Adult stem cell aggregate tissue structure and its manufacturing equipment

Abstract

The present invention relates to tissue structures, instruments for manufacturing the same, and methods for collecting adult stem cells from tissue structures having a core and a sparse fibrous aggregate of adult stem cells, wherein the core has a recess and is composed of fibrous connective tissue, and the sparse fibrous aggregate of adult stem cells is formed in the recess and contains type III collagen and adult stem cells.

Description

Technical Field

[0001] This invention relates to an adult stem cell-accumulated tissue construct (somatic stem cell-accumulated tissue construct) and the apparatus for manufacturing it. Background Technology

[0002] The self-defense response to eliminate foreign substances causes macrophages and other cells to gather around the foreign substance that has invaded the organism. Macrophages adhere to the surface of the foreign substance and break it down, while simultaneously producing TGF-β through monocytes, which in turn stimulates fibroblasts to produce collagen. As a result, the foreign substance that has invaded the organism is encapsulated by connective tissue containing fibroblasts and collagen, thus isolating it within the organism (encapsulation). As a technique for forming tissue from a biological source using this phenomenon, techniques for forming connective tissue by embedding a substrate that serves as a foreign substance within the organism have been reported (Patent Documents 1-6, etc.). For example, Patent Document 5 discloses embedding a spiral-shaped peripheral component along the periphery of a rod-shaped structural component within the organism, thereby creating a connective tissue body formed on the surface of the rod-shaped structural component and encapsulating the peripheral component. Patent Document 6 discloses embedding a component with a scaffold embedded in the periphery of a rod-shaped body into the organism, thereby forming a connective tissue body around the periphery of the component, removing it from the organism, pulling out the rod-shaped body, and thus obtaining a scaffold encapsulated by the connective tissue body. However, the tissue formed on the substrate surface through encapsulation is generally only tens to hundreds of micrometers thick, and the cells contained therein are almost all fibroblasts.

[0003] In recent years, regenerative medicine, which involves transplanting cultured cells or tissues created in vitro into living organisms to regenerate the function of tissues or organs damaged by disease or injury, has attracted considerable attention. Stem cells are crucial materials for generating target cells or constructing tissues in regenerative medicine. On the other hand, the ethical implications of embryonic stem cells and the establishment of safe artificial pluripotent stem cells have not yet been established for use in regenerative medicine. Therefore, there is a need for technologies that efficiently collect adult stem cells with higher safety and fewer ethical concerns.

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: Japanese Patent Application Publication No. 2007-312821

[0007] Patent Document 2: Japanese Patent Application Publication No. 2008-237896

[0008] Patent Document 3: Japanese Patent Application Publication No. 2010-094476

[0009] Patent Document 4: Japanese Patent Application Publication No. 2012-105860

[0010] Patent Document 5: International Publication No. WO2016 / 076416

[0011] Patent Document 6: Japanese Patent Application Publication No. 2006-255288 Summary of the Invention

[0012] The problem that the invention aims to solve

[0013] The subject of this invention is to provide a technology for efficiently collecting adult stem cells.

[0014] Technical solutions for solving the problem

[0015] The inventors conducted in-depth research to solve the above-mentioned problems, and as a result, they discovered that it was possible to create tissue structures from the aggregation of adult stem cells, thus completing this invention.

[0016] That is, the present invention includes the following contents.

[0017] [1] A tissue structure having a core and a sparsely fibrous adult stem cell aggregate, wherein the core has a recess and is composed of fibrous connective tissue, and the sparsely fibrous adult stem cell aggregate is formed in the recess and contains type III collagen and adult stem cells.

[0018] [2] According to the tissue structure described in [1] above, the core portion has one or more of the recesses.

[0019] [3] According to the tissue structure described in [1] or [2] above, wherein the core portion has at least 3 of the recesses.

[0020] [4] The tissue structure according to any one of [1] to [3] above, wherein the recess has an opening width of at least 2.5 mm.

[0021] [5] The tissue structure according to any one of [1] to [4] above, wherein the core portion has a diameter of at least 2.5 mm.

[0022] [6] The tissue structure according to any one of [1] to [5] above, wherein the adult stem cells include at least one of mesenchymal stem cells and pluripotent stem cells.

[0023] [7] The tissue structure according to any one of [1] to [6] above, wherein the tissue structure is rod-shaped and more than one of the recesses is arranged circumferentially.

[0024] [8] The tissue structure according to any one of [1] to [7] above, wherein the tissue structure is rod-shaped and more than one of the recesses is arranged axially.

[0025] [9] The tissue structure according to any one of [1] to [6] above, wherein the shape of the tissue structure is approximately polyhedral and more than one of the recesses is located on different faces.

[0026]

[10] The tissue structure according to any one of [1] to [9] above, wherein the shape of the tissue structure is neither tubular nor sheet-like.

[0027]

[11] A tissue structure, which is a tissue structure according to any one of [1] to

[10] above, wherein the tissue structure is formed by placing a tissue structure manufacturing instrument having a hollow portion in an environment in which biological tissue material is present.

[0028] The tissue structure manufacturing apparatus has a frame that forms the hollow portion.

[0029] The frame has an opening that connects the hollow portion to the external space of the tissue structure manufacturing instrument.

[0030] The frame defines the shape of the tissue structure formed in the hollow portion.

[0031]

[12] According to the tissue structure described in

[11] above, wherein the tissue structure is in a state of filling the hollow part in the tissue structure manufacturing device, and the core part is in close contact with the surface of the frame.

[0032]

[13] A method for collecting adult stem cells, the method comprising: separating an aggregate of adult stem cells or adult stem cells from the tissue structure described in any one of [1] to

[12] above.

[0033]

[14] A tissue structure manufacturing apparatus, the tissue structure manufacturing apparatus comprising:

[0034] Hollow portion, and frame forming said hollow portion,

[0035] The frame has an opening that connects the hollow portion to the external space of the tissue structure manufacturing instrument.

[0036] The tissue structure has a core and a sparsely fibrous aggregate of adult stem cells. The core has a recess and is composed of fibrous connective tissue. The sparsely fibrous aggregate of adult stem cells is formed in the recess and contains type III collagen and adult stem cells.

[0037]

[15] The tissue structure manufacturing device according to

[14] above is used to form a tissue structure having a core and sparsely fibrous adult stem cell aggregate by placing it in an environment in which biological tissue material is present, wherein the core has a recess and is composed of fibrous connective tissue, and the sparsely fibrous adult stem cell aggregate is formed in the recess and contains type III collagen and adult stem cells.

[0038] The tissue structure manufacturing instrument has a hollow portion and a frame forming the hollow portion.

[0039] The frame has an opening that connects the hollow portion to the external space of the tissue structure manufacturing instrument.

[0040] The frame defines the shape of the tissue structure formed in the hollow portion.

[0041] When the tissue structure manufacturing instrument is placed in an environment in which biological tissue material is present, it forms fibrous connective tissue in a manner that extends from the surface of the frame into the interior of the hollow portion, thereby generating a core portion having a recessed portion that extends from the opening into the hollow portion, and forming sparse fibrous tissue in the recessed portion in which adult stem cells are aggregated.

[0042]

[16] The tissue structure manufacturing instrument according to

[14] or

[15] above, wherein the opening has an opening width of at least 2.5 mm.

[0043]

[17] The tissue structure manufacturing device according to any one of

[14] to

[16] above, wherein the opening has an opening shape capable of incorporating a circle with a diameter of 2.5 mm or more.

[0044]

[18] The tissue structure manufacturing device according to any one of

[14] to

[17] above, wherein the frame is composed of a columnar component and a support portion, the support portion fixing the columnar component to maintain the shape of the frame.

[0045]

[19] The tissue structure manufacturing device according to

[18] above, wherein more than one columnar member is fixed at both ends by a support and is circumferentially arranged on a virtual cylindrical surface between the supports.

[0046]

[20] The tissue structure manufacturing device according to

[18] or

[19] above, wherein the opening is formed by a columnar component and a support, or by more than one columnar component.

[0047]

[21] The tissue structure manufacturing device according to any one of

[14] to

[20] above, wherein the opening has a polygonal, rectangular, trapezoidal, spherical, circular or elliptical opening edge.

[0048]

[22] A tissue structure manufacturing device according to any one of

[18] to

[21] above, wherein the support portion further has an opening that connects the hollow portion to the external space of the device.

[0049]

[23] A method for manufacturing a tissue structure, the method comprising: placing a tissue structure manufacturing apparatus as described in any one of

[14] to

[22] above in an environment in which biological tissue material is present, for example in a living organism or in isolated biological tissue, the tissue structure having a core and a sparsely fibrous aggregate of adult stem cells, the core having a recess and being composed of fibrous connective tissue, the sparsely fibrous aggregate of adult stem cells being formed in the recess and containing type III collagen and adult stem cells.

[0050] This specification contains the disclosure of Japanese Patent Application No. 2020-103747, which forms the basis of the priority claim of this application.

[0051] Invention Effects

[0052] According to the present invention, adult stem cells can be collected efficiently. Brief description of the attached diagram

[0054] Figure 1 It is a three-dimensional diagram showing one embodiment of an apparatus for manufacturing tissue structures.

[0055] Figure 2 It is a cross-sectional view showing one embodiment of an apparatus for manufacturing tissue structures.

[0056] Figure 3 It is a schematic diagram illustrating the way adult stem cells are aggregated through tissue structure manufacturing instruments in the order of A, B, C, and D.

[0057] Figure 4 A to 4D are three-dimensional diagrams showing one embodiment of a tissue structure manufacturing apparatus.

[0058] Figure 5 A and Figure 5 B is a three-dimensional view showing one embodiment of a tissue structure manufacturing apparatus.

[0059] Figure 6 A to 6C are perspective views showing one embodiment of a tissue structure manufacturing apparatus.

[0060] Figure 7 It is a three-dimensional diagram showing the three-dimensional structure of the manufacturing instrument for the tissue structure in the modified example.

[0061] Figure 8 It is a three-dimensional diagram showing the three-dimensional structure of the manufacturing instrument for the tissue structure in the modified example.

