 opening into the inner surface 14, and a tissue structure is formed in the hollow portion 11 as cells in the biological tissue invade the hollow portion 11 through the through holes, and the tissue structure comprises a membranous fibrous connective tissue having one side detached from the inner surface 14, and a sparse fibrous tissue that spreads over the entire other side opposite the one side and accumulates pluripotent stem cells.

Description

[Technical field]

[0001] The present disclosure relates to a tissue structure in which pluripotent stem cells are accumulated, a tissue structure forming device, and a method for forming a tissue structure. [Background technology]

[0002] The body's self-defense mechanism accumulates macrophages around foreign bodies that have invaded the body. Macrophages adhere to the surface of foreign bodies and promote collagen production by fibroblasts through the production of TGF-β by monocytes. Foreign bodies that have invaded the body are isolated within the body by being covered with connective tissue that contains fibroblasts and collagen.

[0003] One of the regenerative medical technologies, which is a medical technology that revives lost tissues and organs, replaces a damaged tissue structure with a tissue structure derived from a living body. One of the technologies for forming a tissue structure derived from a living body involves placing a tissue structure forming device, which is a foreign body, in living tissue, and then using the self-defense function of living tissue to form a tissue structure derived from a living body on the tissue structure forming device (see, for example, Patent Documents 1 to 4).

[0004] An example of such a tissue structure forming device includes two connective tissue forming surfaces facing each other. The two connective tissue forming surfaces separate a hollow portion having various shapes such as a flat space, a cylindrical space, a valve-shaped space, etc. Fibroblasts that invade between the two connective tissue forming surfaces form a connective tissue body so as to fill the space (see, for example, Patent Documents 5 to 8).

[0005] Stem cells are important materials for regenerating and repairing target tissues in regenerative medicine technology. On the other hand, the basis for using embryonic stem cells, which require ethical consideration, and induced pluripotent stem cells, which require safety confirmation, in regenerative medicine technology has not yet been established. On the other hand, pluripotent stem cells present in the body, although present in small numbers in living tissues, have passed through the development stage and are moving to the clinical stage. One technique for accumulating pluripotent stem cells present in the body involves placing a tissue structure formation device, which is a foreign body, in the living tissue, and then accumulating the pluripotent stem cells in the living tissue in the tissue structure formation device.

[0006] An example of such a tissue structure forming device accumulates pluripotent stem cells in a hollow portion of the device. The tissue structure forming device includes a frame for surrounding the hollow portion. The frame defines an opening extending in the direction in which the columnar members extend by using two columnar members. The hollow portion surrounded by the frame communicates with the environment inside the living body through the opening. The opening width of 2.5 mm or more in the frame and the depth of 2 mm or more in the hollow portion cause fibroblasts to fill the hollow portion with fibrous connective tissue so as to form a recess in the portion of the fibrous connective tissue facing the opening. The portion of the fibrous connective tissue facing the opening is recessed from the opening toward the hollow portion. Pluripotent stem cells present in the living body are accumulated in such a recess (see, for example, Patent Document 9). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2007-37763 A [Patent Document 2] JP 2007-312821 A [Patent Document 3] JP 2008-237896 A [Patent Document 4] JP 2010-094476 A [Patent Document 5] JP 2014-030598 A [Patent Document 6] JP 2017-169778 A [Patent Document 7] Patent No. 6727637 [Patent Document 8] Patent No. 6755052 [Patent Document 9] Patent No. 6813923 Summary of the Invention [Problem to be solved by the invention]

[0008] As mentioned above, regenerative medicine technologies that utilize the functions of pluripotent stem cells present in the body for disease treatment have passed through the development stage and are moving to the clinical stage. Information on the beneficial and undesirable effects of various doses is essential to ensure efficacy and safety. In this regard, in the above-mentioned technologies, the accumulation of pluripotent stem cells present in the body is limited to the depressions of fibrous connective tissue, so further improvements are needed to make it easier to administer pluripotent stem cells to disease sites in the clinical stage. [Means for solving the problem]

[0009] The tissue structure for solving the above problem is a tissue structure formed by a partition wall placed in an environment containing biological tissue, the partition wall having an inner surface surrounding a hollow portion and a plurality of through holes opening onto the inner surface, the tissue structure being formed in the hollow portion as cells within the biological tissue invade the hollow portion through the through holes, the pluripotent stem cells including stem cells expressing at least one of the pluripotent stem cell markers SSEA3 and SSEA4, and mesenchymal stem cells, and the tissue structure comprises a membranous fibrous connective tissue having one side detached from the inner surface, and a membranous sparse fibrous tissue extending over the entire other side opposite the one side and accumulating the pluripotent stem cells.

[0010] The tissue structure forming device for solving the above problems is a tissue structure forming device that includes a partition wall having an inner surface surrounding a hollow portion and a plurality of through holes opening on the inner surface, and that is placed in an environment containing biological tissue, and that forms a tissue structure in the hollow portion by allowing cells in the biological tissue to invade the hollow portion through the through holes. The tissue structure includes a membranous fibrous connective tissue having one side in contact with the inner surface, and a membranous loose fibrous tissue that spreads over the other side opposite to the one side and accumulates pluripotent stem cells. The pluripotent stem cells include stem cells that express at least one of the pluripotent stem cell markers SSEA3 and SSEA4, and mesenchymal stem cells. The thickness of the partition wall is 0.1 mm or more and 2.0 mm or less, the opening dimension of the through hole is 0.3 mm or more and 3.0 mm or less, the hole distance between the through holes is 0.3 mm or more and 5.0 mm or less, the opening occupancy rate of the through holes on the inner surface is 30% or more and 70% or less, and the depth of the hollow portion is 2 mm or more and 10 mm or less.

[0011] A method for forming a tissue structure to solve the above problem is a method for forming a tissue structure in a partition wall by placing the partition wall in an environment containing biological tissue, wherein the partition wall has an inner surface surrounding a hollow portion and a plurality of through holes opening to the inner surface, and the pluripotent stem cells include stem cells expressing at least one of the pluripotent stem cell markers SSEA3 and SSEA4 and mesenchymal stem cells, and the method includes the steps of: placing the partition wall in the environment so that cells in the biological tissue enter the hollow portion through the through holes; placing the partition wall in the environment so that a membranous fibrous connective tissue having one side in contact with the inner surface is formed and a membranous loose fibrous tissue in which the pluripotent stem cells have accumulated spreads over the entire other side opposite to the one side; and removing the partition wall from the biological tissue when a predetermined period has elapsed before the fibrous connective tissue fills the hollow portion, and peeling off the one side from the inner surface of the partition wall.

[0012] According to each of the above configurations, in the compartment wall placed in an environment containing biological tissue, cells derived from the biological tissue, plasma proteins, etc., enter the hollow portion separated by the compartment wall through the through holes. The cells derived from the biological tissue that enter the hollow portion form sparse fibrous tissue using the inner surface of the compartment wall as a scaffold. The production of collagen over the course of the placement period forms fibrous connective tissue from the sparse fibrous tissue. Then, over the course of the placement period, one side of the membranous fibrous connective tissue is formed on the inner surface of the compartment wall, and the membranous sparse fibrous tissue continues to be formed on the other side of the fibrous connective tissue. The biological tissue-derived pluripotent stem cells that enter the hollow portion continue to accumulate in the sparse fibrous tissue and grow in the sparse fibrous tissue. As a result, the accumulation site of the pluripotent stem cells spreads over the entire other side of the membranous fibrous connective tissue. The attachment of a membranous tissue structure, etc., administers many pluripotent stem cells to a wide disease site.

[0013] In the above tissue structure, the inner surface may be a flattened elliptical cylindrical surface, and the fibrous connective tissue may have a membrane-like shape cut open in the axial direction of the elliptical cylindrical surface. In the above-described method for forming a tissue structure, the inner surface may be a flattened elliptical cylindrical surface, and the method may further include cutting open the fibrous connective tissue detached from the inner surface in the axial direction of the elliptical cylindrical surface.

[0014] According to each of the above configurations, the area required for accumulating pluripotent stem cells in a planar view can be reduced by the amount of the inner surface being an elliptical cylindrical surface compared to when the inner surface spreads out in a flat plane, and therefore a large area can be secured as a site for accumulating pluripotent stem cells even in an environment where the area in a planar view is limited.

[0015] In the above tissue structure, the depth of the hollow portion may be 2 mm or more and 10 mm or less, and the thickness of the fibrous connective tissue may be 0.05 mm or more and 0.5 mm or less. In the above-mentioned method for forming a tissue structure, the depth of the hollow portion may be 2 mm or more and 10 mm or less, and leaving the partition wall in place for a predetermined period of time may result in a thickness of the fibrous connective tissue of 0.05 mm or more and 0.5 mm or less.

[0016] According to the above-mentioned configuration, the strength of the membranous fibrous connective tissue is ensured, so that the tissue structure can be easily handled when administering pluripotent stem cells. In addition, since the depth of the hollow portion is sufficiently greater than the thickness of the fibrous connective tissue, a space for accumulating pluripotent stem cells can be easily secured within the hollow portion.

[0017] In the above tissue structure, the thickness of the partition wall is 0.1 mm or more and 2.0 mm or less, the opening dimension of the through hole on the inner surface is 0.3 mm or more and 3.0 mm or less, the length between adjacent through holes on the inner surface is 0.3 mm or more, the occupancy rate of the openings on the inner surface is 30% or more and 70% or less, and the one side may have capillaries at a site corresponding to the through hole.

