Tissue structure, tissue structure formation device, and tissue structure formation method

Abstract

To provide a tissue structure that facilitates administration of pluripotent stem cells to a disease site, and a method for forming the tissue structure.SOLUTION: A tissue structure formed by a partition wall 12 indwelled in an environment including a biological tissue, wherein the partition wall 12 includes an inner surface 14 surrounding a hollow portion 11 and a plurality of through-holes 13 opened in the inner surface 14, cells in the biological tissue enter the hollow portion 11 through the through-holes to form the tissue structure in the hollow portion 11, and the tissue structure includes a membranous fibrous connective tissue having one side surface peeled from the inner surface 14 and a sparse fibrous tissue spreading over the entire other side surface opposite to the one side surface and having pluripotent stem cells accumulated therein.SELECTED DRAWING: Figure 1

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 substances that have invaded the body. Macrophages adhere to the surface of the foreign substance and stimulate fibroblasts to produce collagen through the production of TGF-β by monocytes. Foreign substances that have invaded the body are isolated within the body by being covered with connective tissue containing fibroblasts and collagen.

[0003] One type of regenerative medicine technology, which is a medical technology for reviving lost tissues and organs, replaces damaged tissue structures with tissue structures derived from living organisms. One technology for forming tissue structures derived from living organisms 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 living organisms 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 define a hollow space having various shapes, such as a flat space, a cylindrical space, or a valve-shaped space. Fibroblasts that invade between the two connective tissue forming surfaces form a connective tissue structure 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. However, the basis for using embryonic stem cells, which require ethical considerations, and induced pluripotent stem cells, which require safety confirmation, in regenerative medicine has yet to be established. Meanwhile, pluripotent stem cells present in vivo, although present in small numbers in living tissues, have progressed through the development stage and are now moving to the clinical stage. One technique for accumulating pluripotent stem cells present in vivo involves placing a tissue structure formation device, a foreign body, into the living tissue, and then accumulating the pluripotent stem cells in the living tissue onto 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 of extension of 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 the frame is 2.5 mm or more and the hollow portion is 2 mm or more deep, allowing fibroblasts to fill the hollow portion with fibrous connective tissue so as to form a depression in the area of ​​the fibrous connective tissue facing the opening. The area 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 depression (see, for example, Patent Document 9). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2007-37763 [Patent Document 2] Japanese Patent Application Laid-Open No. 2007-312821 [Patent Document 3] Japanese Patent Application Laid-Open No. 2008-237896 [Patent Document 4] Japanese Patent Application Laid-Open No. 2010-094476 [Patent Document 5] Japanese Patent Application Laid-Open No. 2014-030598 [Patent Document 6] Japanese Patent Application Laid-Open No. 2017-169778 [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 to treat diseases have passed through the development stage and are now 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, the accumulation of pluripotent stem cells present in the body is limited to the depressions in fibrous connective tissue in the above-mentioned technologies. Therefore, further improvements are needed to facilitate the administration of 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 comprising stem cells expressing at least one of the pluripotent stem cell markers SSEA3 and SSEA4, and mesenchymal stem cells, and the tissue structure comprising a membranous fibrous connective tissue having one side detached from the inner surface, and a membranous loose fibrous tissue extending over the entire other side opposite the one side and accumulating the pluripotent stem cells.

[0010] A tissue structure forming device for solving the above problems includes a compartment wall having an inner surface surrounding a hollow portion and a plurality of through-holes opening into the inner surface. The compartment wall is placed in an environment containing biological tissue, and cells from the biological tissue invade the hollow portion through the through-holes to form a tissue structure in the hollow portion. 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 extends over the other side opposite the one side and contains an accumulation of 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 inter-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 compartment wall by placing the compartment wall in an environment containing biological tissue, wherein the compartment wall has an inner surface surrounding a hollow portion and a plurality of through holes opening into 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 compartment wall in the environment so that cells in the biological tissue invade the hollow portion through the through holes; placing the compartment 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 compartment wall from the biological tissue after a predetermined period of time has passed before the fibrous connective tissue fills the hollow portion and peeling off the one side from the inner surface of the compartment wall.