[0062] Figure 9 It is a three-dimensional diagram showing the three-dimensional structure of the manufacturing instrument for the tissue structure in the modified example.

[0063] Figure 10 These are photographs showing the results of an experiment using tissue fabrication equipment to create tissue structures from aggregated adult stem cells. A shows the equipment used; B shows the equipment removed after being implanted in the organism, with tissue structures formed inside; C shows a cross-section of the resulting tissue structure.

[0064] Figure 11 These are photographs showing the results of staining experiments on tissue structures. A is HE staining; B is MT staining; C is EVG staining; D is SR staining.

[0065] Figure 12 These are polarized light microscope images of Sirius red-stained sections. A shows the area around the columnar components; B shows the center; and C shows the space between the columnar components.

[0066] Figure 13 These are photographs showing the results of immunohistochemical staining of tissue structures for the mesenchymal stem cell markers CD90 and CD105. The left column shows CD90 / DAPI; the right column shows CD105 / DAPI. A and B are pockets; C and D are around columnar components; E and F are the central portion.

[0067] Figure 14 These are photographs showing the results of immunohistochemical staining of tissue structures for the pluripotent stem cell markers SSEA4 and SSEA3. The left column shows SSEA4 / DAPI; the right column shows SSEA3 / DAPI. A and B are pocket regions; C and D are around columnar components; E and F are the central regions.

[0068] Figure 15 These are photographs showing the results of immunohistochemical staining of tissue structures for growth factors, namely vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). The left column shows VEGF / DAPI; the right column shows HGF / DAPI. A and B are pockets; C and D are around columnar components; E and F are the central parts.

[0069] Figure 16These are photographs showing the results of double staining of the stem cell marker SSEA3 and other markers in tissue structures. A represents CD90 / SSEA3 / DAPI; B represents CD105 / SSEA3 / DAPI; C represents SSEA3 / SSEA4 / DAPI; and D represents VEGF / SSEA3 / DAPI.

[0070] Figure 17 This is a graph representing the results of flow cytometry analysis.

[0071] Figure 18 This graph shows the increase in the proportion of CD90-positive cells as indicated by flow cytometry analysis of cell populations before and after cell separation.

[0072] Figure 19 This graph shows the increase in the proportion of SSEA3-positive cells as indicated by flow cytometry analysis of cell populations before and after cell separation.

[0073] Figure 20 These are photographs showing the cell morphology of the pre-isolation cell populations (control group, left column), CD90-positive cell population (middle column), and SSEA3-positive cell population (right column) at day 7 (top) and day 14 (bottom) from the start of culture. Cells isolated using the MACS method were cultured, and fibroblast-like cells adhered to and proliferated within the wells.

[0074] Figure 21 These are photographs and diagrams illustrating an experiment using scaffolds to create tissue structures.

[0075] Figure 22 It is a cross-sectional photograph of a tissue structure formed inside a tissue structure manufacturing instrument.

[0076] Figure 23 This is a graph that uses observed images to show the relationship between the number of adult stem cells and the placement period.

[0077] Figure 24 It means in Figure 4 A photograph of an HE-stained section of a tissue structure formed within a tissue structure manufacturing instrument.

[0078] Figure 25 It means in Figure 4 Photographs of HE-stained sections of tissue structures formed within a tissue structure manufacturing instrument of B.

[0079] Figure 26 It means in Figure 4 Photographs of HE-stained sections of tissue structures formed within a tissue structure manufacturing instrument of C.

[0080] Figure 27 It means in Figure 5 A photograph of an HE-stained section of a tissue structure formed within a tissue structure manufacturing instrument.

[0081] Figure 28 It means in Figure 5 Photographs of HE-stained sections of tissue structures formed within a tissue structure manufacturing instrument of B.

[0082] Figure 29 Is Figure 5 Enlarged HE-stained image of a section of tissue structure formed in the tissue structure manufacturing instrument of B, with a cross section along the axial direction Ax.

[0083] Figure 30 It means in Figure 6 Photographs of the appearance of the tissue structure formed in the tissue structure manufacturing instrument of C and the cross-sectional positions of its two sections.

[0084] Figure 31 It means in Figure 6 Photographs of HE-stained sections of tissue structures with different cross sections formed in the tissue structure manufacturing apparatus of C. Detailed Implementation

[0085] The present invention will now be described in detail.

[0086] This invention relates to a tissue structure manufacturing device capable of efficiently aggregating adult stem cells from biological tissues, and to a tissue structure rich in adult stem cells that can be fabricated using the device.

[0087] This invention relates, for example, to a tissue structure manufacturing apparatus, comprising:

[0088] Hollow portion, and frame forming said hollow portion,

[0089] The frame has an opening, preferably an opening that connects the hollow portion to the external space of the tissue structure manufacturing instrument.

[0090] The tissue structure has a core and a sparsely fibrous aggregate of adult stem cells. The core has a recess and is composed of fibrous connective tissue. The sparsely fibrous aggregate of adult stem cells is formed in the recess and contains type III collagen and adult stem cells. The invention also relates to tissue structures manufactured using such a tissue structure manufacturing apparatus.

[0091] According to the present invention, by placing the above-mentioned tissue structure manufacturing device in an environment where biological tissue material is present (e.g., in a living organism), cells can be aggregated within the tissue structure manufacturing device, and tissue structures containing aggregated adult stem cells can be induced to form within the tissue structure manufacturing device.

[0092] Cells or the like from the biological tissue material enter the hollow portion (opening to the external space of the device) of the tissue structure manufacturing instrument of the present invention from the opening, placed in an environment containing biological tissue material, and fibrous connective tissue is formed in the hollow portion. Directly below the opening of the tissue structure manufacturing instrument, a recess of fibrous connective tissue is formed, indenting from the opening into the hollow portion of the device. A core portion, having this recess and composed of fibrous connective tissue, forms from the device surface facing the hollow portion into the hollow portion of the device, and a large number of adult stem cells accumulate within this recess, forming an adult stem cell aggregate. By placing the tissue structure manufacturing instrument in an environment containing biological tissue material for a sufficient time to form tissue (including fibrous connective tissue and the adult stem cell aggregate within its recess) in the hollow portion of the tissue structure manufacturing instrument to fill the hollow portion, a tissue structure is formed within the device. Adult stem cells can be easily and efficiently recovered from the adult stem cell aggregate in the obtained tissue structure. This invention is based on the insight discovered by the inventors.

[0093] More specifically, the tissue structure manufacturing apparatus of the present invention has a frame (e.g., constituted by the frame) forming a hollow portion within the apparatus, the frame having an opening communicating from the hollow portion to the external space of the tissue structure manufacturing apparatus. The tissue structure manufacturing apparatus is preferably configured such that, when placed in an environment where biological tissue material is present (e.g., within a living organism), fibrous connective tissue is formed in a manner extending from the surface of the frame into the interior of the hollow portion, thereby generating a core portion having a recessed portion extending from the opening into the hollow portion, and sparse fibrous tissue containing aggregated adult stem cells is formed in the recessed portion. When a tissue structure is formed within the tissue structure manufacturing apparatus in a manner that fills the hollow portion formed by the frame, the frame defines the shape of the tissue structure formed within the hollow portion (overall shape, shape of the core portion, position and depth of the aggregated adult stem cell tissue / recess, etc.).

[0094] In one embodiment, the frame may include columnar components and supporting portions, for example, it may be composed of columnar components and supporting portions. The supporting portions fix the columnar components to maintain the shape of the frame and thus maintain the shape of the hollow portion. For example, more than one columnar component may be fixed by the supporting portions at both ends. More than one columnar component may also be fixed by the supporting portions at its middle portion. The frame preferably includes more than one columnar component, for example, it may include, but is not limited to, the following number of columnar components: 3 to 50, 4 to 40, 4 to 30, 5 to 30, 3 to 20, 3 to 15, 3 to 10, 4 to 15, or 5 to 15. The columnar components may have any shape, such as cylindrical, elliptical cylindrical, prismatic, frustum-shaped, twisted, or threaded. The supporting portions may have any shape, such as annular, disc-shaped, elliptical annular, elliptical disc-shaped, rectangular frame-shaped, or plate-shaped (e.g., polygonal, trapezoidal, or irregularly shaped plate-shaped). The aforementioned frame may have one, two, three, four, or more support portions. The frame may be a structure in which more than one columnar member is fixed to its two ends and, depending on the situation, its middle portion (e.g., one or more locations in the middle portion) by support portions connected in the extending direction of the columnar member. In this case, one support portion may fix, for example, but not limited to, the following number of columnar members: 3 to 20, 3 to 15, 3 to 10, 4 to 15, or 5 to 15.

[0095] In one embodiment, in the aforementioned frame, more than one columnar member can be fixed to its two ends and, depending on the situation, its middle portion by support portions, and is circumferentially arranged on a virtual cylindrical surface (e.g., a cylindrical surface, an elliptical cylindrical surface, or a prismatic cylindrical surface) between the support portions. In this case, the support portion can be annular, disc-shaped, elliptical annular, elliptical disc-shaped, rectangular frame-shaped, or plate-shaped (e.g., polygonal, trapezoidal, or irregularly shaped plate-shaped), but is not limited thereto. The more than one columnar member can be arranged at equal intervals or unequal intervals on the virtual cylindrical surface between the support portions. The aforementioned frame can include two or more structures connected in the extending direction of the columnar members, each structure including more than one columnar member fixed to its two ends and, depending on the situation, its middle portion by support portions and circumferentially arranged on the virtual cylindrical surface between the support portions. In this case, one support portion can be fixed with, for example, but not limited to, the following number of columnar members: 3 to 20, 3 to 15, 3 to 10, 4 to 15, or 5 to 15.

[0096] In another embodiment, the frame may include more than one columnar member, for example, it may be composed of more than one columnar member, and these columnar members are fixed to each other to maintain the shape of the frame.

[0097] The columnar components constituting the frame may have a diameter of at least 0.5 mm, 0.5 mm to 5 mm, for example 0.7 mm to 3 mm or 0.7 mm to 1.5 mm, but are not limited thereto.