[0018] According to the above configuration, since the opening size is 0.3 mm or more, cells derived from biological tissues and plasma proteins can easily enter the hollow space partitioned by the partition walls through the through-holes in the partition walls. Since the opening size is 3.0 mm or less, depressions and holes are unlikely to be formed in the fibrous connective tissue at the site facing the through-holes. Furthermore, since the length between adjacent through-holes is 0.3 mm or more and the occupancy rate of the openings is 30% or more and 70% or less, the fibrous connective tissue is likely to grow using the inner surface as a scaffold. Therefore, an environment suitable for cells derived from biological tissues is easily established in the hollow space through the capillaries at the site corresponding to the through-holes.

[0019] In the above-described method for forming a tissue structure, the partition wall may be a hollow tubular member, and the hollow portion may be defined only by the partition wall. According to the above configuration, the hollow portion has a sufficient depth, so that a space for accumulating pluripotent stem cells can be easily ensured within the hollow portion.

[0020] The method for forming the tissue structure may further include grinding and fluidizing the fibrous connective tissue and the loose fibrous tissue detached from the inner surface of the compartment wall. According to the above configuration, the tissue structure in which pluripotent stem cells have accumulated is administered to the diseased site by injection or the like. Effect of the Invention

[0021] According to the tissue structure, tissue structure forming apparatus, and tissue structure forming method disclosed herein, pluripotent stem cells can be easily administered to a diseased site. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 is a perspective view showing a tissue structure forming device. [Diagram 2] FIG. 2 is a cross-sectional view showing a tissue structure. [Diagram 3] FIG. 3 is a cross-sectional view showing the process of forming a tissue structure. [Figure 4] FIG. 4 is a cross-sectional view showing the process of forming a tissue structure. [Diagram 5] FIG. 5 is a cross-sectional view showing the process of forming a tissue structure. [Figure 6] FIG. 6 is a cross-sectional view of the tissue structure forming device. [Figure 7] FIG. 7 is a perspective view showing a tissue structure. [Figure 8] FIG. 8 is a graph showing the thickness of the tissue construct. [Figure 9] FIG. 9 is an image showing the results of HE staining of fibrous connective tissue. [Figure 10] FIG. 10 is an image showing the results of MT staining of fibrous connective tissue. [Figure 11] FIG. 11 is an image showing the results of SR staining of fibrous connective tissue. [Figure 12] FIG. 12 is an image showing the results of HE staining of sparse fibrous tissue. [Figure 13] FIG. 13 is an image showing the results of MT staining of sparse fibrous tissue. [Figure 14] FIG. 14 is an image showing the results of SR staining of sparse fibrous tissue. [Figure 15] FIG. 15 is an image showing the results of CD90 staining of fibrous connective tissue. [Figure 16] FIG. 16 is an image showing the results of CD105 staining of fibrous connective tissue. [Figure 17] FIG. 17 is an image showing the results of CD90 staining of sparse fibrous tissue. [Figure 18] FIG. 18 is an image showing the results of CD105 staining of sparse fibrous tissue. [Figure 19] FIG. 19 is an image showing the results of SSEA3 staining of fibrous connective tissue. [Figure 20] FIG. 20 is an image showing the results of SSEA4 staining of fibrous connective tissue. [Figure 21] FIG. 21 is an image showing the results of SSEA3 staining of sparse fibrous tissue. [Figure 22] FIG. 22 is an image showing the results of SSEA4 staining of sparse fibrous tissue. [Figure 23] FIG. 23 is a perspective view showing a tissue structure forming device according to a modified example. [Figure 24] FIG. 24 is a perspective view showing a tissue structure forming device according to a modified example. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] [Outline of tissue structure forming device 10] As shown in Fig. 1, the tissue structure forming device 10 has a partition wall 12 that divides a hollow portion 11. The partition wall 12 has a plurality of through holes 13. An inner surface 14 of the partition wall 12 borders the opening of the through holes 13. The tissue structure forming device 10 is left in an environment in which biological tissue is present for a predetermined period of time. The period for which the tissue structure forming device 10 is left in is set in advance based on tests for forming a tissue structure 20, etc.

[0024] When the tissue structure forming device 10 is placed in an environment where biological tissue is present, cells derived from the biological tissue, plasma proteins, and the like enter the hollow portion 11 separated by the partition wall 12 from the environment through the through-holes 13 of the partition wall 12. The cells derived from the biological tissue, etc. that have entered the hollow portion 11 grow a membranous fibrous connective tissue 21 toward the inside of the hollow portion 11, using the inner surface 14 that borders the opening of the through-hole 13 as a scaffold. In addition, the cells derived from the biological tissue, etc. that have entered the hollow portion 11 grow a membranous loose fibrous tissue 22 further inward than the fibrous connective tissue 21, and accumulate pluripotent stem cells 23 in the loose fibrous tissue 22.

[0025] As shown in FIG. 2, the tissue structure 20 includes a fibrous connective tissue 21 and a loose fibrous tissue 22. The membranous fibrous connective tissue 21 has a first surface 21A that is peeled off from the inner surface 14 of the partition wall 12. The membranous loose fibrous tissue 22 is formed over the entire second surface 21B of the fibrous connective tissue 21, which is opposite to the first surface 21A. The placement of the tissue structure forming device 10 for a predetermined placement period forms the membranous fibrous connective tissue 21 in the hollow portion 11, and also forms the membranous loose fibrous tissue 22 supported by the fibrous connective tissue 21. A cell population including pluripotent stem cells 23, or the pluripotent stem cells 23, are accumulated in the loose fibrous tissue 22. The present disclosure is based on such findings found by the present inventors.

[0026] The environment including the biological tissue may be a living body including biological tissue. The living body including biological tissue may be the inside of a disease model animal. The environment including the biological tissue may be an in vitro culture system including biological tissue taken out of a living body. The environment including the biological tissue may be an artificial environment constructed outside the living body by imitating biological tissue in a living body. The biological tissue may be an ectodermal tissue, a mesodermal tissue, or an endodermal tissue. The biological tissue may be a tissue in which pluripotent stem cells 23 exist in a healthy state. The biological tissue may be a tissue in which pluripotent stem cells 23 required for tissue repair exist, such as an injured part or a defective part. The biological tissue may be a subcutaneous tissue including fibroblasts. The biological tissue may be that of a human or any animal other than a human that may be a source of obtaining the pluripotent stem cells 23. The living body including the biological tissue may be the inside of a human body or the inside of a non-human organism. The non-human organism may be a non-human primate such as a monkey or a chimpanzee. The non-human organism may be a mammal such as a dog, cat, cow, pig, horse, goat, sheep, rat, or mouse, or may be a bird, fish, or amphibian. The inside of a living body containing a biological tissue may be subcutaneously in the limbs, shoulder, back, or abdomen, or may be the abdominal cavity.

[0027] The tissue structure 20 is attached to an appropriate living tissue or a living tissue having a decreased function without inducing differentiation so that the loose fibrous tissue 22 comes into contact with the appropriate living tissue or a living tissue having a decreased function. In other words, the cell population containing the pluripotent stem cells 23 in the tissue structure 20 or the pluripotent stem cells 23 is directly administered to the appropriate living tissue or a living tissue having a decreased function.

[0028] The tissue structure 20, the cell population containing the pluripotent stem cells 23, and the pluripotent stem cells 23 may be induced to differentiate into target cells outside of a living tissue and then administered to a living tissue or the like. The tissue structure 20, the cell population containing the pluripotent stem cells 23, or the pluripotent stem cells 23 may be administered at a desired time after cryopreservation. The pluripotent stem cells 23 may also be isolated from the sparse fibrous tissue 22. The isolation of the pluripotent stem cells 23 may be performed by enzyme treatment of the tissue structure forming device 10 taken out from the environment containing the living tissue. The isolation of the pluripotent stem cells 23 may be performed by filtration using a membrane filter, a mesh filter, or the like. The pluripotent stem cells 23 isolated from the sparse fibrous tissue 22 may be cultured and expanded, or may not be cultured and expanded. The cell population containing the pluripotent stem cells 23 partially purified from the sparse fibrous tissue 22 may be cultured and expanded, or may not be cultured and expanded.

[0029] The tissue structure 20, the cell population containing the pluripotent stem cells 23, or the pluripotent stem cells 23 may be administered to a disease site and used for regenerative medicine such as tissue repair, tissue regeneration, repair of tissue or organ damage, and repair of reduced function of tissue or organ. The tissue structure 20, the cell population containing the pluripotent stem cells 23, or the pluripotent stem cells 23 may be used for tissue construction or cell differentiation in an in vitro culture system. The tissue structure 20, the cell population containing the pluripotent stem cells 23, or the pluripotent stem cells 23 may be used for tissue construction or cell differentiation in an ex vivo system.

[0030] The tissue structure 20 may be used as a fluid obtained by grinding the fibrous connective tissue 21 and the loose fibrous tissue 22. The tissue structure 20 may be used as a fluid obtained by grinding only the loose fibrous tissue 22. The fluidized tissue structure 20 may be administered to the diseased site by injection or the like.