[0012] According to each of the above configurations, when a compartment wall is placed in an environment containing biological tissue, cells derived from the biological tissue, plasma proteins, etc., enter the hollow space defined by the compartment wall through the through-holes. The cells derived from the biological tissue that enter the hollow space form loose fibrous tissue using the inner surface of the compartment wall as a scaffold. Over the course of the placement period, collagen production and other processes lead to the formation of fibrous connective tissue from the loose fibrous tissue. Over the course of the placement period, one side of membranous fibrous connective tissue forms on the inner surface of the compartment wall, and membranous loose fibrous tissue continues to form on the other side of the fibrous connective tissue. The biological tissue-derived pluripotent stem cells that enter the hollow space continue to accumulate in the loose fibrous tissue and proliferate in the loose fibrous tissue. This allows the accumulation site of pluripotent stem cells to spread across the entire other side of the membranous fibrous connective tissue. By attaching a membranous tissue structure, a large number of pluripotent stem cells can be administered to a wide diseased area.

[0013] In the 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. The above-described method for forming a tissue structure may further comprise the step of: the inner surface being a flattened elliptical cylindrical surface; and the step of 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 pluripotent stem cell accumulation in plan view can be reduced by the amount of the inner surface being an elliptical cylindrical surface compared to when the inner surface is flat, and a large area can be secured as a site for pluripotent stem cell accumulation even in an environment where the area in plan view is limited.

[0015] In the 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 method for forming the 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 configuration, the strength of the membranous fibrous connective tissue is ensured, making the tissue construct easy to handle when administering pluripotent stem cells. Furthermore, because the depth of the hollow portion is sufficiently greater than the thickness of the fibrous connective tissue, it is easy to ensure a space within the hollow portion for accumulating pluripotent stem cells.

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

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

[0019] In the method for forming a tissue structure described above, 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 secured 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. [Effects of the Invention]

[0021] The tissue structure, tissue structure forming device, and tissue structure forming method disclosed herein facilitate the administration of pluripotent stem cells to a diseased site. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 is a perspective view showing a tissue structure forming device. [Figure 2] FIG. 2 is a cross-sectional view showing the tissue structure. [Figure 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. [Figure 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 structure. [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 loose 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 INVENTION

[0023] [Outline of tissue structure forming device 10] As shown in Figure 1, the tissue structure forming device 10 has a partition wall 12 that separates 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 openings of the through holes 13. The tissue structure forming device 10 is left in an environment where biological tissue is present for a predetermined period of time. The period for which the tissue structure forming device 10 is left in place is set in advance based on tests for forming the tissue structure 20, etc.

[0024] When the tissue structure forming device 10 is placed in an environment where biological tissue is present, cells, plasma proteins, and the like derived from the biological tissue enter the hollow portion 11 separated by the partition wall 12 from the environment through the through-holes 13 in the partition wall 12. The cells, etc. derived from the biological tissue that have entered the hollow portion 11 grow 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. Furthermore, the cells, etc. derived from the biological tissue that have entered the hollow portion 11 grow 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 comprises fibrous connective tissue 21 and loose fibrous tissue 22. The membranous fibrous connective tissue 21 has a first surface 21A that is detached from the inner surface 14 of the compartment wall 12. The membranous loose fibrous tissue 22 is formed over the entire second surface 21B of the fibrous connective tissue 21, opposite the first surface 21A. When the tissue structure forming device 10 is left in place for a predetermined period of time, the membranous fibrous connective tissue 21 is formed in the hollow portion 11, and also the membranous loose fibrous tissue 22 supported by the fibrous connective tissue 21 is formed. A cell population containing 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 discovered by the present inventors.

[0026] The environment containing biological tissue may be an in vivo environment containing biological tissue. The in vivo environment containing biological tissue may be the inside of a disease model animal. The environment containing biological tissue may be an in vitro culture system containing biological tissue removed from a living organism. The environment containing biological tissue may be an artificial environment constructed outside the body to mimic biological tissue in a living organism. The biological tissue may be ectodermal tissue, mesodermal tissue, or endodermal tissue. The biological tissue may be tissue in which pluripotent stem cells 23 exist in a healthy state. The biological tissue may be tissue in which pluripotent stem cells 23 required for tissue repair exist, such as in an injured or defective area. The biological tissue may be subcutaneous tissue containing fibroblasts. The biological tissue may be from a human or any non-human animal that can be a source of pluripotent stem cells 23. The in vivo environment containing 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 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 biological tissue may be subcutaneously located 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 the living tissue having a decreased function. That is, 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 the 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 living tissue and then administered to living tissue, etc. The tissue structure 20, the cell population containing the pluripotent stem cells 23, or the pluripotent stem cells 23 may be cryopreserved and then administered at a desired time. The pluripotent stem cells 23 may also be isolated from the loose fibrous tissue 22. The pluripotent stem cells 23 may be isolated by enzymatic treatment of the tissue structure forming device 10 removed from the environment containing the living tissue. The pluripotent stem cells 23 may also be isolated by filtration using a membrane filter, mesh filter, or the like. The pluripotent stem cells 23 isolated from the loose fibrous tissue 22 may or may not be cultured and expanded. The cell population containing the pluripotent stem cells 23 partially purified from the loose fibrous tissue 22 may or may not be cultured and expanded.