[0098] The supporting part constituting the frame can have a diameter of 5mm to 30mm, for example 5mm to 25mm or 6mm to 20mm, but is not limited thereto.

[0099] In another embodiment, the frame may include wire-like components such as mesh or coil shapes, for example, it may be composed of wire-like components. The wire-like components may have a diameter of 0.5mm to 5mm, for example 0.7mm to 3mm or 0.7mm to 1.5mm, but are not limited thereto. The frame may include wire-like components and a support portion, for example, it may be composed of wire-like components and a support portion. The support portion fixes the wire-like components to maintain the shape of the frame.

[0100] In this invention, unless otherwise specified, "diameter" (length from one side to the other) is defined as the longest distance between two parallel lines connecting the two sides of a cross-section (generally a cross-section in the width direction) of a target component or region. It should be noted that the cross-section is not limited to a circle or ellipse, but can also be a polygon, trapezoid, or irregular shape, etc.

[0101] The length of the manufacturing instruments, frames and their constituent parts, namely columnar parts, in the longitudinal direction can be 5mm to 100mm, for example 15mm to 90mm, 20mm to 80mm, 15mm to 70mm or 20mm to 60mm, but is not limited to this.

[0102] The instruments, frames, and constituent parts of tissue structures, such as columnar components, supports, and linear components, preferably contain or are made of biocompatible materials. Examples of biocompatible materials include, but are not limited to: resins such as acrylic resin and silicone resin; ceramics; stainless steel; cobalt-chromium alloys; titanium; titanium alloys such as nickel-titanium; and metals such as platinum and gold. The biocompatible materials used herein preferably possess rigidity sufficient to prevent deformation within a living organism, at least during the fabrication of the tissue structure.

[0103] The aforementioned frame may have one, two, three, or four or more (e.g., four, five, six, seven, eight, nine, or ten or more) openings that connect the hollow portion of the instrument to the external space of the tissue structure manufacturing instrument and form a recessed portion of fibrous connective tissue directly below it. For example, it may have 1 to 50, 3 to 50, 4 to 40, 4 to 30, 5 to 30, 3 to 20, 3 to 15, 3 to 10, 4 to 15, or 5 to 15 openings. These openings are preferably formed by columnar members and support portions, or by more than one columnar member. Alternatively, these openings may also be formed by linear members, or by linear members and support portions.

[0104] The support portion of the aforementioned frame may also have an opening that connects the hollow portion inside the instrument to the external space of the instrument. For example, when the support portion is annular, elliptical, or rectangular, it may have such an opening.

[0105] The opening of the frame only needs to have a sufficient width to form a fibrous connective tissue / core, a recess of the fibrous connective tissue / core, and an aggregation of adult stem cells within the recess. In this invention, the "opening width" of the opening of the frame is defined as the width of the opening in the width direction. Preferably, the opening of the frame has an opening width of at least 2.5 mm, for example, it can have an opening width of 3.0 mm or more. In one embodiment, the opening can have an opening width of 2.5 mm to 28 mm, 2.5 mm to 25 mm, 2.5 mm to 20 mm, or 3.0 mm to 10 mm. The opening width of the opening of the frame is also referred to as the opening width W in this specification.

[0106] It should be noted that, in relation to this invention, the terms "length direction" and "width direction" are used for objects of any shape or having any shape. For example, in addition to rectangles or cuboids, they are also used for various shapes or objects including other polygons, circles, ellipses, cylinders, prisms, cones, pyramids, frustums, and frames having these shapes. In partial shapes or objects such as circles, squares, or cubes, the length in the "length direction" and the "width direction" may be the same.

[0107] The opening width of the frame can be determined by the opening edge on the component forming the opening (column component, support component, linear component, etc.), at a position defined on the same plane with the smallest opening area (e.g., refer to...). Figure 2(10F). When the frame is composed of more than one columnar member and a support, or more than one columnar member or linear member, the opening width of the aforementioned opening can also be calculated as the shortest distance between adjacent columnar members or linear members on the cross-section in the width direction of the opening, which includes the frame. In this case, the cross-section in the width direction of the frame can be a cross-section parallel to the support. When adjacent columnar members or linear members are arranged in parallel, the opening width of the aforementioned opening can also be calculated as the single shortest distance between the columnar members or linear members.

[0108] Alternatively, in addition to the above, the opening of the frame preferably has an opening shape (shape and size) capable of incorporating a circle with a diameter of 2.5 mm or more (e.g., 3.0 mm or more). The opening of the frame can, for example, have an opening shape capable of incorporating a circle with a diameter of 2.5 mm to 28 mm, 2.5 mm to 25 mm, 2.5 mm to 20 mm, or 3.0 mm to 10 mm. Frame openings with this opening shape are particularly suitable for recesses forming fibrous connective tissue and for adult stem cell aggregation tissue within such recesses. Here, regarding the opening of the frame, "an opening shape capable of incorporating a circle" means that the inner side of the opening shape (e.g., rectangular or polygonal) can connect and accommodate a circle with a specified diameter at more than one point. For example, in an opening with a rectangular shape having a long side of 20 mm and a short side of 2.5 mm, a circle with a maximum diameter of 2.5 mm can be incorporated. In this invention, the opening shape of the opening of the frame can be determined at the position where the opening area is the smallest, for example, it can be determined on the surface (e.g., a plane) that includes the opening edge.

[0109] In one embodiment, the opening of the frame can have an opening end of any shape, such as polygonal, rectangular, trapezoidal, spherical, circular, or elliptical.

[0110] Regarding at least one of the openings in the frame of the tissue structure manufacturing instrument according to the present invention, the opposing edges of each opening may have different opening widths in the extending direction of the edges. According to this configuration of the tissue structure manufacturing instrument, an opening may include opening widths of different sizes.

[0111] The hollow portion formed by the frame preferably has sufficient depth to form the core. In the frame, the depth of the hollow portion from the aforementioned opening can be at least 2 mm. For example, the depth of the hollow portion from the aforementioned opening can be 2 mm to 30 mm, 2 mm to 26 mm, or 2 mm to 12 mm. The depth of the hollow portion in the frame from the aforementioned opening (denoted as depth D in this specification) can be determined as the distance from the midpoint of the straight line connecting the points on the opening edges of the individual openings to the deepest point of the hollow portion on a cross-section in the width direction of the frame. Figure 2 In the frame, when more than one columnar member is fixed at both ends and, depending on the situation, at its middle portion by a support, and is circumferentially (preferably in the form of surrounding the virtual cylindrical surface) arranged on the virtual cylindrical surface sandwiched between the supports (e.g., inline or outlined to the virtual cylindrical surface), the center point of the cross-section in the width direction of the cylindrical surface can be taken as the deepest point of the hollow portion. The center point of the cross-section in the width direction can be a point equidistant from each columnar member circumferentially arranged on the virtual cylindrical surface. The center point of the cross-section in the width direction can be the center of the circular cross-section of the virtual cylindrical surface. The tissue structure manufacturing instrument of the present invention can have at least one opening with a depth D of 2 mm or more (preferably when the depth D of the opening is constant) or a maximum depth D of 2 mm or more (when the depth D of the opening is not constant).

[0112] It should be noted that at least one of the openings in the frame of the tissue structure manufacturing instrument of the present invention is the above-mentioned opening, and more preferably all the openings in the frame are the above-mentioned openings.

[0113] In the frame of the tissue structure manufacturing instrument of the present invention, more than one of the aforementioned openings may exist adjacent to each other, separated by columnar members. More than one of the aforementioned openings may also be arranged circumferentially along the frame. Alternatively, more than one of the aforementioned openings may be arranged axially along the frame. Furthermore, more than one of the aforementioned openings may be arranged both circumferentially and axially along the frame. According to this arrangement of openings, the density of recesses formed on the outer surface of the tissue structure for aggregating adult stem cells can be increased; in a preferred embodiment, the aggregation efficiency of adult stem cells can also be improved, thereby increasing the number of adult stem cells collected from one aggregation device. When using this tissue structure manufacturing instrument, the core portion of the manufactured tissue structure may have more than one recess divided by the periphery of the recesses, in which case each recess will be adjacent to other recesses separated by at least one periphery.

[0114] The tissue structure manufacturing apparatus of the present invention preferably does not have rod-shaped or other components in the hollow portion formed by the frame, or preferably is configured such that rod-shaped or other components are contained in the hollow portion, and is not placed in an environment where biological tissue material is present.

[0115] In one embodiment, the tissue structure manufacturing apparatus of the present invention includes, for example... Figure 1 As shown. Figure 1 As shown, the tissue structure manufacturing instrument 10 (hereinafter also referred to as instrument 10) has three annular support portions 11. Each support portion 11 may contain, or be made of, a biocompatible resin such as acrylic resin or silicone resin, but is not limited thereto. The three support portions 11 have two first support portions 11A and one second support portion 11B sandwiched between the two first support portions 11A, arranged along an axial direction Ax. One first support portion 11A and the second support portion 11B are located at both ends of a virtual cylindrical surface S. The two first support portions 11A have through holes 11H communicating between the outside and inside of the instrument along the axial direction Ax. The second support portion 11B has through holes 11H communicating between the inside of one cylindrical surface S and the inside of another cylindrical surface S along the axial direction Ax. When the device 10 is embedded or placed in an environment where biological tissue material exists, such as in biological tissue within a living organism, cells, such as fibroblasts, will flow into the hollow part of the device through these through-holes 11H.

[0116] A first support portion 11A and a second support portion 11B fix three cylindrical columnar components 13. The three columnar components 13 are equally spaced on each cylindrical surface S along their circumferential direction Ci, forming three openings 10H together with the first support portion 11A and the second support portion 11B. The columnar components 13, the two first support portions 11A, and the second support portion 11B constitute the frame of the device 10. Before the device 10 is implanted in an environment containing biological tissue material, such as biological tissue within a living organism, the space enclosed by the columnar components 13, the first support portion 11A, and the second support portion 11B is a hollow portion 10S. Each opening 10H connects the hollow portion 10S within the device to the external space of the device. When the device 10 is implanted in an environment containing biological tissue material, such as biological tissue within a living organism, cells, such as fibroblasts, flow from the biological tissue into the hollow portion 10S within the device through each opening 10H.