[0031] [Configuration of tissue structure forming device 10] 1, the partition wall 12 is a cylindrical member having a flattened elliptical cylindrical shape. An inner surface 14 surrounding the hollow portion 11 has a flattened elliptical cylindrical surface shape.

[0032] The shape of the partition wall 12 may be a cylinder or a flat hollow rectangular parallelepiped. The inner surface 14 surrounding the hollow portion 11 may have a cylindrical surface or a flat rectangular parallelepiped. Since the size of the space of the biological tissue is limited, when the accumulation range of the pluripotent stem cells 23 is required to be expanded, the shape of the partition wall 12 is preferably a cylinder or a flat rectangular parallelepiped in which the membranous fibrous connective tissue 21 spreads three-dimensionally, compared to a flat plate shape. In addition, when the position of the partition wall 12 is required to be stable in an environment containing biological tissue, the shape of the partition wall 12 is preferably a flat elliptical cylinder, compared to a cylindrical shape.

[0033] The cylindrical end of the cylindrical partition wall 12 is closed by a cap. The cap has a plurality of air holes. The air holes are used to reduce the pressure in the hollow portion 11 that is placed in an environment containing biological tissue. When the shape of the partition wall 12 needs to be stable in an environment containing biological tissue, a configuration in which the cylindrical end of the partition wall 12 is supported by the cap is preferable. When the thickness of the membranous fibrous connective tissue 21 needs to be uniform, it is preferable that the air holes of the cap function as through holes 13 in the vicinity of the partition wall 12.

[0034] The constituent material of the partition wall 12 is compatible with biological tissues. The constituent material of the partition wall 12 may be a metal material such as stainless steel, titanium, a titanium-nickel alloy, or a cobalt-chromium alloy, or a synthetic resin such as a silicone resin, a polymethylpentene resin, a polytetrafluoroethylene resin, a polypropylene resin, a polyethylene resin, PEEK, an acrylic resin, nylon, a polycarbonate resin, or a polysulfone resin. The constituent material of the partition wall 12 may be a laminate of a metal material and a synthetic resin.

[0035] The inner surface 14 of the compartment wall 12 may have softness and a water contact angle for promoting the formation of fibrous connective tissue 21. Promoting the formation of fibrous connective tissue 21 stabilizes the support of the loose fibrous tissue 22 by the fibrous connective tissue 21, making it easier to handle the loose fibrous tissue 22 in the tissue structure 20. When promotion of the formation of fibrous connective tissue 21 is required, the constituent material of the inner surface 14 is preferably any one selected from the group consisting of polymethylpentene resins, polyfluoroethylene resins, polypropylene resins, polyethylene resins, silicone resins, and mixed resins of these resins with other resin components.

[0036] The through holes 13 are circular holes penetrating from the inner surface 14 to the outer surface of the partition wall 12. The through holes 13 are scattered throughout the partition wall 12. The openings of the through holes 13 have a circular shape on the inner surface 14, and are scattered throughout the inner surface 14. The through holes 13 are disposed at equal intervals in the circumferential direction of the partition wall 12 and in the extension direction of the partition wall 12.

[0037] The through hole 13 is not limited to a circular hole, and may be an elliptical hole, a polygonal hole, or an irregular hole. The opening of the through hole 13 may have an elliptical shape, a polygonal shape, or an irregular shape on the inner surface 14. When smooth peeling of the fibrous connective tissue 21 is required, the inner surface of the through hole 13 is preferably a curved surface, and the through hole 13 is preferably a circular hole.

[0038] The openings of the through-holes 13 may be regularly or randomly arranged on the inner surface 14 and outer surface of the partition wall 12. When the thickness of the membranous fibrous connective tissue 21 is required to be uniform within a predetermined range, or when the distribution of the pluripotent stem cells 23 is required to be uniform within a predetermined range, it is preferable that the openings of the through-holes 13 are regularly arranged within a predetermined range on the inner surface 14.

[0039] It is believed that the tissue structure forming device 10 placed in an environment containing biological tissue proceeds with tissue formation as follows, however, tissue formation by the tissue structure forming device 10 is not limited to this reasoning.

[0040] 3, when the tissue structure forming device 10 is placed in an environment containing biological tissue, the biological tissue is located outside the hollow portion 11. Liquid components 25 such as extracellular matrix derived from the biological tissue enter the hollow portion 11 through the through-holes 13. The hollow portion 11 of the tissue structure forming device 10 is filled with the liquid components 25 derived from the biological tissue.

[0041] When the hollow portion 11 of the tissue structure forming device 10 is filled with the liquid component 25, cells such as pluripotent stem cells 23 derived from biological tissue and fibroblasts invade the hollow portion 11 through the through-holes 13. The fibroblasts that have invaded the hollow portion 11 begin to form loose fibrous tissue 22 from the surface of the partition wall 12 toward the inside of the hollow portion 11, using the inner surface 14 bordering the opening of the through-hole 13 as a scaffold.

[0042] 4, the loose fibrous tissue 22 formed using the inner surface 14 as a scaffold promotes the production of collagen and the like by cells accumulated in the loose fibrous tissue 22 on the inner surface 14, which is easily recognized as a foreign body. As a result, the loose fibrous tissue 22 formed using the inner surface 14 as a scaffold grows fibrous connective tissue 21 that is denser than the loose fibrous tissue 22 from the inner surface 14 toward the inside of the hollow portion 11, starting from the opening edge of the through hole 13.

[0043] The inside of the hollow portion 11, which is more easily recognized as a foreign body than the outside of the hollow portion 11, further promotes the invasion of fibroblasts and pluripotent stem cells 23. The fibroblasts and pluripotent stem cells 23 that continue to invade the hollow portion 11 form loose fibrous tissue 22 inward of the fibrous connective tissue 21 during the process of growth of the fibrous connective tissue 21. The pluripotent stem cells 23 that invade the hollow portion 11 continue to accumulate in the loose fibrous tissue 22.

[0044] 5, the fibroblasts and pluripotent stem cells 23 that continue to enter the hollow portion 11 form a membranous fibrous connective tissue 21 that spreads over the entire inner surface 14 so as to connect the opening edges of the through-holes 13 with the fibrous connective tissue 21. The fibroblasts and pluripotent stem cells 23 that continue to enter the hollow portion 11 form a membranous loose fibrous tissue 22 and capillaries 24 that follow the spread of the fibrous connective tissue 21.

[0045] The inside of the hollow portion 11, which is more easily recognized as a foreign body than the outside of the hollow portion 11, continues to accumulate pluripotent stem cells 23 in the sparse fibrous tissue 22 even while the fibrous connective tissue 21 is being formed in a membrane-like shape. The pluripotent stem cells 23 accumulated in the sparse fibrous tissue 22 proliferate within the sparse fibrous tissue 22. As a result, cells derived from biological tissues that have entered the hollow portion 11 grow the membranous sparse fibrous tissue 22 further inward than the membranous fibrous connective tissue 21, and accumulate many pluripotent stem cells 23 in the sparse fibrous tissue 22.

[0046] The tissue structure forming device 10 is removed from the environment containing biological tissue before the fibrous connective tissue 21 fills up the hollow portion 11. The tissue structure forming device 10 placed in the environment containing biological tissue continues to form loose fibrous tissue 22 and accumulate pluripotent stem cells 23 in the loose fibrous tissue 22 during the process of growth of the fibrous connective tissue 21 for a predetermined placement period.

[0047] [Configuration of partition wall 12] 6, the thickness T12 of the partition wall 12 is the depth of the through-hole 13. The thickness T12 of the partition wall 12 has a size for suppressing deformation of the partition wall 12 in the living tissue. The thickness T12 of the partition wall 12 has a substantially uniform size in the range in which the through-hole 13 is arranged. The thickness T12 of the partition wall 12 has a size for allowing cells and the like in the living tissue to enter the hollow portion 11.

[0048] The opening dimension WT of the through hole 13 is the dimension that the opening of the through hole 13 has on the inner surface 14. The opening dimension WT of the through hole 13 has a size that allows cells in biological tissue to pass through the through hole 13. The minimum value of the opening dimension WT through which cells in biological tissue can pass is about 0.01 mm. When the through hole 13 is a circular hole, the opening dimension WT of the through hole 13 is the diameter of the circular hole. When the through hole 13 is an elliptical hole, the opening dimension WT of the through hole 13 is the major axis of the elliptical hole. When the opening of the through hole 13 is polygonal or has an irregular shape, the opening dimension WT is the diameter of a circle circumscribing the opening.

[0049] The multiple through holes 13 in one partition wall 12 may have the same opening dimension WT. The multiple through holes 13 in one partition wall 12 may have different opening dimensions WT. The multiple through holes 13 in one partition wall 12 having the same opening dimension WT may be arranged in the partition wall 12 so as to be adjacent to each other as a group. In one partition wall 12, the multiple through holes 13 having different opening dimensions WT may be arranged so that the opening dimensions WT are alternately different in the arrangement direction of the through holes 13.

[0050] When the tissue structure forming device 10 is placed in an environment where biological tissue is present, the inner surface 14 of the partition wall 12 has a size that allows it to function as a scaffold for cells derived from the biological tissue. The inter-hole distance on the inner surface 14 is the shortest length on the inner surface 14 between adjacent through-holes 13. The inter-hole distance on the inner surface 14 has a size that allows the inner surface 14, which is the plane between adjacent through-holes 13, to function as a scaffold for cells and the like in the biological tissue.