[0029] The tissue construct 20, a cell population containing 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 tissue or organ function. The tissue construct 20, a cell population containing 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 construct 20, a cell population containing 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 compartment wall 12 may be cylindrical 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 shape. Since the spatial size of biological tissue is limited, when an expanded accumulation range of pluripotent stem cells 23 is required, the shape of the compartment wall 12 is preferably cylindrical or flat rectangular, in which the membranous fibrous connective tissue 21 spreads three-dimensionally, rather than a flat plate. Furthermore, when a stable position of the compartment wall 12 is required in an environment containing biological tissue, the shape of the compartment wall 12 is preferably a flat elliptical cylinder, rather than 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 vents. The air vents are used to reduce the pressure in the hollow portion 11 that is placed in an environment containing biological tissue. If the shape of the partition wall 12 needs to be stable in an environment containing biological tissue, it is preferable that the cylindrical end of the partition wall 12 is supported by the cap. If the thickness of the membranous fibrous connective tissue 21 needs to be uniform, it is preferable that the air vents in the cap function as through-holes 13 near 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 also 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 to promote 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 construct 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 resin, polyfluoroethylene resin, polypropylene resin, polyethylene resin, silicone resin, and mixed resins of these with other resin components.

[0036] The through holes 13 are circular holes that penetrate 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 arranged at equal intervals in both the circumferential direction of the partition wall 12 and 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 arranged regularly or randomly on the inner surface 14 and outer surface of the partition wall 12. When the thickness of the membranous fibrous connective tissue 21 needs to be uniform within a predetermined range, or when the distribution of the pluripotent stem cells 23 needs to be uniform within a predetermined range, it is preferable that the openings of the through-holes 13 be arranged regularly 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 will proceed with tissue formation as follows, but tissue formation by the tissue structure forming device 10 is not limited to this theory.

[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 that borders 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 other substances 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, which is denser than the loose fibrous tissue 22, from the opening edge of the through-hole 13 toward the inside of the hollow portion 11, starting from the inner surface 14.

[0043] The inside of the hollow portion 11, which is more likely to be recognized as a foreign body than the outside of the hollow portion 11, further promotes the invasion of fibroblasts and pluripotent stem cells 23. 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 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 likely to be recognized as a foreign body than the outside of the hollow portion 11, continues to accumulate pluripotent stem cells 23 in the loose 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 loose fibrous tissue 22 proliferate within the loose fibrous tissue 22. As a result, cells derived from biological tissues that have entered the hollow portion 11 grow the membranous loose fibrous tissue 22 further inward than the membranous fibrous connective tissue 21, and accumulate many pluripotent stem cells 23 in the loose 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 the hollow portion 11. The tissue structure forming device 10, which is left 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 over a predetermined retention period.

[0047] [Configuration of partition wall 12] As shown in Figure 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 that suppresses deformation of the partition wall 12 in the biological tissue. The thickness T12 of the partition wall 12 has a size that is approximately uniform throughout the range in which the through-holes 13 are arranged. The thickness T12 of the partition wall 12 has a size that allows cells and the like in the biological tissue to enter the hollow portion 11.

[0048] The opening dimension WT of the through-hole 13 is the dimension of the opening of the through-hole 13 on the inner surface 14. The opening dimension WT of the through-hole 13 is large enough to allow cells in biological tissue to pass through the through-hole 13. The minimum opening dimension WT that allows cells in biological tissue to pass through is approximately 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 plurality of through holes 13 in one partition wall 12 may have the same opening dimension WT. The plurality of through holes 13 in one partition wall 12 may have different opening dimensions WT. The plurality of 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 one another as a group. The plurality of through holes 13 in one partition wall 12 having different opening dimensions WT may be arranged so that the opening dimensions WT are alternately different in the direction in which the through holes 13 are arranged.