[0117] Figure 2 Indicates inclusion Figure 1 A schematic diagram of the cross-section in the width direction of the opening of the device shown. Figure 2As shown, the opening width W can be the length of the straight line connecting the points on the opening edge 10F of the cylindrical surface S, which is the shortest distance between adjacent columnar members 13 in the circumferential direction. When adjacent columnar members 13 are parallel, the opening width W is constant in the axial direction Ax. When adjacent columnar members 13 are not parallel, the opening width W is different at different locations in the axial direction Ax. The opening of the device 10 can have an opening width W of 2.5 mm or more, for example, 2.5 mm or more but less than 28 mm, 2.5 mm or more but less than 25 mm, 2.5 mm or more but less than 20.0 mm, or 3.0 mm or more but less than 10 mm. From the viewpoint of further improving the aggregation efficiency of adult stem cells, the opening width W is preferably at least 2.5 mm across the entire axial direction Ax of the device 10. Alternatively, in addition to the above, the opening of the device 10 preferably has an opening shape that can accommodate a circle with a diameter of 2.5 mm or more and 28 mm or less, 2.5 mm or more and 25 mm or less, 2.5 mm or more and 20 mm or less, or 3.0 mm or more and 10 mm or less.

[0118] Figure 2 The distance between the center (center point) Ct of the cylindrical surface S on the cross section shown and the midpoint of the straight line connecting the point located on the opening edge 10F is equivalent to the depth D of the hollow portion 10S from the opening 10H. The device 10 may have an opening 10H with a depth D or a maximum depth of at least 2 mm, for example, 2 mm to 30 mm, 2 mm to 26 mm, or 2 mm to 12 mm.

[0119] When implanted in an environment containing biological tissue materials, such as in a tissue structure manufacturing device 10 within a living organism, it will resemble... Figure 3 The organization is formed as outlined, but the invention is not limited to this theory. Figure 3 As shown in Figure A, when the device 10 is implanted in an environment where biological tissue material is present, such as biological tissue within a living organism, the biological tissue 21 will be located on the outer side of the hollow portion 10S. Next, as... Figure 3 As shown in Figure B, liquid components from biological tissue will penetrate into the hollow portion 10S through the opening 10H and the through hole 11H. After the device 10 is implanted into the biological tissue, after an appropriate period of time, such as 3 to 6 hours, the hollow portion 10S of the device 10 will be filled with liquid components from the biological tissue. Figure 3 As shown in Figure C, when the hollow portion 10S of the device 10 is filled with liquid, fibroblasts 22C from the biological tissue invade the hollow portion 10S through the opening 10H and the through-hole 11H. The fibroblasts 22C that have invaded the hollow portion 10S will begin to form fibrous connective tissue 22 from the surface of the columnar component 13 toward the interior of the hollow portion 10S.

[0120] like Figure 3 As shown in Figure D, the fibrous connective tissue that begins to form on the surface of the hollow portion 10S facing the columnar member 13 extends and grows into the interior of the hollow portion 10S. Then, the fibrous connective tissue 22 extending from the surfaces of each columnar member 13 connects with each other inside the hollow portion 10S, thereby forming a recess 22H on the surface of the fibrous connective tissue 22, which indents from the opening 10H into the hollow portion 10S. It should be noted that the fibrous connective tissue 22 can also be used to manufacture the supporting portion 11, a constituent element of the device 10, from the tissue structure, for example... Figure 1 The surfaces of the first support portion 11A and the second support portion 11B of the device shown form and grow into the interior of the hollow portion 10S, and connect with the fibrous connective tissue 22 extending from the surface of the columnar member 13. From each columnar member 13 and the support portion 11 (e.g. Figure 1 The fibrous connective tissue 22 formed by the expansion of the surfaces of the first support portion 11A and the second support portion 11B of the device shown connects to each other to form a core portion 25. That is, the core portion 25, which is composed of fibrous connective tissue 22, has a recess on its outer surface. As the fibrous connective tissue 22 with the recess and the core portion 25 are continuously formed, the adult stem cells 23C contained in the biological tissue will move into the recess 22H through the opening 10H. When the tissue structure manufacturing device 10 is properly placed in an environment where biological tissue material is present, such as in a biological tissue in a living organism, an aggregate of adult stem cells containing adult stem cells 23C (also called a pocket portion) will be formed in the recess.

[0121] In another embodiment of the tissue structure manufacturing apparatus 10, such as Figure 4 As shown in Figure A, the device 10 may include: a columnar component 13; two annular first support portions 11A with an outer diameter (diameter of the outer circle) of 6 mm; and a second annular support portion 11B with an outer diameter of 6 mm, sandwiched between the two first support portions 11A. Between the upper first support portion 11A and the second support portion 11B, three columnar components 13 with a diameter of 1 mm (25 mm long) are fixed, resulting in an opening width W of 3.0 mm and a depth D of 2 mm. Between the lower first support portion 11A and the second support portion 11B, four columnar components 13 with a diameter of 1 mm (25 mm long) are fixed, resulting in an opening width W of 2.5 mm and a depth D of 2.2 mm. Figure 4In the device 10 shown in A, each opening formed by the first support portion 11A, the second support portion 11B, and the columnar member 13 in the upper section has an opening shape that can accommodate a circle with a maximum diameter of 3.0 mm (equal to the opening width W); each opening formed by the first support portion 11A, the second support portion 11B, and the columnar member 13 in the lower section has an opening shape that can accommodate a circle with a maximum diameter of 2.5 mm (equal to the opening width W).

[0122] In another embodiment of the tissue structure manufacturing apparatus, such as Figure 4 As shown in Figure B, the device 10 may include: a columnar component 13; two annular first support portions 11A with an outer diameter of 11 mm; and two annular second support portions 11B with an outer diameter of 11 mm, sandwiched between the two first support portions 11A. Six columnar components 13 (20 mm long) with a diameter of 1 mm are fixed between the upper first support portion 11A and the second support portion 11B, resulting in an opening width W of 4.0 mm and a depth D of 4.6 mm. Eight columnar components 13 (20 mm long) with a diameter of 1 mm are supported between the lower first support portion 11A and the second support portion 11B, resulting in an opening width W of 3.0 mm and a depth D of 4.8 mm. Four columnar components 13 (20 mm long) with a diameter of 1 mm are fixed between the two second support portions 11B in the middle section, resulting in an opening width W of 6.0 mm and a depth D of 4.0 mm. Figure 4 In the device 10 shown in B, each opening formed by the first support portion 11A, the second support portion 11B, and the columnar member 13 in the upper section has an opening shape that can accommodate a circle with a maximum diameter of 4.0 mm (equal to the opening width W); each opening formed by the first support portion 11A, the second support portion 11B, and the columnar member 13 in the lower section has an opening shape that can accommodate a circle with a maximum diameter of 3.0 mm (equal to the opening width W); and each opening formed by the two second support portions 11B and the columnar member 13 in the middle section has an opening shape that can accommodate a circle with a maximum diameter of 6.0 mm (equal to the opening width W).

[0123] In another embodiment of the tissue structure manufacturing apparatus, such as Figure 4As shown in Figure C, the device 10 may include: columnar components 13; two annular first support portions 11A with an outer diameter of 16 mm; and two annular second support portions 11B with an outer diameter of 16 mm, sandwiched between the two first support portions 11A. Between the upper first support portion 11A and the second support portion 11B, eight columnar components 13 with a diameter of 1 mm (20 mm long) are fixed, resulting in an opening width W of 5.0 mm and a depth D of 7.1 mm. Between the lower first support portion 11A and the second support portion 11B, ten columnar components 13 with a diameter of 1 mm (20 mm long) are fixed, resulting in an opening width W of 3.5 mm and a depth D of 7.3 mm. Between the two second support portions 11B in the middle section, six columnar components 13 with a diameter of 1 mm (20 mm long) are fixed, resulting in an opening width W of 6.5 mm and a depth D of 6.8 mm. Figure 4 In the device 10 shown in C, each opening formed by the first support portion 11A, the second support portion 11B, and the columnar member 13 in the upper section has an opening shape that can accommodate a circle with a maximum diameter of 5.0 mm (equal to the opening width W); each opening formed by the first support portion 11A, the second support portion 11B, and the columnar member 13 in the lower section has an opening shape that can accommodate a circle with a maximum diameter of 3.5 mm (equal to the opening width W); and each opening formed by the two second support portions 11B and the columnar member 13 in the middle section has an opening shape that can accommodate a circle with a maximum diameter of 6.5 mm (equal to the opening width W).

[0124] In another embodiment of the tissue structure manufacturing apparatus 10, such as Figure 4 As shown in Figure D, the device 10 may include: columnar components 13; two annular first support portions 11A with an outer diameter of 11 mm; and a second annular second support portion 11B with an outer diameter of 10 mm, sandwiched between the two first support portions 11A. Between the two first support portions 11A, six columnar components 13 with a diameter of 1 mm (60 mm in length) are fixed, such that the opening width W is 4.0 mm and the depth D is 4.6 mm. These columnar components 13 are also fixed approximately at the center of their length direction by the second support portion 11B. Figure 4 In the device 10 shown in D, each opening formed by the first support portion 11A, the second support portion 11B, and the columnar member 13 has an opening shape that can accommodate a circle with a maximum diameter of 4.0 mm.

[0125] In another embodiment of the tissue structure manufacturing apparatus, such as Figure 5As shown in Figure A, the device 10 may have columnar components 13 and two annular first support portions 11A with an outer diameter of 11 mm. Between the two first support portions 11A, from one first support portion 11A to the other, eight columnar components 13 with a diameter of 1 mm are fixed in a manner where the opening width W continuously varies from 1.0 mm to 5.0 mm and the depth D continuously varies from 5.0 mm to 4.3 mm. The opening width W is measured on a cross-section of the device 10 in the width direction parallel to the support portions 11A. Figure 5 The length (length) of the device 10 shown in A is 40 mm.