[0051] The gaps between adjacent through holes 13 in one partition wall 12 may have the same hole distance. The gaps between adjacent through holes 13 in one partition wall 12 may have different hole distances. A plurality of through holes 13 having the same hole distance in one partition wall 12 may be arranged in the partition wall 12 as one group. A plurality of through holes 13 having different hole distances in one partition wall 12 may be arranged so that the hole distances are alternately different in the arrangement direction of the through holes 13.

[0052] The density of the openings on the inner surface 14 of the partition wall 12 is large enough that the entry of cells derived from biological tissue is not inhibited by the fibrous connective tissue 21. The density of the openings on the inner surface 14 of the partition wall 12 is large enough that the fibrous connective tissue 21 forms a membrane. The opening occupancy rate of the inner surface 14 is the ratio of the total area occupied by the openings of the through holes 13 to the unit area of ​​the inner surface 14.

[0053] The depth of the hollow portion 11 is a hollow depth D11. The hollow depth D11 is large enough so that in the process of the growth of the fibrous connective tissue 21, the invasion of fibroblasts and pluripotent stem cells 23 is promoted, and the formation of the sparse fibrous tissue 22 is more likely to proceed than the formation of the fibrous connective tissue 21.

[0054] The hollow depth D11 is the depth of the hollow portion 11 extending from the opening of the through hole 13 on the inner surface 14 in the depth direction of the through hole 13. The depth direction of the through hole 13 is the thickness direction of the partition wall 12 around the through hole 13. When a foreign object such as a support member 15 is present in the hollow portion 11, the hollow depth D11 is the distance between the inner surface 14 and the foreign object in the depth direction of the through hole 13. When a foreign object is not present in the hollow portion 11 and a part of the inner surface 14 faces another part of the inner surface 14 in the depth direction of the through hole 13, such as a cylindrical inner surface 14, the hollow depth D11 is half the distance between the part of the inner surface 14 and another part of the inner surface 14 in the depth direction of the through hole 13.

[0055] The partition wall 12 may satisfy the following conditions 1 to 5. For example, the thickness T12 of the partition wall 12 may be 0.1 mm, the opening dimension WT may be 0.3 mm, the distance between the holes may be 0.3 mm, the opening occupancy rate may be 30%, and the hollow depth D11 may be 2 mm. For example, the thickness T12 of the partition wall 12 may be 2.0 mm, the opening dimension WT may be 3.0 mm, the distance between the holes may be 5.0 mm, the opening occupancy rate may be 70%, and the hollow depth D11 may be 10 mm. For example, the thickness T12 of the partition wall 12 may be 1.0 mm, the opening dimension WT may be 1.0 mm, the distance between the holes may be 3.0 mm, the opening occupancy rate may be 50%, and the hollow depth D11 may be 4 mm.

[0056] The opening dimension WT and the hole distance within one partition wall 12 may be a combination of different values ​​within a range that satisfies conditions 2 and 3. For example, one partition wall 12 may have a through hole 13 having an opening dimension WT of 0.5 mm and a through hole 13 having an opening dimension WT of 1.5 mm. For example, one partition wall 12 may have a plurality of through holes 13 having a hole distance of 0.5 mm and a plurality of through holes 13 having a hole distance of 1.5 mm. For example, one partition wall 12 may have a plurality of through holes 13 having a hole distance of 0.5 mm and an opening dimension WT of 0.5 mm, and a plurality of through holes 13 having a hole distance of 1.5 mm and an opening dimension WT of 1.5 mm. For example, one partition wall 12 may include a plurality of through holes 13 having an opening dimension WT of 0.5 mm with a hole distance of 1.5 mm, and a plurality of through holes 13 having an opening dimension WT of 1.5 mm with a hole distance of 0.5 mm. For example, one partition wall 12 may include through holes 13 as a plurality of circular holes having an opening dimension WT of 0.5 mm with a hole distance of 0.3 mm, and may further include through holes 13 as a plurality of elliptical holes having an opening dimension WT of 1.5 mm with a hole distance of 5.0 mm.

[0057] (Condition 1) The thickness T12 of the partition wall 12 is not less than 0.1 mm and not more than 2.0 mm. (Condition 2) The opening dimension WT of the through hole 13 is equal to or greater than 0.3 mm and equal to or less than 3.0 mm. (Condition 3) The distance between holes on the inner surface 14 is 0.3 mm or more and 5.0 mm or less. (Condition 4) The opening occupancy rate of the inner surface 14 is 30% or more and 70% or less. (Condition 5) The hollow depth D11 is equal to or greater than 2 mm and equal to or less than 10 mm.

[0058] In the tissue structure forming device 10, collagen production may occur through competition between the outer surface of the partition wall 12 and in the hollow portion 11. Even if the opening occupancy rates are equal to each other in a structure, if the number of openings is small and the size of the openings is large, the outer surface of the partition wall 12 is likely to be recognized as a foreign body due to an excessively large inter-pore distance, and the outer surface of the partition wall 12 is likely to be covered with connective tissue. As a result, the openings of the through holes 13 are likely to be closed by connective tissue before the tissue structure 20 is formed in the hollow portion 11. Alternatively, pluripotent stem cells 23 such as plasma proteins, fibroblasts, vascular endothelial cells, and mesenchymal cells have difficulty entering the hollow portion 11, which slows down the formation of fibrous connective tissue 21 and the formation of loose fibrous tissue 22.

[0059] In contrast, when conditions 1 to 5 are satisfied, the number of through holes 13 is appropriately large and the opening dimension WT is appropriately small. Therefore, on the outer surface of partition wall 12, the openings of through holes 13 are less likely to be covered with collagen because the inter-hole distance is small. Also, on the inner surface 14 of partition wall 12, there are secured a large number of starting points for the formation of fibrous connective tissue 21 to the extent that the fibrous connective tissue 21 can be formed into a membrane.

[0060] The accumulation of the pluripotent stem cells 23 is realized by the invasion of the pluripotent stem cells 23 in the living tissue into the hollow portion 11, and the proliferation of the invaded pluripotent stem cells 23 in the hollow portion 11. The invasion of the pluripotent stem cells 23 is promoted by the through-holes 13 remaining open during most of the predetermined retention period, and the space in which the loose fibrous tissue 22 is formed remaining in the hollow portion 11. When the conditions 1 to 5 are satisfied, the growth of the loose fibrous tissue 22 can be promoted more than the growth of the fibrous connective tissue 21, and the invasion of the fibroblasts and the pluripotent stem cells 23 can be promoted to such an extent that a large depression or hole opposite the opening of the through-hole 13 is not formed in the fibrous connective tissue 21. Then, the fibrous connective tissue 21 having a membrane shape is easily formed, and as the growth of the fibrous connective tissue 21 is promoted, the pluripotent stem cells 23 are easily accumulated inward of the fibrous connective tissue 21. In particular, when the environment containing the biological tissue is the same as that in the body of a disease model animal, the growth of the fibrous connective tissue 21 is likely to be delayed, and therefore the probability of accumulating the pluripotent stem cells 23 increases.

[0061] When the thickness T12 of the partition wall 12 is 2.0 mm or less and the opening dimension WT of the through-hole 13 is 0.3 mm or more, even if the through-hole 13 is slightly narrowed by the fibrous connective tissue 21, cells and the like in the biological tissue can enter the hollow portion 11. Because the through-hole 13 is not easily blocked by the fibrous connective tissue 21, the fibrous connective tissue 21 tends to grow to a thickness of 0.05 mm or more and 0.5 mm or less, and pluripotent stem cells 23 tend to accumulate in the loose fibrous tissue 22.

[0062] When the thickness T12 of the partition wall 12 is 2.0 mm or less and the opening dimension WT of the through-hole 13 is 3.0 mm or less, large depressions or holes are unlikely to be formed in the fibrous connective tissue 21 at the site facing the through-hole 13. Suppressing the formation of large depressions in the fibrous connective tissue 21 suppresses the formation of partial weak sites in the fibrous connective tissue 21, making the membranous tissue structure 20 easier to handle.

[0063] When the thickness T12 of the partition wall 12 is 0.1 mm or more, the opening dimension WT of the through-hole 13 is 3.0 mm or less, and the opening occupancy rate is 70% or less, the mechanical strength of the partition wall 12 is easily obtained. Therefore, deformation of the partition wall 12 is easily suppressed in the living tissue.

[0064] When the inter-hole distance is 0.3 mm or more, the fibrous connective tissue 21 is likely to grow using the inner surface 14 as a scaffold. When the inter-hole distance is 5.0 mm or less, the fibrous connective tissue 21 growing from the periphery of the opening of one through-hole 13 is likely to be connected to the fibrous connective tissue 21 growing from the periphery of the opening of another through-hole 13. The connection between the peripheries of the openings of the through-holes 13 in the fibrous connective tissue 21 suppresses the formation of partial weak sites in the fibrous connective tissue 21, making it easier to handle the membranous tissue structure 20. Furthermore, when the opening occupancy rate is 30% or more and 70% or less, the fibrous connective tissue 21 is likely to spread in a membranous manner over the entire inner surface 14.