[0050] When the tissue structure forming device 10 is placed in an environment where biological tissue is present, the inner surface 14 of the compartment wall 12 has a size that allows it to function as a scaffold for cells derived from the biological tissue. The distance between holes on the inner surface 14 is the shortest distance on the inner surface 14 between adjacent through-holes 13. The distance between holes 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 inter-hole distance. The gaps between adjacent through holes 13 in one partition wall 12 may have different inter-hole distances. A plurality of through holes 13 having the same inter-hole distance in one partition wall 12 may be arranged as a single group in the partition wall 12. A plurality of through holes 13 having different inter-hole distances in one partition wall 12 may be arranged so that the inter-hole distances are alternately different in the direction in which the through holes 13 are arranged.

[0052] The density of the openings on the inner surface 14 of the partition wall 12 is large enough to prevent the entry of cells derived from biological tissues from being 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 to allow the fibrous connective tissue 21 to form 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 growth of the fibrous connective tissue 21, the invasion of fibroblasts and pluripotent stem cells 23 is promoted, and the formation of the loose 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 one 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 in the case of a cylindrical inner surface 14, the hollow depth D11 is half the distance between one 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-to-hole distance within one partition wall 12 may be a combination of different values ​​within ranges that satisfy conditions 2 and 3. For example, one partition wall 12 may have a through hole 13 with an opening dimension WT of 0.5 mm and a through hole 13 with an opening dimension WT of 1.5 mm. For example, one partition wall 12 may have a plurality of through holes 13 with an inter-hole distance of 0.5 mm and a plurality of through holes 13 with an inter-hole distance of 1.5 mm. For example, one partition wall 12 may have a plurality of through holes 13 with an inter-hole distance of 0.5 mm and an opening dimension WT of 0.5 mm, and a plurality of through holes 13 with an inter-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 and a hole spacing of 1.5 mm, and a plurality of through holes 13 having an opening dimension WT of 1.5 mm and a hole spacing 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 and a hole spacing of 0.3 mm, and further include through holes 13 as a plurality of elliptical holes having an opening dimension WT of 1.5 mm and a hole spacing of 5.0 mm.

[0057] (Condition 1) The thickness T12 of the partition wall 12 is 0.1 mm or more and 2.0 mm or less. (Condition 2) The opening dimension WT of the through-hole 13 is 0.3 mm or more and 3.0 mm or less. (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 2 mm or more and 10 mm or less.

[0058] In the tissue structure forming device 10, collagen production may occur competitively between the outer surfaces of the compartment walls 12 and within the hollow portion 11. Even if the opening occupancy rates are equal, if the number of openings is small and the openings are large, the outer surfaces of the compartment walls 12 are likely to be recognized as foreign bodies due to excessively large inter-pore distances, and the outer surfaces of the compartment walls 12 are 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, slowing the formation of fibrous connective tissue 21 and 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 the partition wall 12, the openings of the through-holes 13 are less likely to be covered with collagen since the distance between the holes is small. Also, on the inner surface 14 of the partition wall 12, there are secured a sufficient number of starting points for the formation of the fibrous connective tissue 21 to form the fibrous connective tissue 21 in a membrane-like form.

[0060] The accumulation of pluripotent stem cells 23 is achieved by the pluripotent stem cells 23 in the biological tissue invading the hollow portion 11 and the proliferation of the invaded pluripotent stem cells 23 in the hollow portion 11. The invasion of pluripotent stem cells 23 is promoted by the through-holes 13 remaining open for most of the predetermined retention period and by the continued existence of spaces in the hollow portion 11 for the formation of loose fibrous tissue 22. When conditions 1 to 5 are satisfied, the growth of loose fibrous tissue 22 can be promoted more than the growth of fibrous connective tissue 21, and the invasion of fibroblasts and pluripotent stem cells 23 can be promoted to an extent that large depressions or holes opposite the openings of the through-holes 13 are not formed in the fibrous connective tissue 21. Furthermore, membrane-like fibrous connective tissue 21 is easily formed, and as the growth of the fibrous connective tissue 21 is promoted, the pluripotent stem cells 23 are more likely to accumulate 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 tends 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 unlikely to be 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 form 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 biological tissue.