[0126] In another embodiment of the tissue structure manufacturing apparatus, such as Figure 5 As shown in Figure B, the device 10 may have columnar components 13 and two annular first support portions 11A with an outer diameter of 16 mm. Between the two first support portions 11A, from one first support portion 11A to the other, eight columnar components 13 with a diameter of 1 mm are fixed in a manner where the opening width W continuously varies from 1.0 mm to 8.0 mm and the depth D continuously varies from 7.5 mm to 6.3 mm. The opening width W is measured on a cross-section of the device 10 in the width direction parallel to the support portions 11A. Figure 5 The length (length) of the device 10 shown in B is 60 mm.

[0127] In another embodiment of the tissue structure manufacturing apparatus, such as Figure 6 As shown in A, the device 10 may have a generally polyhedral shape consisting of a large rectangular frame 11C as an example of a support, a small rectangular frame 11D as an example of a support, and a columnar component 13 (1 mm in diameter). The large rectangular frame 11C has a size of 20 mm (vertical) × 15 mm (horizontal) [both are internal dimensions, the thickness of the rectangular frame is 1 mm, and the external dimensions are 22 mm (vertical) × 17 mm (horizontal)]; the small rectangular frame 11D has a size of 3 mm (vertical) × 15 mm (horizontal) [both are internal dimensions, the thickness of the rectangular frame is 1 mm, and the external dimensions are 5 mm (vertical) × 17 mm (horizontal)]. Figure 6 The device 10 shown in Figure A has four columnar components 13 with four vertex portions fixed to a large rectangular frame 11C and four vertex portions fixed to a small rectangular frame 11D. Additionally, Figure 6 The device 10 shown in A also has a columnar component 13 that connects the midpoint of one long side of the large rectangular frame 11C to the midpoint of one short side of the small rectangular frame 11D.

[0128] Figure 6The device 10 shown in A has two trapezoidal openings 10H, which are formed by a long side of a large rectangular frame 11C, a short side of a small rectangular frame 11D, and three columnar members 13 connected to these sides (the shortest columnar member 13, which is connected at right angles to the large rectangular frame 11C and the small rectangular frame 11D, has a length of 55 mm). Figure 6 The device 10 shown in Figure A has a trapezoidal opening 10H formed by a long side of a large rectangular frame 11C, a short side of a small rectangular frame 11D, and two columnar members 13 connected to these sides. Additionally, Figure 6 The device 10 shown in A also has a rectangular opening 10H formed by a short side on a large rectangular frame 11C, a long side on a small rectangular frame 11D, and two columnar members 13 connected to these sides. Figure 6 The device 10 shown in A also has two through openings 12H formed by dividing the large rectangular frame 11C by its two long sides and two short sides, and the small rectangular frame 11D by its two long sides and two short sides. Figure 6 In the device 10 shown in A, the opening width is 1.0 mm to 20.0 mm, and the depth D is 2.0 mm to 10.5 mm. The opening width W is measured on a cross-section of the device 10 in the width direction parallel to the long side of the large rectangular frame 11C.

[0129] like Figure 6 As shown in B, device 10 and Figure 6 The device 10 shown in A is the same and can have a generally polyhedral shape consisting of a large rectangular frame 11C as a support, a small rectangular frame 11D as a support, and a columnar component 13 (1 mm in diameter). The large rectangular frame 11C has a size of 20 mm (vertical) × 15 mm (horizontal) [both are internal dimensions, the thickness of the rectangular frame is 1 mm, and the external dimensions are 22 mm (vertical) × 17 mm (horizontal)]; the small rectangular frame 11D has a size of 3 mm (vertical) × 15 mm (horizontal) [both are internal dimensions, the thickness of the rectangular frame is 1 mm, and the external dimensions are 5 mm (vertical) × 17 mm (horizontal)]. Figure 6 The device 10 shown in B has two columnar components 13, one with two vertices fixed to a large rectangular frame 11C and the other with two vertices fixed to a small rectangular frame 11D. Additionally, Figure 6 The device 10 shown in B connects a short side of the large rectangular frame 11C and a long side of the small rectangular frame 11D through a plate-like body 26 with a slit 26S (1 mm wide).

[0130] Figure 6The device 10 shown in B has a trapezoidal opening 10H formed by a long side on a large rectangular frame 11C, a short side on a small rectangular frame 11D, and two columnar members 13 connected to these sides. Figure 6 The device 10 shown in B also has a rectangular opening 10H formed by a short side of a large rectangular frame 11C, a long side of a small rectangular frame 11D, and two columnar members 13 connected to these sides. Additionally, Figure 6 The device 10 shown in B has two trapezoidal openings 10H formed by a long side on a large rectangular frame 11C, a short side on a small rectangular frame 11D, the edge of a plate-like body 26 connecting these sides, and a columnar component 13. Figure 6 The device 10 shown in B also has two through openings 12H formed by dividing the large rectangular frame 11C by its two long sides and two short sides, and the small rectangular frame 11D by its two long sides and two short sides. Figure 6 In the device 10 shown in B, the opening width is 1.0 mm to 20.0 mm, and the depth D is 2.0 mm to 20.5 mm. The opening width W is measured on a cross-section of the device 10 in the width direction parallel to the long side of the large rectangular frame 11C.

[0131] like Figure 6 As shown in C, the device 10 may have a generally polyhedral shape consisting of a large rectangular frame 11C (as an example of a support), a small rectangular frame 11D (as an example of a support), a medium rectangular frame 11E (as an example of a support), and a columnar component 13 (1 mm in diameter). The large rectangular frame 11C has a size of 20 mm (vertical) × 15 mm (horizontal) [both are internal dimensions, the thickness of the rectangular frame is 1 mm, and the external dimensions are 22 mm (vertical) × 17 mm (horizontal)]; the medium rectangular frame 11E has a size of 15 mm (vertical) × 15 mm (horizontal) [both are internal dimensions, the thickness of the rectangular frame is 1 mm, and the external dimensions are 17 mm (vertical) × 17 mm (horizontal)]; and the small rectangular frame 11D has a size of 3 mm (vertical) × 15 mm (horizontal) [both are internal dimensions, the thickness of the rectangular frame is 1 mm, and the external dimensions are 5 mm (vertical) × 17 mm (horizontal)]. Figure 6 The device 10 shown in C has four columnar components 13 with four vertex portions fixed on a large rectangular frame 11C and four vertex portions fixed on a small rectangular frame 11D. The four columnar components 13 are also fixed by a middle rectangular frame 11E. Figure 6 The device 10 shown in C also has a columnar component 13 that connects the midpoint of one long side of the large rectangular frame 11C and the midpoint of one short side of the small rectangular frame 11D, and is fixed in the middle by a middle rectangular frame 11E.

[0132] Figure 6The device 10 shown in C has a rectangular opening 10H formed by a short side on a large rectangular frame 11C, a side on a medium rectangular frame 11E, and two columnar members 13 connected to these sides. Figure 6 The device 10 shown in C has a rectangular opening 10H formed by one side of the middle rectangular frame 11E, one long side of the small rectangular frame 11D, and two columnar members 13 connected to these sides. Additionally, Figure 6 The device 10 shown in C has two trapezoidal openings 10H formed by a long side on a large rectangular frame 11C, a side on a medium rectangular frame 11E, and three columnar members 13 connected to these sides. Figure 6 The device 10 shown in C has two trapezoidal openings 10H formed by a side on a middle rectangular frame 11E, a short side on a small rectangular frame 11D, and three columnar members 13 connected to these sides. Figure 6 The device 10 shown in C has a rectangular opening 10H formed by a long side on a large rectangular frame 11C, a side on a medium rectangular frame 11E, and two columnar members 13 connected to these sides. Figure 6 The device 10 shown in C has a rectangular opening 10H formed by a side on a middle rectangular frame 11E, a short side on a small rectangular frame 11D, and two columnar members 13 connected to these sides. Figure 6 The device 10 shown in C also has two through openings 12H formed by dividing the large rectangular frame 11C by its two long sides and two short sides, and the small rectangular frame 11D by its two long sides and two short sides. Figure 6 In the device 10 shown in C, the opening width is 1.0 mm to 20.0 mm, and the depth D is 2.0 mm to 10.5 mm. The opening width W is measured on a cross-section of the device 10 in the width direction parallel to the long side of the large rectangular frame 11C.

[0133] The position of the opening 10H is not limited to its arrangement in the circumferential and / or axial direction Ax of the instrument 10, but can also be arranged in a periodically fixed position on a spiral extending in the length direction of the instrument 10. In addition, the position of the opening 10H is not limited to the repeated arrangement of each opening 10H in a specified direction. For example, more than one opening 10H can form an opening group, and more than one opening group can be arranged in a repeated arrangement in a specified direction.

[0134] In another embodiment of the tissue structure manufacturing apparatus, the shape of the apparatus 10 or the frame is not particularly limited; it can be rod-shaped, or approximately polyhedral (e.g., cube-shaped, cuboid-shaped, etc.), or approximately spherical or approximately hemispherical. The shape of the apparatus 10 or the frame is not limited to a straight rod shape; for example, it can also be curved.

[0135] In another embodiment of the tissue structure manufacturing apparatus, such as Figure 7 As shown, the frame constituting the device 10 can be coil-shaped, and can be one or more spirals with the opening edge 10F connected in the circumferential direction of the coil. The opening width W can be measured as the shortest distance between adjacent circumferential portions of the coil-shaped linear component 16. The device 10 preferably has an opening with an opening width W of at least 2.5 mm, for example, 2.5 mm to 28 mm, 2.5 mm to 25 mm, 2.5 mm to 20 mm, or 3.0 mm to 10 mm. In addition, the shortest distance between the central axis of the coil-shaped device 10 and the coil-shaped linear component 16 can also be calculated as the depth D, and the device 10 can also have a region with a depth D of at least 2 mm, for example, 2 mm to 30 mm, 2 mm to 26 mm, or 2 mm to 12 mm.