[0065] The retention period of the tissue structure forming device 10 is preferably a short period of time, about 4 weeks from the start of retention, during which inflammation caused by the incision required for retention of the tissue structure forming device 10 subsides. On the other hand, when the environment containing the living tissue is the inside of a disease model animal, the growth of the fibrous connective tissue 21 is likely to be slower than in a healthy state. When the thickness T12 of the partition wall 12 is 2.0 mm or less and the opening dimension WT of the through hole 13 is 3.0 mm or less, even in such an environment, large depressions or holes are unlikely to be formed in the fibrous connective tissue 21 at the site facing the through hole 13 after the retention period has elapsed. Furthermore, when the opening occupancy rate is 30% or more and 70% or less, the fibrous connective tissue 21 is likely to spread in a membrane-like manner over the entire inner surface 14. Furthermore, the through hole 13 is unlikely to be blocked by the fibrous connective tissue 21.

[0066] Furthermore, when the hollow depth D11 is 2 mm or more, the hollow portion 11 is significantly prevented from being buried in the fibrous connective tissue 21 during a suitable retention period, making it easy to obtain a sparse fibrous tissue 22 of sufficient thickness and pluripotent stem cells 23 accumulated in the sparse fibrous tissue 22. In particular, when the environment including the living tissue is the in vivo environment of a disease model animal, the growth of the fibrous connective tissue 21 is likely to be delayed, increasing the likelihood that the pluripotent stem cells 23 can be accumulated.

[0067] Returning to FIG. 1 , the tissue structure forming device 10 includes a support member 15 in the hollow portion 11. The support member 15 may support both ends of the partition wall 12 in the extension direction. The support member 15 may be fitted to the tubular ends of the partition wall 12 so as to close both ends of the hollow portion 11 in the extension direction of the partition wall 12, for example. The support member 15 may be inserted into the tubular interior of the partition wall 12 so as to abut against a portion of the inner surface 14 from within the hollow portion 11, for example. Support of the partition wall 12 by the support member 15 stabilizes the shape of the hollow portion 11 in the biological tissue.

[0068] The support member 15 has a flattened elliptical cylindrical shape so that the outer surface of the support member 15 is aligned with the inner surface 14 of the partition wall 12. When the inner surface 14 of the partition wall 12 has a cylindrical surface shape, the support member 15 may have a cylindrical shape. When the inner surface 14 of the partition wall 12 has a flattened rectangular parallelepiped shape, the support member 15 may have a rectangular parallelepiped shape. The support member 15 provides a predetermined hollow depth D11 between the inner surface 14 of the partition wall 12 and the outer surface of the support member 15. When the partition wall 12 has a strength that makes it difficult to deform in biological tissue, the tissue structure forming device 10 may omit the support member 15.

[0069] The constituent material of the support member 15 is compatible with biological tissue. The constituent material of the support member 15 may be the same as the constituent material of the partition wall 12, or may be different from the constituent material of the partition wall 12. The constituent material of the support member 15 may be a metal material such as stainless steel, titanium, titanium-nickel alloy, cobalt-chromium alloy, or a synthetic resin such as silicon, PEEK, acrylic, nylon, polycarbonate, polysulfone, polymethylpentene, or polytetrafluoroethylene. The constituent material of the partition wall 12 may be a laminate of a metal material and a constituent resin.

[0070] The through-holes 13 may be formed by etching the partition walls 12 or by laser drilling the partition walls 12 . [Organizational structure 20] Returning to FIG. 2, the fibrous connective tissue 21 has a higher fiber density than the sparse fibrous tissue 22 and contains fewer cells than the sparse fibrous tissue 22. The fibrous connective tissue 21 contains collagen fibers such as fibrous collagen and fibroblasts. The fibrous connective tissue 21 may contain collagen fiber bundles. The collagen fibers of the fibrous connective tissue 21 preferably contain type I collagen. The ratio of type I collagen in the total collagen contained in the fibrous connective tissue 21 may be 65% by weight or more and 90% by weight or less. The ratio of type I collagen in the total collagen contained in the fibrous connective tissue 21 is higher than the ratio of type I collagen in the total collagen contained in the sparse fibrous tissue 22.

[0071] The thickness H21 of the fibrous connective tissue 21 may be 0.05 mm or more and 0.5 mm or less. When the thickness of the fibrous connective tissue 21 is 0.05 mm or more and 0.5 mm or less, the strength of the fibrous connective tissue 21 having a membrane shape is ensured, so that the tissue structure 20 is easy to handle when administering the pluripotent stem cells 23. When the depth of the hollow portion 11 is 2 mm or more and 10 mm or less and the thickness of the fibrous connective tissue 21 is 0.05 mm or more and 0.5 mm or less, the depth of the hollow portion 11 is sufficiently larger than the thickness of the fibrous connective tissue 21. Therefore, a space for accumulating the pluripotent stem cells 23 is easily ensured in the hollow portion 11. When improved accuracy is required for accumulating the pluripotent stem cells 23, the depth of the hollow portion 11 is preferably 3 mm or more and 10 mm or less, more preferably 4 mm or more and 10 mm or less, and even more preferably 5 mm or more and 10 mm or less.

[0072] The fibrous connective tissue 21 may have a protrusion 21T conforming to the shape of the through hole 13 at a site corresponding to the through hole 13. The thickness HT of the protrusion 21T is equal to or less than the depth of the through hole 13. The fibrous connective tissue 21 may omit the protrusion 21T. The fibrous connective tissue 21 may have capillaries 24 extending to the sparse fibrous tissue 22 at a site corresponding to the through hole 13. The capillaries 24 are newly generated in the hollow portion 11 through the through hole 13 when the tissue structure 20 is formed.

[0073] The loose fibrous tissue 22 has a lower fiber density than the fibrous connective tissue 21, and has more cells scattered among the fibers than the fibrous connective tissue 21. The loose fibrous tissue 22 contains fibroblasts, collagen fibers such as fibrous collagen, vascular endothelial cells, fibrin, and pluripotent stem cells 23 such as mesenchymal cells. The loose fibrous tissue 22 may contain type III collagen or fibrous collagen other than type III collagen. The content of type III collagen in the loose fibrous tissue 22 is higher than the content of type III collagen in the fibrous connective tissue 21.

[0074] The pluripotent stem cells 23 contained in the sparse fibrous tissue 22 express a pluripotent stem cell marker. The pluripotent stem cells 23 may express at least one pluripotent stem cell marker or may express at least one mesenchymal stem cell marker. The pluripotent stem cells 23 contained in the sparse fibrous tissue 22 may contain mesenchymal stem cells.

[0075] The pluripotent stem cells 23 contained in the sparse fibrous tissue 22 may express both the pluripotent stem cell marker SSEA3 and the pluripotent stem cell marker SSEA4. The pluripotent stem cells 23 contained in the sparse fibrous tissue 22 may express the pluripotent stem cell marker SSEA3 or the pluripotent stem cell marker SSEA4.

[0076] The pluripotent stem cells 23 contained in the sparse fibrous tissue 22 may express both the mesenchymal stem cell marker CD90 and the mesenchymal stem cell marker CD105. The pluripotent stem cells 23 contained in the sparse fibrous tissue 22 may express the mesenchymal stem cell marker CD90 or the mesenchymal stem cell marker CD105. The pluripotent stem cells 23 contained in the sparse fibrous tissue 22 may express both the mesenchymal stem cell marker CD90 and the pluripotent stem cell marker SSEA3.

[0077] The loose fibrous tissue 22 may contain stem cells expressing the mesenchymal stem cell marker CD105, and stem cells expressing both the mesenchymal stem cell marker CD105 and the pluripotent stem cell marker SSEA3.

[0078] The sparse fibrous tissue 22 may contain stem cells having high angiogenic potential. The sparse fibrous tissue 22 may contain, as the stem cells having high angiogenic potential, stem cells expressing the growth factor marker VEGF, or stem cells expressing both the growth factor marker VEGF and the pluripotent stem cell marker SSEA3.

[0079] The loose fibrous tissue 22 may contain stem cells expressing the growth factor marker HGF. The ratio of the total number of pluripotent stem cells 23 contained in the sparse fibrous tissue 22 to the total number of cells contained in the sparse fibrous tissue 22 is the stem cell ratio. The stem cell ratio can be calculated as the total cell number, which is the number of cells recovered from the decomposed tissue when the sparse fibrous tissue 22 is treated in a 0.25% collagenase type I solution at 37°C for 1.5 hours. The stem cell ratio can be calculated as the number of cells expressing at least one selected from the group consisting of mesenchymal stem cell marker CD90, mesenchymal stem cell marker CD105, pluripotent stem cell marker SSEA3, and pluripotent stem cell marker SSEA4. The stem cell ratio of the sparse fibrous tissue 22 is 5% or more, preferably 10% or more, more preferably 20% or more, and even more preferably 30% or more. The stem cell ratio of the sparse fibrous tissue 22 may be 60% or less, 50% or less, 40% or less, or 30% or less.

[0080] The ratio of the number of mesenchymal stem cells contained in the sparse fibrous tissue 22 to the number of pluripotent stem cells contained in the sparse fibrous tissue 22 is the mesenchymal stem cell ratio. The mesenchymal stem cell ratio can be calculated as the number of cells expressing the mesenchymal stem cell marker CD90 or the mesenchymal stem cell marker CD105 as the number of mesenchymal stem cells. The mesenchymal stem cell ratio can be calculated as the number of cells expressing the mesenchymal stem cell marker CD90, the mesenchymal stem cell marker CD105, the pluripotent stem cell marker SSEA3, or the pluripotent stem cell marker SSEA4 as the number of pluripotent stem cells. The mesenchymal stem cell ratio may be 5% or more, or 10% or more. The mesenchymal stem cell ratio may be 60% or less, or 50% or less.