[0064] When the inter-hole distance is 0.3 mm or more, the fibrous connective tissue 21 tends 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 around the opening of one through-hole 13 tends to connect with the fibrous connective tissue 21 growing from around the opening of another through-hole 13. Connecting the fibrous connective tissues 21 around the openings of the through-holes 13 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 tends to spread in a membrane-like manner over the entire inner surface 14.

[0065] The placement period of the tissue structure forming device 10 is preferably a short period of time, about four weeks from the start of placement, during which inflammation caused by the incision required for placement of the tissue structure forming device 10 has subsided. On the other hand, when the environment containing the biological tissue is the inside of a disease model animal, the growth of the fibrous connective tissue 21 tends 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 form in the fibrous connective tissue 21 at the site facing the through-hole 13 after the placement period has elapsed. Furthermore, when the opening occupancy rate is 30% or more and 70% or less, the fibrous connective tissue 21 tends 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 easier to obtain loose fibrous tissue 22 of sufficient thickness and pluripotent stem cells 23 that accumulate in the loose fibrous tissue 22. In particular, when the environment containing the biological tissue is the same as 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 of accumulating pluripotent stem cells 23.

[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. For example, 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. Support of the partition wall 12 by the support member 15 stabilizes the shape of the hollow portion 11 within the biological tissue.

[0068] The support member 15 has a flattened elliptical cylindrical shape so that the outer surface of the support member 15 fits snugly against the inner surface 14 of the partition wall 12. If the inner surface 14 of the partition wall 12 has a cylindrical surface, the support member 15 may have a cylindrical shape. If 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 has a predetermined hollow depth D11 between the inner surface 14 of the partition wall 12 and the outer surface of the support member 15. If the partition wall 12 has a strength that makes it difficult for it 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 tissues. The constituent material of the support member 15 may be the same as or 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, or 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 loose fibrous tissue 22 and contains fewer cells than the loose 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 proportion 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 proportion of type I collagen in the total collagen contained in the fibrous connective tissue 21 is higher than the proportion of type I collagen in the total collagen contained in the loose 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 membrane-like fibrous connective tissue 21 is ensured, making it easy to handle the tissue structure 20 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 greater than the thickness of the fibrous connective tissue 21. This makes it easier to ensure a space within the hollow portion 11 for accumulating the pluripotent stem cells 23. When improved accuracy in accumulating the pluripotent stem cells 23 is required, 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 protrusions 21T corresponding to the shape of the through-holes 13 at locations corresponding to the through-holes 13. The thickness HT of the protrusions 21T is equal to or less than the depth of the through-holes 13. The fibrous connective tissue 21 may omit the protrusions 21T. The fibrous connective tissue 21 may have capillaries 24 extending to the sparse fibrous tissue 22 at locations corresponding to the through-holes 13. The capillaries 24 are newly generated in the hollow portion 11 through the through-holes 13 during the formation of the tissue structure 20.

[0073] The loose fibrous tissue 22 has a lower fiber density than the fibrous connective tissue 21, and more cells are dispersed 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, fibrins, and pluripotent stem cells 23 such as mesenchymal cells. The loose fibrous tissue 22 may contain type III collagen or may contain fibrous collagen other than type III collagen. The type III collagen content in the loose fibrous tissue 22 is higher than the type III collagen content in the fibrous connective tissue 21.