[0136] In another embodiment of the tissue structure manufacturing apparatus, such as Figure 8 As shown, the device 10 may also be constructed from a cubic or cuboid frame with a hollow portion. The cubic or cuboid side 17 has an opening 10H, and the opening width W can be determined as the shortest distance between adjacent columnar members constituting the side 17. The device 10 or frame preferably has at least one opening with a width W of at least 2.5 mm, for example, 2.5 mm to 28 mm, 2.5 mm to 25 mm, 2.5 mm to 20 mm, or 3.0 mm to 10 mm. Alternatively, the depth D can be calculated as the shortest distance between the center point of the cubic or cuboid device 10 or frame and the side of the device 10. The device 10 may also have a region with a depth D of at least 2 mm, for example, 2 mm to 30 mm, 2 mm to 26 mm, or 2 mm to 12 mm.

[0137] The shape of the frame of device 10 can be, for example, Figure 8 The cube or cuboid structure shown is connected on one face.

[0138] In another embodiment of the tissue structure manufacturing apparatus, the apparatus 10 may also be in the form of a support. Figure 9 The illustrated support-shaped device 10 can have an expandable and contractible mesh-shaped cylindrical form, with the mesh forming an opening 10H and end openings 14H for inserting a balloon. The present invention, for example, can... Figure 9The scaffold-shaped device shown is used as a tissue structure manufacturing instrument. In a typical implementation, in the presence of biological tissue material, such as biological tissue in a living organism, a balloon catheter carrying a retractable scaffold is inserted, water pressure is applied subcutaneously to inflate the balloon, increasing the diameter of the scaffold, and then the water pressure is released, causing the balloon to contract and be removed, thereby facilitating the implantation of the scaffold.

[0139] This invention also relates to a method for manufacturing tissue structures composed of aggregated adult stem cells using the tissue structure manufacturing device of the present invention. The method for manufacturing tissue structures composed of aggregated adult stem cells using the tissue structure manufacturing device includes: placing the tissue structure manufacturing device in an environment in the presence of biological tissue material, such as within a living organism or biological tissue; more specifically, it includes: configuring (e.g., embedding) the tissue structure manufacturing device in an environment in the presence of biological tissue material, such as within a living organism or biological tissue, and leaving it in place for a certain period of time. The environment in the presence of biological tissue material, such as biological tissue or isolated biological tissue within a living organism, may contain fibroblasts. The biological tissue may be ectodermal tissue, mesodermal tissue, or endoderm tissue. The biological tissue in which the tissue structure manufacturing device is placed may be tissue containing adult stem cells in a healthy state, or tissue containing adult stem cells that requires tissue repair, such as an injured or missing area. A preferred example of biological tissue containing fibroblasts is subcutaneous tissue.

[0140] Tissue structure manufacturing instruments can be implanted and placed within biological tissues within a living organism. Alternatively, tissue structure manufacturing instruments can also be placed in an artificial environment that simulates biological tissues within a living organism, i.e., an in vitro culture system containing biological tissues taken from a living organism, or an environment constructed outside a living organism to simulate biological tissues within a living organism. Examples of artificial environments that simulate biological tissues within a living organism include artificial tissues or artificial organs created through the three-dimensional culture of isolated cells or tissues.

[0141] When implanting a tissue structure manufacturing instrument into a biological tissue within a living organism, the organism or tissue is incised under anesthesia to create an insertion port. The tissue structure manufacturing instrument is then inserted into the port. After implantation, the incision is sutured.

[0142] The biological tissue in which the tissue structure manufacturing device is configured (e.g., implanted) can be any animal tissue that can serve as a source of adult stem cells. The tissue structure manufacturing device can be configured (e.g., implanted) within any animal. Examples of such animals include, but are not limited to, primates such as humans, monkeys, and chimpanzees; mammals such as dogs, cats, cattle, pigs, horses, goats, sheep, rats, and mice; and animals such as birds, fish, and amphibians. In one embodiment, these animals can be humans or non-human animals, such as non-human mammals. In one embodiment, the tissue structure manufacturing device can be implanted within the biological tissue or biological tissue isolated from an animal individual (humans or non-human animals, etc., as subject individuals) intended for transplantation from a tissue structure manufactured using the tissue structure manufacturing device, or from cells differentiated from such adult stem cells, or from an animal individual, or from an organism whose genetically closely related individuals. Thus, adult stem cells from the individual or from a closely related individual can be transplanted. In this invention, there are no particular restrictions on the location within the organism in which the tissue structure manufacturing device is implanted; it can be the abdomen, chest, shoulder, back, limbs, or abdominal cavity, etc.

[0143] The tissue structure manufacturing instrument, configured in an environment where biological tissue material is present, is placed in a core portion with a concave portion and composed of fibrous connective tissue, which is formed in the hollow portion of the instrument, and in which adult stem cells are aggregated.

[0144] The placement time of the tissue structure manufacturing device in an environment containing biological tissue material, such as biological tissue within an organism or isolated biological tissue, is preferably a period during which excessive inflammation of the biological tissue is not caused and sufficient individual stem cells can be accumulated. This period can be appropriately set according to the placement site, the type of living organism, and the size of the device. This placement time can generally be from 5 days to 4 months, preferably from 1 week (7 days) to 3 months. For example, it can be from 1 week to 2 months, from 1 week to 5 weeks, from 9 days to 5 weeks, from 9 days to 4 weeks, from 2 weeks to 5 weeks, from 2 weeks to 4 weeks, from 2 weeks to 3 weeks, or from 3 weeks to 5 weeks.

[0145] After the placement period, the tissue structure manufacturing instrument was removed from the environment where biological tissue material was present. Within the removed tissue structure manufacturing instrument, a tissue structure was formed, containing a recess with aggregated adult stem cells and a core containing fibrous connective tissue.

[0146] When removing the instrument 10 from the organism, the implantation site of the tissue structure manufacturing instrument is cut open under anesthesia as needed. After removing the tissue structure manufacturing instrument from the incision, the wound is preferably sutured.

[0147] This allows for the extraction of tissue structures from biological tissue materials, such as those within a living organism, using tissue manufacturing instruments. The tissue structures can also be separated from the extracted tissue structures using these instruments, thus enabling their recovery. In this way, tissue structures can be manufactured using these instruments.

[0148] The present invention also provides a tissue structure of the present invention that can be manufactured using the aforementioned tissue structure manufacturing apparatus. More specifically, the present invention relates to a tissue structure having a core portion and sparsely fibrous tissue (adult stem cell aggregate tissue), the core portion having a recess and being composed of fibrous connective tissue, the sparsely fibrous tissue being formed in the recess and containing type III collagen and adult stem cells.

[0149] In this invention, the fibrous connective tissue 22 constituting the core 25 of the tissue structure is a strong, high-density tissue mainly composed of collagen fibers and fibroblasts. The fibrous connective tissue constituting the core in this invention can also be called dense connective tissue, unlike adipose tissue or loose connective tissue. Preferably, the fibrous connective tissue constituting the core in this invention mainly contains type I collagen. On the other hand, the content of type III collagen in the core of this invention is generally low, especially in its central part, where only a very small amount of type III collagen is present. In a preferred embodiment, the proportion of type I collagen in the total collagen in the core of this invention is about 65% to 90% by weight, for example, about 70% to 85% by weight; the proportion of type III collagen is about 5% to 30% by weight, for example, about 10% to 25% by weight (e.g., ...). Figure 12 A and Figure 12 B). In a preferred embodiment, in the core portion of the invention composed of fibrous connective tissue, adult stem cells may be present near the recess, while the area away from the recess (especially the central portion of the core portion) is completely or substantially devoid of adult stem cells.

[0150] On the other hand, the adult stem cell aggregate 23 (pocket portion) formed in the concave portion is a sparse fibrous tissue containing adult stem cells, particularly adult stem cell 23C enriched. In this invention, the adult stem cell aggregate formed in the concave portion contains type III collagen, with a higher content of type III collagen than in the core portion. The adult stem cell aggregate formed in the concave portion may also contain other cells such as fibroblasts and vascular endothelial cells, and may also contain other types of collagen or other substances. In a preferred embodiment, the proportion of type I collagen in the total collagen of the adult stem cell aggregate in this invention is approximately 30% to 75% by weight, for example, approximately 35% to 65% by weight; the proportion of type III collagen in the total collagen is approximately 20% to 65% by weight, for example, approximately 30% to 60% by weight (e.g., Figure 12 C). The amount of fiber in the adult stem cell aggregate tissue formed in the concave portion is relatively small, and the fibers are randomly oriented. The adult stem cell aggregate tissue 23 formed in the concave portion is a sparsely fibrous soft tissue because it only sparsely contains collagen fibers. It should be noted that the concave portion forming the adult stem cell aggregate tissue 23 is usually located on the outer surface of the core portion.

[0151] In a preferred embodiment, the proportion of type III collagen in the total collagen of the adult stem cell aggregate (pocket portion) formed in the recess of the tissue structure of the present invention is higher than the proportion of type III collagen in the total collagen of the core portion of the tissue structure. In one embodiment, the former (the proportion of type III collagen in the pocket portion) can be 30% to 60% by weight, and the latter (the proportion of type III collagen in the core portion) can be 10% to 25% by weight.

[0152] In a preferred embodiment, the proportion of type I collagen in the total collagen of the adult stem cell aggregate (pocket portion) formed in the recess of the tissue structure of the present invention is lower than the proportion of type I collagen in the total collagen of the core portion of the tissue structure. In one embodiment, the former (the proportion of type I collagen in the pocket portion) can be 35% to 65% by weight, and the latter (the proportion of type I collagen in the core portion) can be 70% to 85% by weight.

[0153] The tissue structures involved in this invention, particularly the core and adult stem cell aggregates, are substantially devoid of elastic fibers. In this invention, "substantially devoid of elastic fibers" means completely undetectable or substantially undetectable by Elastica van Gieson (EVG) staining.