[0081] [Test example] First, the back of a diabetic pig model, which is a disease model animal, or an aged pig was used as an environment containing biological tissue. Next, the pig was anesthetized, and a tissue structure forming device 10 having the following device structure example was embedded subcutaneously in the back, and the pig's back was sutured. After the tissue structure forming device 10 was left in place for a predetermined retention period, the pig was anesthetized and the subcutaneous tissue structure forming device 10 was removed. Almost the entire hollow portion 11 of the tissue structure forming device 10 removed from the subcutaneous tissue was buried in soft tissue. [Equipment structure example] Length in the axial direction of the partition wall 12: 5 cm Circumferential length of partition wall 12: 6 cm - Compartment wall 12 construction material: Stainless steel plate Thickness of partition wall 12: T12: 1mm Hollow depth D11: 1mm, 2mm, 4mm Opening dimension WT: 1.0mm ·Hole distance: 3.0mm ·Aperture occupancy rate: 50% Detention period: 2 weeks, 4 weeks, 6 weeks, 8 weeks, 11 weeks

[0082] As shown in Fig. 7, a membrane-like tissue structure 20 was peeled off from the inner surface 14 of the partition wall 12, which is an elliptical cylindrical surface. The tissue structure 20 was then cut open along a cutting line 20K along the axial direction of the partition wall 12, thereby obtaining a flat membrane-like tissue structure 20. On the outer surface of the tissue structure 20, irregularities 20T including numerous protrusions 21T were observed at positions corresponding to the openings of the through-holes 13.

[0083] [Dyeing evaluation] Using test examples of tissue structure forming devices 10 with different hollow depths D11 and retention periods, paraffin-embedded blocks of tissue structure 20 were prepared for the center and edges in the planar direction of membranous tissue structure 20. Sections were also prepared by cutting the paraffin-embedded blocks of fixed tissue structure 20 with a microtome so that the thickness of the sections in the short direction was 3 μm or more and 5 μm or less. Next, each section was stained with hematoxylin and eosin (HE), Masson's trichrome (MT), Elastica van Gieson (EVG), and Sirius red (SR), and observed under a microscope.

[0084] As a result of the observation, pink color due to HE staining was observed throughout the entire tissue structure 20. Blue color due to MT staining was observed throughout the entire tissue structure 20. This confirmed that collagen fibers were abundantly contained in the tissue structure 20. In addition, red color due to SR staining was observed throughout the entire tissue structure 20. This confirmed that collagen was abundantly contained in the tissue structure 20.

[0085] As shown in Fig. 9, in the thickness direction of the tissue structure 20, deep staining by HE staining was observed over the entire surface direction of the first surface 21A from about 0.1 mm from the first surface 21A. In addition, as shown in Fig. 10, deep staining by MT staining was also observed from about 0.1 mm from the first surface 21A. This confirmed that the fibrous connective tissue 21, which is a dense tissue, was formed from the first surface 21A to about 0.1 mm in the thickness direction of the tissue structure 20. As shown in the light colored portion of Fig. 11, yellow or orange by SR staining was observed from about 0.1 mm from the first surface 21A by observation with a polarizing microscope. This also confirmed that the fibrous connective tissue 21 contains a large amount of type I collagen.

[0086] As shown in Fig. 12, in the thickness direction of the tissue structure 20, light staining was observed in HE staining over the entire surface direction of the first surface 21A from about 2 mm from the fibrous connective tissue 21. Furthermore, as shown in Fig. 13, light staining was also observed in MT staining from about 2 mm from the fibrous connective tissue 21. This confirmed that the sparse fibrous tissue 22 was formed inside the fibrous connective tissue 21. As shown in the light-colored portion of Fig. 14, green by SR staining was observed from about 2 mm from the fibrous connective tissue 21 by observation with a polarizing microscope. This also confirmed that the sparse fibrous tissue 22 contained type III collagen.

[0087] Furthermore, no black color due to EVG staining was observed throughout the tissue structure 20. This demonstrated that the tissue structure 20 contained almost no elastic fibers. Next, mesenchymal stem cell marker CD90 was detected using anti-CD90 antibody (Abcam) as the primary antibody and goat anti-mouse IgG H&L (Abcam) as the secondary antibody. Mesenchymal stem cell marker CD105 was detected using anti-CD105 antibody (Abcam) as the primary antibody and goat anti-rabbit IgG H&L (Abcam) as the secondary antibody.

[0088] The pluripotent stem cell marker SSEA4 was detected using anti-SSEA4 antibody (Abcam) as the primary antibody and ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific) as the secondary antibody. The pluripotent stem cell marker SSEA3 was detected using anti-SSEA3 antibody (Bioss) as the primary antibody and anti-FITC microbeads (Miltenyi Biotec) as the secondary antibody.

[0089] As shown in FIG. 15, in the detection results for the mesenchymal stem cell marker CD90, no staining was observed in the fibrous connective tissue 21. Also, as shown in FIG. 16, in the detection results for the mesenchymal stem cell marker CD105, no staining was observed in the fibrous connective tissue 21. In contrast, as shown in FIG. 17, in the detection results for the mesenchymal stem cell marker CD90, staining was observed in a large number of cells throughout the sparse fibrous tissue 22. Also, as shown in FIG. 18, in the detection results for the mesenchymal stem cell marker CD105, staining was observed in a large number of cells throughout the sparse fibrous tissue 22.

[0090] As shown in FIG. 19, in the detection results for the pluripotent stem cell marker SSEA3, no staining was observed in the fibrous connective tissue 21. Also, as shown in FIG. 20, in the detection results for the pluripotent stem cell marker SSEA4, no staining was observed in the fibrous connective tissue 21. In contrast, as shown in FIG. 21, in the detection results for the pluripotent stem cell marker SSEA3, staining was observed in a large number of cells throughout the sparse fibrous tissue 22. Also, as shown in FIG. 22, in the detection results for the pluripotent stem cell marker SSEA4, staining was observed in a large number of cells throughout the sparse fibrous tissue 22.

[0091] Next, vascular endothelial growth factor (VEGF) was detected using anti-VEGFA antibody (Abcam) as the primary antibody and cell staining buffer (BioLegend) as the secondary antibody. Hepatocyte growth factor (HGF) was detected using anti-HGF antibody (Abcam) as the primary antibody and FITC-labeled anti-CD90 antibody (BioLegend) as the secondary antibody.

[0092] In the detection results of the vascular endothelial growth factor marker, expression was observed in the sparse fibrous tissue 22, and high expression was observed in the sparse fibrous tissue 22 on the opposite side to the fibrous connective tissue 21. In the detection results of the vascular endothelial growth factor marker, expression was observed in the sparse fibrous tissue 22, and high expression was observed in the sparse fibrous tissue 22 on the opposite side to the fibrous connective tissue 21.

[0093] Next, we performed double staining with the pluripotent stem cell marker SSEA3 and the mesenchymal stem cell marker CD90, double staining with the pluripotent stem cell marker SSEA3 and the mesenchymal stem cell marker CD105, double staining with the pluripotent stem cell marker SSEA3 and the pluripotent stem cell marker SSEA4, and double staining with the pluripotent stem cell marker SSEA3 and the vascular endothelial growth factor marker.

[0094] As a result of double staining, in addition to double-positive cells, cells that were single-positive for each marker were observed. It was found that the cells that make up sparse fibrous tissue 22 are a hierarchical cell population with different degrees of undifferentiation. In addition, some cells simultaneously expressed the pluripotent stem cell markers SSEA4 and SSEA3, which is consistent with the characteristics of more undifferentiated pluripotent stem cells such as ES cells. In addition, since VEGF was particularly highly expressed in SSEA3-positive cells, it is thought that the highly undifferentiated stem cells present in the tissue of sparse fibrous tissue 22 have high angiogenic potential.

[0095] [Thickness evaluation] Using test examples of tissue structure forming devices 10 with different hollow depths D11 and retention periods, the thickness of fibrous connective tissue 21 in tissue structure 20 and the thickness of tissue structure 20 were measured. The thickness H21 of fibrous connective tissue 21 and the thickness H22 of sparse fibrous tissue 22 were each the average value between the center and the edge in the planar direction of membrane-like tissue structure 20.

[0096] The measurement results using tissue structure forming devices 10 with hollow depths D11 of 1 mm and 2 mm are shown in Fig. 8. In Fig. 8, the thickness of the fibrous connective tissue 21 using the tissue structure forming device 10 with a hollow depth D11 of 1 mm is shown with a white triangle, and the thickness of the tissue structure 20 is shown with a white circle. In Fig. 8, the thickness of the fibrous connective tissue 21 using the tissue structure forming device 10 with a hollow depth D11 of 2 mm is shown with a black square, and the thickness of the tissue structure 20 is shown with a black circle.

[0097] As shown by the open circle in Fig. 8, when the hollow depth D11 is 1 mm, the thickness of the tissue structure 20 grows to 0.6 mm, which is approximately 2 / 3 of the hollow depth D11, after two weeks of retention. As shown by the open triangle in Fig. 8, the thickness of the fibrous connective tissue 21 grows to approximately 1 / 3 of the hollow depth D11 after two weeks of retention. In other words, after two weeks of retention, the tissue structure 20 has fibrous connective tissue 21 with a thickness of approximately 1 / 3 of the hollow depth D11 and thin sparse fibrous tissue 22 with a thickness of approximately 1 / 3 of the hollow depth D11.