[0074] The pluripotent stem cells 23 contained in the sparse fibrous tissue 22 express pluripotent stem cell markers. The pluripotent stem cells 23 may express at least one pluripotent stem cell marker or 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] Pluripotent stem cells 23 contained in sparse fibrous tissue 22 may express both the pluripotent stem cell marker SSEA3 and the pluripotent stem cell marker SSEA4. Pluripotent stem cells 23 contained in 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 with high angiogenic potential. The stem cells with high angiogenic potential in the sparse fibrous tissue 22 may be 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 by treating the sparse fibrous tissue 22 in a 0.25% collagenase type I solution at 37°C for 1.5 hours and then calculating the number of cells recovered from the decomposed tissue as the total cell number. The stem cell ratio can be calculated by calculating the number of cells expressing at least one marker selected from the group consisting of the mesenchymal stem cell marker CD90, the mesenchymal stem cell marker CD105, the pluripotent stem cell marker SSEA3, and the 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 by counting the number of cells expressing the mesenchymal stem cell marker CD90 or the mesenchymal stem cell marker CD105. The mesenchymal stem cell ratio can be calculated by counting 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. 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, which is a disease model animal, or an elderly 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 implanted subcutaneously in the back of the pig, and the pig's back was sutured. After the tissue structure forming device 10 was left in place for a predetermined period of time, the pig was anesthetized and the tissue structure forming device 10 was removed from the subcutaneous tissue. 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 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 Figure 7, a membrane-like tissue structure 20 was peeled off from the inner surface 14 of the partition wall 12, which was an elliptical cylindrical surface. Then, the tissue structure 20 was 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] [Staining evaluation] Using test examples of tissue structure forming devices 10 with different hollow depths D11 and different retention periods, paraffin-embedded blocks of the tissue structure 20 were prepared from the center and edges of the membranous tissue structure 20 in the planar direction. Furthermore, sections were prepared by cutting the paraffin-embedded blocks of the fixed tissue structure 20 with a microtome so that the thickness of the sections in the transverse direction was 3 μm to 5 μm. 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 Figure 9, deep staining by HE staining was observed throughout the entire surface of the first surface 21A from approximately 0.1 mm from the first surface 21A in the thickness direction of the tissue structure 20. Furthermore, as shown in Figure 10, deep staining by MT staining was also observed from approximately 0.1 mm from the first surface 21A. This confirmed that fibrous connective tissue 21, which is a dense tissue, was formed from the first surface 21A to approximately 0.1 mm in the thickness direction of the tissue structure 20. As shown by the light-colored areas in Figure 11, yellow and orange colors from SR staining were observed from approximately 0.1 mm from the first surface 21A under a polarizing microscope. This also confirmed that the fibrous connective tissue 21 contained a large amount of type I collagen.

[0086] As shown in Figure 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 approximately 2 mm from the fibrous connective tissue 21. Furthermore, as shown in Figure 13, light staining was also observed in MT staining from approximately 2 mm from the fibrous connective tissue 21. This confirmed that loose fibrous tissue 22 was formed inside the fibrous connective tissue 21. As shown in the light-colored area in Figure 14, a green color due to SR staining was observed from observation under a polarizing microscope from approximately 2 mm from the fibrous connective tissue 21. This also confirmed that loose fibrous tissue 22 contained type III collagen.

[0087] Furthermore, the black color due to EVG staining was not observed throughout the tissue structure 20. This indicates that the tissue structure 20 contains 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 Figure 15, in the detection results for the mesenchymal stem cell marker CD90, no staining was observed in the fibrous connective tissue 21. Furthermore, as shown in Figure 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 Figure 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. Furthermore, as shown in Figure 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 Figure 19, in the detection results for the pluripotent stem cell marker SSEA3, no staining was observed in the fibrous connective tissue 21. Furthermore, as shown in Figure 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 Figure 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. Furthermore, as shown in Figure 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 within 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 within the sparse fibrous tissue 22 on the opposite side to the fibrous connective tissue 21.

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

[0094] Double staining revealed double-positive cells as well as cells that were single-positive for each marker. It was confirmed that the cells that make up loose fibrous tissue 22 are a hierarchical cell population with varying degrees of undifferentiation. Furthermore, 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. Furthermore, VEGF was expressed particularly highly in SSEA3-positive cells, suggesting that the highly undifferentiated stem cells present in loose fibrous tissue 22 possess a high angiogenic potential.

[0095] [Thickness evaluation] Using test examples of tissue structure forming devices 10 with different hollow depths D11 and different 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 Figure 8. In Figure 8, the thickness of fibrous connective tissue 21 using tissue structure forming device 10 with hollow depth D11 of 1 mm is shown by an open triangle, and the thickness of tissue structure 20 is shown by an open circle. In Figure 8, the thickness of fibrous connective tissue 21 using tissue structure forming device 10 with hollow depth D11 of 2 mm is shown by an open square, and the thickness of tissue structure 20 is shown by an open circle.