[0154] The tissue structure involved in this invention, when formed in the hollow portion of a tissue structure manufacturing instrument, is formed to fill the hollow portion within the instrument, and therefore takes on a shape that follows the shape of the hollow portion within the instrument. For example, when using... Figure 4 When the tissue structure manufacturing apparatus shown in D manufactures a tissue structure, a core composed of fibrous connective tissue extends radially from the center of the tissue structure to the surface of the six columnar components 13, arranged according to the columnar components 13. A recess is formed directly below each opening between the columnar components 13 (a total of six recesses). Adult stem cell aggregates are formed in these six recesses, such as... Figure 11 A cross-sectional photograph of the tissue structure is shown. Therefore, the shape of the tissue structure involved in this invention is defined by a frame forming a hollow portion. That is, the tissue structure involved in this invention is formed using a tissue structure manufacturing apparatus.

[0155] In this invention, the "core portion" composed of fibrous connective tissue refers to the core of the tissue structure, used to support the adult stem cell aggregation tissue formed in its recesses. In a preferred embodiment, the core portion of the invention extends continuously from the center of the tissue structure to its outermost edge. In this invention, for example, when the tissue structure is rod-shaped, the "center portion" of the tissue structure refers to the entire length or a portion of the central axis (central axis in the length direction) of the tissue structure and / or the area near its circumference; the central axis is the line connecting the center points (geometric centers) of each cross-section (cross-section orthogonal to the length direction) of the tissue structure in the width direction. Alternatively, for example, when the tissue structure is generally polyhedral, the "center portion" of the tissue structure in this invention refers to the entire length or a portion of the central axis (central axis in the length direction) of the tissue structure and / or the area near its circumference; the central axis is the line connecting the center points (geometric centers) of each cross-section in the width direction of the tissue structure.

[0156] The core portion of the tissue structure involved in this invention preferably has a diameter of at least 2.5 mm. The "diameter" of the core portion, as defined above, is defined as the longest distance between two parallel lines connecting both sides of a cross section relative to the width direction of the core portion. Typically, the diameter of the core portion can be the end portions 24E of two recessed peripheral portions 24 existing on opposite sides of the core portion in a cross section along the width direction, separated from the center of the core portion. Figure 22 The longest distance between )

[0157] In one embodiment, the tissue structure or its core portion involved in this invention may or may not be solid. Regarding the tissue structure or its core portion, "solid" means that it is filled with tissue up to the center. It should be noted that in tissue structures removed from a tissue structure manufacturing instrument placed in an environment containing biological tissue material, a thin tissue membrane may sometimes form on the outer side of the columnar component; however, the presence of such a thin tissue membrane does not imply that the tissue structure or its core portion involved in this invention is not solid.

[0158] The tissue structure of the present invention has at least one recess in its core portion in which an aggregate of adult stem cells is formed, preferably two or more, more preferably three or more such recesses. The tissue structure of the present invention may have, for example, 4, 5, 6, 7, 8, 9, or 10 or more recesses in its core portion in which an aggregate of adult stem cells is formed. The number of recesses in the core portion of the tissue structure of the present invention in which an aggregate of adult stem cells is formed may be, for example, 1 to 50, 3 to 50, 4 to 40, 4 to 30, 5 to 30, 3 to 20, 3 to 15, 3 to 10, 4 to 15, or 5 to 15. The recesses are formed of fibrous connective tissue. Typically, the inner surface of each recess in the core portion is configured as a generally curved surface (e.g., a mortar shape).

[0159] In the tissue structure of the present invention, the recess where adult stem cell aggregates are formed preferably has, but is not limited to, an opening width WW of at least 2.5 mm. The opening width WW of the recess can be calculated as being the same as the opening width W of the opening of the frame of the tissue structure manufacturing instrument used to manufacture the tissue structure. Alternatively, the opening width WW of the recess in the tissue structure of the present invention can be determined on a cross-section in the width direction of the tissue structure after the tissue structure manufacturing instrument is removed as: the shortest distance between the peripheral edge 24 of the recess formed around the frame (e.g., columnar member, support portion, linear member, etc.) of the tissue structure manufacturing instrument and the end point (corresponding to the opening edge of the frame opening) on ​​the surface of the frame (e.g., columnar member, support portion, linear member, etc.). In the tissue structure of the present invention, the above-mentioned recess of the core portion preferably has an opening shape with an opening width WW of 2.5 mm or more, for example, 2.5 mm or more and 28 mm or less, 2.5 mm or more and 25 mm or less, 2.5 mm or more and 20.0 mm or less, or 3.0 mm or more and 10 mm or less.

[0160] Alternatively, in addition to the above, the recess in the core portion of the tissue structure of the present invention, where adult stem cell aggregates are formed, preferably has an opening shape (morphology and size) capable of incorporating a circle with a diameter of 2.5 mm or more (e.g., 3.0 mm or more). The opening of the aforementioned recess may, for example, have a shape and size capable of incorporating a circle with a diameter of 2.5 mm to 28 mm, 2.5 mm to 25 mm, 2.5 mm to 20 mm, or 3.0 mm to 10 mm. The opening of the aforementioned recess with such a shape and size corresponds to the opening shape of the recess particularly suitable for forming fibrous connective tissue and the frame of the adult stem cell aggregate within that recess. Here, regarding the opening of the recess in the core portion of the tissue structure of the present invention, "capable of incorporating a circle" means that, inside the opening shape (e.g., rectangular or polygonal), a circle with a predetermined diameter can be connected and accommodated at one or more points of the opening. For example, in the shape of a rectangle with a long side of 20mm and a short side of 2.5mm, a circle with a maximum diameter of 2.5mm can be inscribed.

[0161] In one embodiment, in the tissue structure of the present invention, the fibrous connective tissue or core portion may have more than one recess divided by the periphery of the recess.

[0162] In one embodiment, the outer surface of the fibrous connective tissue or core of the tissue structure of the present invention may be composed of more than one recess and more than one recess periphery.

[0163] The tissue structure involved in this invention can have any shape, such as rod-shaped (e.g., generally cylindrical or generally polygonal), generally polyhedral, generally frustum-shaped, or generally spherical. In one embodiment, the tissue structure of this invention is rod-shaped, and more than one of the aforementioned recesses can be arranged circumferentially thereafter. In another embodiment, the tissue structure of this invention is rod-shaped, and more than one of the aforementioned recesses can also be arranged axially thereafter. In yet another embodiment, the tissue structure of this invention is rod-shaped, and more than one of the aforementioned recesses can also be arranged both axially and circumferentially thereafter.

[0164] In another embodiment, the shape of the tissue structure of the present invention is generally polyhedral, and more than one of the above-mentioned recesses may be located on different faces of the generally polyhedral.

[0165] The shape of the tissue structure of the present invention is preferably non-tubular, and even more preferably non-sheet-like.

[0166] The tissue structure of the present invention has sparse fibrous tissue formed in a recessed portion of the core. Adult stem cells (adult stem cell aggregates) are aggregated in this tissue formed in the recessed portion of the tissue structure of the present invention. The adult stem cells contained in the adult stem cell aggregates preferably include at least one of mesenchymal stem cells and pluripotent stem cells, more preferably simultaneously including both mesenchymal stem cells and pluripotent stem cells. The adult stem cell aggregates may contain stem cells expressing stem cell markers, such as pluripotent stem cells expressing at least one pluripotent stem cell marker and / or mesenchymal stem cells expressing at least one mesenchymal stem cell marker. The adult stem cell aggregates preferably contain pluripotent stem cells simultaneously expressing the pluripotent stem cell markers SSEA4 and SSEA3. The adult stem cell aggregates may also contain stem cells expressing either SSEA3 or SSEA4, such as pluripotent stem cells. The adult stem cell aggregates may also contain mesenchymal stem cells expressing either or both of the mesenchymal stem cell markers CD90 and CD105. The adult stem cell aggregates may also contain stem cells expressing the mesenchymal stem cell marker CD90, or both CD90 and SSEA3. The adult stem cell aggregate may also contain stem cells expressing the mesenchymal stem cell marker CD105, or both CD105 and SSEA3. The adult stem cell aggregate may also contain stem cells expressing the growth factor marker VEGF, or both VEGF and SSEA3. These stem cells are highly angiogenic. The adult stem cell aggregate may also contain stem cells expressing the growth factor marker HGF. In addition to the above-mentioned markers, or as alternatives to the above-mentioned markers, the adult stem cell aggregate of the tissue structure of the present invention may also contain adult stem cells expressing different markers, such as pluripotent stem cells and mesenchymal stem cells.

[0167] In a preferred embodiment, the tissue structure of the present invention may contain more than 1%, more than 5%, more than 10%, or more than 30%, and less than 60%, less than 50%, less than 40%, or less than 30%, for example, 1% to 60%, 5% to 40%, 5% to 30%, or 5% to 20% (stem cell proportion based on cell number) of adult stem cells (including mesenchymal stem cells and pluripotent stem cells) relative to the total number of cells contained therein.

[0168] In a preferred embodiment, the adult stem cell aggregate tissue of the present invention may contain more than 20%, more than 30%, or more than 50%, and less than 90%, less than 80%, or less than 70%, for example, 20% to 90%, 30% to 90%, 30% to 80%, or 50% to 90% (stem cell proportion based on cell number) of adult stem cells (including mesenchymal stem cells and pluripotent stem cells) relative to the total number of cells contained therein.

[0169] In a preferred embodiment, when the tissue structure and / or adult stem cell aggregate of the present invention is treated in a 0.25% collagenase type I solution at 37°C for 1.5 hours as described in Example 4, the cells recovered from the tissue (adult stem cell aggregate and its surroundings) thus decomposed may contain more than 20%, more than 30%, or more than 50%, and less than 90%, less than 80%, or less than 70%, for example, 20% to 90%, 30% to 90%, 30% to 80%, or 50% to 90% (stem cell proportion based on cell number) of adult stem cells (including mesenchymal stem cells and pluripotent stem cells).

[0170] The above stem cell ratio can be calculated by using the number of cells expressing at least one stem cell marker (e.g., but not limited to: at least one selected from the group consisting of CD90, CD105, SSEA3 and SSEA4, or at least one selected from the group consisting of CD90, SSEA3 and SSEA4) as the number of stem cells (adult stem cells).