[0098] On the other hand, after a retention period of four weeks or more has passed, the thickness of the tissue structure 20 grows to the entire hollow depth D11. The thickness of the fibrous connective tissue 21 also grows to the entire hollow depth D11. In other words, after a retention period of four weeks has passed, the tissue structure 20 does not have sparse fibrous tissue 22 and is composed of fibrous connective tissue 21 having a thickness of the hollow depth D11.

[0099] Thus, when the hollow depth D11 is 1 mm, the membranous loose fibrous tissue 22 in which the pluripotent stem cells 23 are accumulated can be obtained by detention for two weeks. However, this detention period is slightly shorter than the period suitable for allowing inflammation caused by detention to subside. The margin for the period during which the loose fibrous tissue 22 can be obtained is also short, such as less than one week. Furthermore, the thickness of the loose fibrous tissue 22 is less than 0.5 mm.

[0100] In contrast, as shown by the solid circle in Fig. 8, when the hollow depth D11 is 2 mm, the thickness of the tissue structure 20 grows to 1.1 mm, which is approximately 1 / 2 of the hollow depth D11, after two weeks of retention. On the other hand, as shown by the solid square in Fig. 8, the thickness of the fibrous connective tissue 21 grows to approximately 1 / 8 of the hollow depth D11 after two weeks of retention. In other words, after two weeks of retention, the tissue structure 20 has fibrous connective tissue 21 with a thickness of 1 / 2 the hollow depth D11 and sparse fibrous tissue 22 with a thickness of 1.0 mm or more.

[0101] After four weeks or more of retention, the thickness of the tissue structure 20 grows to about 9 / 10 of the hollow depth D11. Meanwhile, the thickness of the fibrous connective tissue 21 remains at about 1 / 8 of the hollow depth D11, the same as after two weeks of retention. Thus, the tendency for the thickness of the tissue structure 20 to be almost the entire hollow depth D11, while the thickness of the fibrous connective tissue 21 to remain at about 1 / 4 or less of the hollow depth D11, is observed until eight weeks of retention. After eleven weeks of retention, the tissue structure 20 does not have sparse fibrous tissue 22 and is composed of fibrous connective tissue 21 having a thickness of the hollow depth D11.

[0102] That is, when the hollow depth D11 is 2 mm, (a) the membranous sparse fibrous tissue 22 in which the pluripotent stem cells 23 have accumulated can be obtained during a suitable retention period that allows inflammation due to retention to subside. Also, when the hollow depth D11 is 2 mm, (b) the margin of time during which the sparse fibrous tissue 22 can be obtained is long, such as two weeks or more. And, since the thickness of the sparse fibrous tissue 22 is 1.0 mm or more, (c) many pluripotent stem cells 23 can easily be uniformly accumulated in the membranous sparse fibrous tissue 22.

[0103] When the hollow depth D11 is 4 mm, after a retention period of two weeks, the thickness of tissue structure 20 grows to 1.2 mm and the thickness of fibrous connective tissue 21 grows to about 0.2 mm, similar to the case where the hollow depth D11 is 2 mm. After a retention period of four weeks, the thickness of tissue structure 20 grows to 2.0 mm and the thickness of fibrous connective tissue 21 remains at about 0.2 mm, similar to the case where the hollow depth D11 is 2 mm. Then, during a retention period of six to eleven weeks, the thickness of tissue structure 20 grows to 4.0 mm and the thickness of fibrous connective tissue 21 remains at about 0.2 mm.

[0104] That is, even when the hollow depth D11 is 4 mm, the membranous sparse fibrous tissue 22 in which the pluripotent stem cells 23 have accumulated can be obtained during a suitable retention period that allows inflammation caused by retention to subside. The margin for the period during which the sparse fibrous tissue 22 can be obtained is also long, such as two weeks or more. In addition, since the thickness of the sparse fibrous tissue 22 is 1.0 mm or more, many pluripotent stem cells 23 are likely to accumulate uniformly in the membranous sparse fibrous tissue 22.

[0105] Thus, in the formation of the sparse fibrous tissue 22, setting the hollow depth D11 to 2 mm or more requires a new technical finding that (i) there is a difference in growth rate between the fibrous connective tissue 21 and the sparse fibrous tissue 22. Setting the hollow depth D11 to 2 mm or more also requires a new technical finding that (ii) when a certain hollow depth D11 is exceeded, the correlation between the difference in growth rate and the hollow depth D11 changes. Setting the hollow depth D11 to 2 mm or more is derived for the first time from the viewpoint of setting the hollow depth D11 so as to enhance the growth of the sparse fibrous tissue 22 more than the growth of the fibrous connective tissue 21. The tendency shown in the findings of (i) and (ii) above was more clearly observed in the partition wall 12 that satisfied the conditions 1 to 4.

[0106] Thus, setting the hollow depth D11 to 2 mm or more has the effect of obtaining a membranous sparse fibrous tissue 22 supported by a membranous fibrous connective tissue 21 after a suitable retention period that allows inflammation due to retention to subside. Setting the hollow depth D11 to 2 mm or more has the effect of obtaining a large number of pluripotent stem cells 23 uniformly accumulated in the membranous sparse fibrous tissue 22 after a suitable retention period that allows inflammation due to retention to subside. Setting the hollow depth D11 to 2 mm or more has the unique and outstanding effect of obtaining for the first time a membranous sparse fibrous tissue 22 in which a large number of pluripotent stem cells 23 are uniformly accumulated, with a margin for a suitable retention period that allows inflammation due to retention to subside.

[0107] As described above, according to the above embodiment, the following effects can be obtained. (1) Cells derived from biological tissue that enter the hollow portion 11 form one side of a fibrous connective tissue 21 using the inner surface 14 as a scaffold. As the fibrous connective tissue 21 grows, the pluripotent stem cells 23 in the biological tissue are accumulated in the membranous loose fibrous tissue 22 on the other side of the fibrous connective tissue 21. Since the accumulation site of the pluripotent stem cells 23 spreads over the entire other side of the membranous fibrous connective tissue 21, it is easy to administer the pluripotent stem cells 23 to the diseased site by, for example, attaching a membranous tissue structure 20.

[0108] (2) When the fibrous connective tissue 21 detached from the inner surface 14, which is a flat elliptical cylindrical surface, is cut open in the axial direction of the elliptical cylindrical surface, the accumulation site of pluripotent stem cells 23 is further expanded within the limited biological tissue compared to when a membranous tissue structure 20 is formed on a flat inner surface 14.

[0109] (3) When the hollow depth D11 is 2 mm or more, the membranous sparse fibrous tissue 22 in which the pluripotent stem cells 23 have accumulated can be obtained during a suitable retention period that allows inflammation caused by retention to subside. Furthermore, when the hollow depth D11 is 2 mm, the margin for the period during which the sparse fibrous tissue 22 can be obtained is long, such as 2 weeks or more. Furthermore, since the thickness of the sparse fibrous tissue 22 is 1.0 mm or more, many pluripotent stem cells 23 are likely to accumulate uniformly in the membranous sparse fibrous tissue 22.

[0110] (4) When the depth of the hollow portion 11 is 2 mm or more and 10 mm or less and the thickness of the fibrous connective tissue 21 is 0.05 mm or more and 0.5 mm or less, the strength of the membrane-like fibrous connective tissue 21 is ensured. This makes it easy to handle the tissue structure 20 when administering the pluripotent stem cells 23. In addition, since the depth of the hollow portion 11 is sufficiently greater than the thickness of the fibrous connective tissue 21, a space for accumulating the pluripotent stem cells 23 is easily ensured within the hollow portion 11.

[0111] (5) When the opening dimension WT is 0.3 mm or more, cells derived from biological tissue, plasma proteins, and the like easily enter the hollow portion 11 through the through-holes 13 in the partition wall 12. When the opening dimension WT is 3.0 mm or more, depressions or holes are unlikely to form in the fibrous connective tissue 21 at the sites facing the through-holes 13. When the inter-hole distance is 0.3 mm or more and the occupancy rate of the openings is 30% or more and 70% or less, the fibrous connective tissue 21 is likely to grow using the inner surface 14 as a scaffold. For this reason, an environment suitable for cells derived from biological tissue is likely to be established in the hollow portion 11 through the capillaries 24 at the sites corresponding to the through-holes 13.

[0112] The above embodiment may be modified as follows. The cylindrical end of the cylindrical partition wall 12 may be open and not closed by a cap. Alternatively, the cap closing the cylindrical end of the partition wall 12 may have a large vent hole that opens most of the cylindrical end.