[0097] As shown by the open circles in Figure 8, when the hollow depth D11 is 1 mm, the thickness of the tissue structure 20 grows to 0.6 mm, which is approximately two-thirds of the hollow depth D11, after two weeks of retention. As shown by the open triangles in Figure 8, the thickness of the fibrous connective tissue 21 grows to approximately one-third 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 one-third of the hollow depth D11 and thin sparse fibrous tissue 22 with a thickness of approximately one-third 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 cover the entire hollow depth D11. The thickness of the fibrous connective tissue 21 also grows to cover the entire hollow depth D11. In other words, after a retention period of four weeks has passed, the tissue structure 20 does not contain loose 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 containing the accumulated pluripotent stem cells 23 can be obtained by detention for two weeks. However, this detention period is slightly shorter than the preferred detention period 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, at 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 black circles in Figure 8, when the hollow depth D11 is 2 mm, the thickness of tissue structure 20 grows to 1.1 mm, which is approximately half of the hollow depth D11, after two weeks of retention. On the other hand, as shown by the black squares in Figure 8, the thickness of fibrous connective tissue 21 grows to approximately one-eighth of the hollow depth D11 after two weeks of retention. In other words, after two weeks of retention, tissue structure 20 has fibrous connective tissue 21 with a thickness of half the hollow depth D11 and loose fibrous tissue 22 with a thickness of 1.0 mm or more.

[0101] Furthermore, after a retention period of four weeks or more, the thickness of the tissue structure 20 grows to approximately 9 / 10 of the hollow depth D11. Meanwhile, the thickness of the fibrous connective tissue 21 remains approximately 1 / 8 of the hollow depth D11, the same as after a retention period of two weeks. Thus, the tendency for the thickness of the tissue structure 20 to cover almost the entire hollow depth D11 while the thickness of the fibrous connective tissue 21 to remain at or below approximately 1 / 4 of the hollow depth D11 is observed until a retention period of eight weeks has elapsed. Then, after a retention period of 11 weeks has elapsed, the tissue structure 20 does not contain loose 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) a membranous loose fibrous tissue 22 containing accumulated pluripotent stem cells 23 can be obtained within a suitable retention period that allows inflammation due to retention to subside. Furthermore, when the hollow depth D11 is 2 mm, (b) the period during which the loose fibrous tissue 22 can be obtained is also long, such as two weeks or more. Furthermore, because the thickness of the loose fibrous tissue 22 is 1.0 mm or greater, (c) many pluripotent stem cells 23 can easily be uniformly accumulated in the membranous loose 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 approximately 0.2 mm, just as when 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 approximately 0.2 mm, just as when the hollow depth D11 is 2 mm. Then, over 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 approximately 0.2 mm.

[0104] That is, even when the hollow depth D11 is 4 mm, a membranous loose fibrous tissue 22 containing accumulated pluripotent stem cells 23 can be obtained within a suitable retention period that allows inflammation due to retention to subside. The period during which the loose fibrous tissue 22 can be obtained is also long, such as two weeks or more. Furthermore, because the thickness of the loose fibrous tissue 22 is 1.0 mm or greater, many pluripotent stem cells 23 are likely to accumulate uniformly in the membranous loose fibrous tissue 22.

[0105] Thus, in forming the loose fibrous tissue 22, setting the hollow depth D11 to 2 mm or more requires the new technical knowledge that (i) there is a difference in growth rate between the fibrous connective tissue 21 and the loose fibrous tissue 22. Setting the hollow depth D11 to 2 mm or more also requires the new technical knowledge that (ii) the correlation between the difference in growth rate and the hollow depth D11 changes once the hollow depth D11 exceeds a certain value. Setting the hollow depth D11 to 2 mm or more can only be derived from the perspective of determining the hollow depth D11 so as to promote the growth of the loose fibrous tissue 22 over the growth of the fibrous connective tissue 21. The trends shown in the above findings (i) and (ii) were more clearly observed in the partition walls 12 that satisfied conditions 1 to 4.

[0106] Thus, setting the hollow depth D11 to 2 mm or more has the effect of obtaining, after a suitable retention period that allows inflammation due to placement to subside, a membranous loose fibrous tissue 22 supported by membranous fibrous connective tissue 21. Setting the hollow depth D11 to 2 mm or more also has the effect of obtaining, after a suitable retention period that allows inflammation due to placement to subside, a large number of pluripotent stem cells 23 uniformly accumulated in the membranous loose fibrous tissue 22. Setting the hollow depth D11 to 2 mm or more also has the unique and distinctive effect of obtaining, for the first time, a membranous loose fibrous tissue 22 in which a large number of pluripotent stem cells 23 uniformly accumulated, with ample time to spare, during a suitable retention period that allows inflammation due to placement 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 fibrous connective tissue 21 using the inner surface 14 as a scaffold. As the fibrous connective tissue 21 grows, pluripotent stem cells 23 in the biological tissue are accumulated in membranous loose fibrous tissue 22 on the other side of the fibrous connective tissue 21. Because the accumulation site of pluripotent stem cells 23 spreads over the entire other side of the membranous fibrous connective tissue 21, it is easy to administer pluripotent stem cells 23 to the diseased area by, for example, attaching a membranous tissue structure 20.