[0113] In this modified example, when a foreign body such as the support member 15 is present in the hollow portion 11, the outer surface of the support member 15 functions as a scaffold for cells derived from biological tissues that enter the hollow portion 11 from the end of the tube. Then, fibrous connective tissue 21 is formed from the inner surface 14 toward the hollow portion 11, and is also formed from the outer surface of the support member 15 toward the hollow portion 11. In this case, the tissue structure 20 formed in the hollow portion 11 has a three-layer structure in which the loose fibrous tissue 22 is sandwiched between the fibrous connective tissue 21 in contact with the inner surface 14 and the fibrous connective tissue 21 in contact with the outer surface of the support member 15. That is, the membranous loose fibrous tissue 22 in which the pluripotent stem cells 23 have accumulated may be sandwiched between the membranous fibrous connective tissue 21. When most of the end of the tube is opened and the foreign body such as the support member 15 functions as a scaffold for forming the fibrous connective tissue 21, the hollow depth D11 is half the distance between the inner surface 14 and the foreign body in the depth direction of the through hole 13.

[0114] The tissue structure 20 may be processed into a two-layer structure of the fibrous connective tissue 21 in contact with the outer surface of the support member 15 and the loose fibrous tissue 22 in contact with the fibrous connective tissue 21, through intra-layer peeling of the loose fibrous tissue 22 in the three-layer structure. The tissue structure 20 may be processed into a two-layer structure of the fibrous connective tissue 21 in contact with the inner surface 14 and the loose fibrous tissue 22 in contact with the fibrous connective tissue 21, through intra-layer peeling of the loose fibrous tissue 22 in the three-layer structure.

[0115] 23, the partition wall 12 having a cylindrical shape may have one or more protruding ribs extending in the axial direction. The one or more protruding ribs partially increase the axial thickness of the partition wall 12. The one or more protruding ribs increase the mechanical durability of the partition wall 12 against stress applied thereto, and stabilize the shape of the hollow portion 11.

[0116] 24, the partition wall 12 having a cylindrical shape may be provided with a protruding rib that extends in the axial direction and rotates in the circumferential direction. This protruding rib also partially increases the axial thickness of the partition wall 12. The spiral protruding rib also increases the mechanical durability of the partition wall 12 against stress applied thereto, and stabilizes the shape of the hollow portion 11.

[0117] That is, the partition wall 12 may have a thick reinforcing rib in a part thereof. The reinforcing rib may be arranged so as to avoid the through holes 13, or may be arranged so as to cover a part of the through holes 13. When uniformity in the thickness of the tissue structure 20 is required, it is preferable that the reinforcing rib is arranged so as to avoid the through holes 13 so as not to impair the regularity in the arrangement of the through holes 13. [Explanation of symbols]

[0118] WT…Opening dimensions 10…Tissue structure forming device 11...Hollow part 12...Partition wall 13...Through hole 14…Inner 20…Organizational structure 21...Fibrous connective tissue 21A…Side 1 21B…Second side 22...Lose fibrous tissue 23…Pluripotent stem cells 24...Capillaries

Claims

1. A tissue structure formed by a partition wall placed in an environment containing biological tissue, The partition wall includes an inner surface surrounding the hollow portion and a plurality of through holes opening into the inner surface, The partition wall is configured so that cells in the biological tissue enter the hollow portion through the through-hole, The pluripotent stem cells include stem cells expressing at least one of the pluripotent stem cell markers SSEA3 and SSEA4, and mesenchymal stem cells; The tissue structure comprises: disposing the partition wall in the environment such that cells in the biological tissue enter the hollow portion through the through-holes; placing the partition wall in the environment so that a membranous fibrous connective tissue having one side in contact with the inner surface is formed and the membranous loose fibrous tissue in which the pluripotent stem cells have been accumulated spreads over the entire other side opposite to the one side; removing the partition wall from the biological tissue after a predetermined period of time has elapsed before the fibrous connective tissue fills the hollow portion, and peeling off the one side surface from the inner surface of the partition wall; The method includes the steps of: The tissue structure comprises: The fibrous connective tissue is in the form of a membrane having one side in contact with the inner surface; The membrane-like loose fibrous tissue extends over the entire other side opposite to the one side and accumulates the pluripotent stem cells; The thickness of the partition wall is 0.1 mm or more and 2.0 mm or less, The opening dimension of the through hole on the inner surface is 0.3 mm or more and 3.0 mm or less, A length between adjacent through holes on the inner surface is 0.3 mm or more and 5.0 mm or less, The occupancy rate of the openings on the inner surface is 30% or more and 70% or less, The one side surface of the fibrous connective tissue has a protrusion having a shape corresponding to the through-hole, The depth of the hollow portion is 2 mm or more and 10 mm or less, the thickness of the fibrous connective tissue is 0.05 mm or more and 0.5 mm or less, the thickness of the loose fibrous tissue, which has a lower fiber density than the fibrous connective tissue, is 1.0 mm or more, the ratio of type I collagen in the total collagen contained in the fibrous connective tissue is higher than the ratio of type I collagen in the total collagen contained in the loose fibrous tissue, and the content of type III collagen in the loose fibrous tissue is higher than the content of type III collagen in the fibrous connective tissue. A tissue structure characterized by:

2. The inner surface is a flattened elliptical cylindrical surface, The fibrous connective tissue has a membrane-like shape cut in the axial direction of the elliptical cylindrical surface. The tissue structure of claim 1 .

3. The thickness of the fibrous connective tissue is 0.05 mm or more and 0.5 mm or less. The tissue structure of claim 1 .

4. An inner surface surrounding the hollow portion; A partition wall having a plurality of through holes opening on the inner surface, A tissue structure forming device configured such that the partition wall is placed in an environment containing biological tissue, and cells in the biological tissue invade the hollow portion through the through-hole, The organizational structure is A membrane-like fibrous connective tissue having one side in contact with the inner surface; A membrane-like loose fibrous tissue that spreads over the entire other side opposite to the one side and accumulates pluripotent stem cells; The pluripotent stem cells are Stem cells expressing at least one of the pluripotent stem cell markers SSEA3 and SSEA4; and mesenchymal stem cells, The thickness of the partition wall is 0.1 mm or more and 2.0 mm or less, The opening dimension of the through hole is 0.3 mm or more and 3.0 mm or less, The hole distance between the through holes is 0.3 mm or more and 5.0 mm or less, an opening occupancy rate of the through holes on the inner surface is 30% or more and 70% or less; The depth of the hollow portion is 2 mm or more and 10 mm or less, The through-hole is configured to be embedded in a protrusion having a shape corresponding to the shape of the through-hole on the one side surface of the fibrous connective tissue, the thickness of the fibrous connective tissue is 0.05 mm or more and 0.5 mm or less, the thickness of the loose fibrous tissue, which has a lower fiber density than the fibrous connective tissue, is 1.0 mm or more, the ratio of type I collagen in the total collagen contained in the fibrous connective tissue is higher than the ratio of type I collagen in the total collagen contained in the loose fibrous tissue, and the content of type III collagen in the loose fibrous tissue is higher than the content of type III collagen in the fibrous connective tissue. A tissue structure forming device comprising:

5. 1. A method for forming a tissue structure, comprising forming a tissue structure on a compartment wall by placing the compartment wall in a non-human environment including a living tissue, the method comprising: The partition wall includes an inner surface surrounding the hollow portion and a plurality of through holes opening into the inner surface, The pluripotent stem cells include stem cells expressing at least one of the pluripotent stem cell markers SSEA3 and SSEA4, and mesenchymal stem cells; disposing the partition wall in the environment such that cells in the biological tissue enter the hollow portion through the through-holes; placing the partition wall in the environment so that a membranous fibrous connective tissue having one side in contact with the inner surface is formed and the membranous loose fibrous tissue in which the pluripotent stem cells have been accumulated spreads over the entire other side opposite to the one side; and removing the partition wall from the biological tissue after a predetermined period of time has elapsed before the fibrous connective tissue fills the hollow portion, and peeling off the one side surface from the inner surface of the partition wall. The thickness of the partition wall is 0.1 mm or more and 2.0 mm or less, The opening dimension of the through hole on the inner surface is 0.3 mm or more and 3.0 mm or less, A length between adjacent through holes on the inner surface is 0.3 mm or more and 5.0 mm or less, The occupancy rate of the openings on the inner surface is 30% or more and 70% or less, The one side surface of the fibrous connective tissue has a protrusion having a shape corresponding to the through-hole, The depth of the hollow portion is 2 mm or more and 10 mm or less, the thickness of the fibrous connective tissue is 0.05 mm or more and 0.5 mm or less, the thickness of the loose fibrous tissue, which has a lower fiber density than the fibrous connective tissue, is 1.0 mm or more, the ratio of type I collagen in the total collagen contained in the fibrous connective tissue is higher than the ratio of type I collagen in the total collagen contained in the loose fibrous tissue, and the content of type III collagen in the loose fibrous tissue is higher than the content of type III collagen in the fibrous connective tissue. A method for forming a tissue structure comprising:

6. The inner surface is a flattened elliptical cylindrical surface, and cutting open the fibrous connective tissue detached from the inner surface in the axial direction of the elliptical cylindrical surface. A method for forming the tissue structure according to claim 5 .

7. The depth of the hollow portion is 2 mm or more and 10 mm or less, Retaining the partition wall for a predetermined period of time reduces the thickness of the fibrous connective tissue to 0.05 mm or more and 0.5 mm or less. A method for forming the tissue structure according to claim 5 .

8. The partition wall is a hollow tubular member, The hollow portion is defined only by the partition wall. A method for forming the tissue structure according to any one of claims 5 to 7.

9. The method further comprises grinding and fluidizing the fibrous connective tissue and the loose fibrous tissue detached from the inner surface of the compartment wall. A method for forming the tissue structure according to claim 5 .

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