[0108] (2) When fibrous connective tissue 21 peeled off 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, a membranous loose fibrous tissue 22 containing accumulated pluripotent stem cells 23 can be obtained within a suitable retention period that allows inflammation due to retention to subside. Furthermore, when the hollow depth D11 is 2 mm, the period during which the loose fibrous tissue 22 can be obtained is long, such as 2 weeks or more. Furthermore, since the thickness of the loose fibrous tissue 22 is 1.0 mm or more, many pluripotent stem cells 23 are likely to accumulate uniformly in the membranous loose 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 construct 20 when administering the pluripotent stem cells 23. Furthermore, because the depth of the hollow portion 11 is sufficiently greater than the thickness of the fibrous connective tissue 21, it is easy to ensure a space within the hollow portion 11 for accumulating the pluripotent stem cells 23.

[0111] (5) When the opening dimension WT is 0.3 mm or more, cells derived from biological tissue, plasma proteins, and the like can 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 locations 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% to 70%, the fibrous connective tissue 21 can easily grow using the inner surface 14 as a scaffold. Therefore, an environment suitable for cells derived from biological tissue can easily be established in the hollow portion 11 through the capillaries 24 at the locations 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 a 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 tissue that enter the hollow portion 11 from the end of the tube. Fibrous connective tissue 21 is formed from the inner surface 14 toward the hollow portion 11, and also 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 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 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 open and a 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 fibrous connective tissue 21 in contact with the outer surface of the support member 15 and loose fibrous tissue 22 in contact with the fibrous connective tissue 21, via intra-layer peeling of the loose fibrous tissue 22 in the three-layer structure. The tissue structure 20 may also be processed into a two-layer structure of fibrous connective tissue 21 in contact with the inner surface 14 and loose fibrous tissue 22 in contact with the fibrous connective tissue 21, via intra-layer peeling of the loose fibrous tissue 22 in the three-layer structure.

[0115] As shown in Fig. 23, the cylindrical partition wall 12 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] As shown in Figure 24, the cylindrical partition wall 12 may be provided with a protruding rib that extends in the axial direction and turns 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 so as to partially block the through holes 13. When uniformity in the thickness of the tissue structure 20 is required, it is preferable that the reinforcing rib be 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 compartment wall placed in an environment containing biological tissue, The partition wall has an inner surface surrounding the hollow portion and a plurality of through holes opening into the inner surface, cells in the biological tissue invade the hollow portion through the through-holes, thereby forming the tissue structure in the hollow portion; 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: a membrane-like fibrous connective tissue having one side peeled from the inner surface; and a membrane-like loose fibrous tissue that extends over the entire other side opposite to the one side and accumulates the pluripotent stem cells. 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 open in the axial direction of the elliptical cylindrical surface. The tissue structure of claim 1 .

3. 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 tissue structure of claim 1 .

4. 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, The one side has capillaries at positions corresponding to the through holes. The tissue structure of claim 3 .

5. 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 in which the compartment wall is placed in an environment containing biological tissue, and cells in the biological tissue invade the hollow portion through the through-holes to form a tissue structure in the hollow portion, The tissue structure comprises: a membrane-like fibrous connective tissue having one side in contact with the inner surface; a membranous loose fibrous tissue that extends 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; 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, which is the 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. Tissue structure forming device.

6. A method for forming a tissue structure, comprising: forming a tissue structure on a compartment wall by placing the compartment wall in an environment containing biological tissue, the method comprising: The partition wall has 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 so that cells in the biological tissue enter the hollow portion through the through-holes; placing the compartment 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 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. A method for forming a tissue structure comprising:

7. 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. The method for forming the tissue structure according to claim 6 .

8. The depth of the hollow portion is 2 mm or more and 10 mm or less, Leaving the partition wall in place 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. The method for forming the tissue structure according to claim 6 .

9. the partition wall is a hollow tubular member, The hollow portion is separated only by the partition wall. A method for forming the tissue structure according to any one of claims 6 to 8.

10. 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. The method for forming the tissue structure according to claim 6 .

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