[0171] In one embodiment, the tissue structure and adult stem cell aggregate of the present invention may contain mesenchymal stem cells and pluripotent stem cells in a ratio of mesenchymal stem cells: pluripotent stem cells = 60-95: 5-40, 60-80: 10-30, or 65-75: 15-25, but are not limited to the above ratios. This ratio of mesenchymal stem cells to pluripotent stem cells can be calculated by using the number of cells expressing at least one mesenchymal stem cell marker (e.g., but not limited to: CD90 or CD105) as the number of mesenchymal stem cells, and the number of cells expressing at least one pluripotent stem cell marker (e.g., but not limited to: SSEA3 or SSEA4) as the number of pluripotent stem cells.

[0172] In the tissue structure of this invention, the adult stem cell aggregation tissue formed in the concave portion may contain type III collagen in addition to the adult stem cells. Furthermore, it may contain other cells such as fibroblasts and vascular endothelial cells, and may also contain other types of collagen or other substances. The presence of vascular endothelial cells can be studied, for example, by detecting the expression of the vascular endothelial cell marker vWF. The presence of fibroblasts can be studied, for example, by detecting the expression of the fibroblast marker vimentin.

[0173] High VEGF expression can be observed, particularly in the relatively outer regions of the tissue structure of the present invention. On the other hand, typically, VEGF expression is weaker in the fibroblasts that are more abundant in the central part of the tissue structure of the present invention.

[0174] The tissue structure of the present invention can be formed by placing the above-mentioned tissue structure manufacturing device in an environment where biological tissue material is present, such as in biological tissue within a living organism.

[0175] The tissue structure of the present invention can be manufactured using the aforementioned tissue structure manufacturing instrument in the presence of biological tissue material, and then kept within the instrument. In this case, the tissue structure fills the hollow portion within the tissue structure manufacturing instrument, and its core portion is preferably in close contact with the surface of the instrument's frame (e.g., columnar component, support portion, linear component, etc.). Alternatively, the tissue structure of the present invention can also be a tissue structure that has been separated from the tissue structure manufacturing instrument after formation.

[0176] The adult stem cell aggregate formed in the recess of the fibrous connective tissue (core portion) of the tissue structure of the present invention can be easily collected from the fibrous connective tissue. For example, the adult stem cell aggregate 23 can be easily scraped from the recess 22H of the fibrous connective tissue 22 (core portion 25) of the tissue structure of the present invention. Alternatively, the adult stem cell aggregate 23 or adult stem cells 23C can be recovered from the instrument 10, which is taken from an environment in which biological tissue material is present, such as a biological body, by scraping the adult stem cell aggregate 23 containing aggregated adult stem cells 23C from the recess 22H, or by enzymatically digesting the adult stem cell aggregate 23. Alternatively, the instrument 10 taken from an environment in which biological tissue material is present can be directly treated with collagenase, elastase, trypsin, or TrypLE. TM (Thermo Fisher Scientific), Accutase (R) (Innovative Cell Technologies, Inc.) Enzymatic treatment with dispersants and other enzymes is used to separate adult stem cells 23C from the adult stem cell aggregate 23 formed in the recess within the hollow portion of the instrument. Alternatively, the adult stem cell aggregate 23 scraped from the recess 22H can be treated with collagenase, elastase, trypsin, and TrypLE. TM (Thermo Fisher Scientific), Accutase (R) Adult stem cells 23C are isolated from adult stem cell aggregates 23 by enzymatic treatment with enzymes such as dispersants (Innovative Cell Technologies, Inc.) and dispersants. The enzyme treatment time depends on the amount of material being treated, and typically ranges from 0.5 to 2 hours. Alternatively, the cells remaining after enzyme treatment can be isolated from fibrous connective tissue 22, from which adult stem cells 23C can be recovered.

[0177] The purification degree of adult stem cell 23C can also be increased through further separation and purification steps. For example, cells containing adult stem cell 23C can be separated by filtration using membrane filters or mesh filters. The separation and purification of adult stem cell 23C can be performed using markers specifically expressed on the cell surface of adult stem cells, such as stem cell markers (pluripotent stem cell markers and / or mesenchymal stem cell markers). For example, one or both of the pluripotent stem cell markers SSEA4 and SSEA3, and one or both of the mesenchymal stem cell markers CD105 and CD105 can be used as stem cell markers. For example, adult stem cells can be easily separated and / or concentrated from adult stem cell aggregates using conventional methods such as magnetic cell sorting (MACS) and fluorescence-activated cell sorting (FACS).

[0178] The present invention also relates to a method for collecting adult stem cells 23C, the method comprising: separating adult stem cell aggregates or adult stem cells from the tissue structure of the present invention.

[0179] Adult stem cells isolated from the adult stem cell aggregate of the tissue structure of the present invention may or may not be cultured and proliferated as needed. Alternatively, the adult stem cell aggregate or cell population containing partially purified adult stem cells from the adult stem cell aggregate may or may not be cultured and proliferated as needed. The adult stem cell aggregate, cell population containing adult stem cells, or adult stem cells can be used for tissue construction or cell differentiation in vitro or ex vivo. The adult stem cell aggregate, cell population containing adult stem cells, or adult stem cells can be administered to a subject (e.g., a patient) for regenerative medicine, such as tissue repair (tissue regeneration), prevention or treatment (e.g., repair or improvement) of tissue or organ disorders (e.g., congenital or acquired absence, congenital or acquired dysfunction or abnormality, injury, etc.) or functional impairment; or for enhancing the function of tissues or organs. That is, the adult stem cell aggregates, cell populations containing adult stem cells, or cell preparations containing adult stem cells obtained in this invention can be used in regenerative medicine, such as tissue repair (tissue regeneration), prevention or treatment (e.g., repair or improvement) of tissue or organ disorders or functional impairments; or to enhance the function of tissues or organs. This invention also provides cell preparations (or pharmaceutical compositions) containing adult stem cell aggregates obtained from the tissue structure of this invention, or adult stem cells derived from such adult stem cell aggregates, or cell populations containing adult stem cells. This invention also provides a method for manufacturing a cell preparation (or pharmaceutical composition), comprising: preparing a cell preparation (or pharmaceutical composition) using adult stem cell aggregates obtained from the tissue structure of this invention, or adult stem cells derived from such adult stem cell aggregates, or cell populations containing adult stem cells. The cell preparations or pharmaceutical compositions of this invention may also contain pharmaceutically acceptable additives (e.g., but not limited to: carriers, solvents, excipients, wetting agents, stabilizers, isotonic agents, buffers, preservatives, colorants, cryoprotectants, etc.).

[0180] The adult stem cell aggregates, cell populations containing adult stem cells, or adult stem cells obtained in this invention can be administered to a subject (e.g., a patient) after inducing differentiation into target cells outside of biological tissues, or can be administered directly to appropriate biological tissues (sites with tissue or organ disorders or functional impairments, etc.) without differentiation induction. The adult stem cell aggregates, cell populations containing adult stem cells, or adult stem cells obtained in this invention can be cryopreserved using conventional methods and administered when necessary. Differentiation induction treatment can be performed using conventional methods for cell differentiation induction or any known method. This invention also provides cell preparations (or pharmaceutical compositions) containing adult stem cell aggregates of this invention that have undergone differentiation induction treatment, or adult stem cells derived from such adult stem cell aggregates, or cell populations containing adult stem cells (e.g., cells differentiated from adult stem cells derived from adult stem cell aggregates of this invention). This invention also provides a method for manufacturing a cell preparation (or pharmaceutical composition), comprising: preparing a cell preparation (or pharmaceutical composition) using adult stem cell aggregates of this invention that have undergone differentiation induction treatment, or adult stem cells derived from such adult stem cell aggregates, or cell populations containing adult stem cells. These cell preparations or pharmaceutical compositions may also contain pharmaceutically acceptable additives (e.g., but not limited to: carriers, solvents, excipients, wetting agents, stabilizers, isotonic agents, buffers, preservatives, colorants, cryoprotectants, etc.).

[0181] This invention also relates to methods for administering regenerative medicine to a subject (e.g., a patient), including applying the adult stem cell aggregate of the invention, adult stem cells from the aggregate, or a cell population containing adult stem cells, or cells differentiated from the adult stem cells. Examples of methods include tissue repair (tissue regeneration), prevention or treatment (e.g., repair or improvement) of tissue or organ disorders or functional impairments, or methods for enhancing tissue or organ function. Subjects (e.g., patients) may include, but are not limited to, primates such as humans, monkeys, and chimpanzees, and mammals such as dogs, cats, cattle, pigs, horses, goats, sheep, rats, and mice; as well as animals such as birds, fish, and amphibians. In one embodiment, the subject (e.g., patient) may be a human or a non-human animal, such as a non-human mammal. In one embodiment, the subject (e.g., patient) may require regenerative medicine, such as tissue repair (tissue regeneration), prevention or treatment (e.g., repair or improvement) of tissue or organ disorders or functional impairments, or enhancement of tissue or organ function. Subjects (e.g., patients) may have tissue or organ disorders or functional impairments that they expect to be treated by transplantation of adult stem cells or cells differentiated from adult stem cells.

[0182] According to the present invention, tissue structures in which adult stem cells are aggregated can be manufactured, thereby enabling efficient collection of adult stem cells and making the acquisition and utilization of adult stem cells easier.

[0183] Example

[0184] The present invention will be described in more detail below using examples. However, the scope of the present invention is not limited to these examples.

[0185] [Materials and Instruments]

[0186] The embodiments described in this application used the following test animals, reagents, and instruments.

[0187] -Test animal: female beagle, 3 years old, weighing 10 kg

[0188] - Figures 4-6 The tissue structure manufacturing apparatus shown

[0189] - Figure 9 The stent and balloon catheter shown are of the following shapes.

[0190] -HypoThermosol tissue preservation solution (R) FRS (BioLIfe Solutions, 101102)

[0191] - Collagenase type I (Worthington, LS004214)

[0192] -Dalberg's Modified Eagle's Medium (DMEM) [DMEM-LG (Low Glucose); Wako Pure Chemical Industries, Ltd., Japan, 041-29775]

[0193] -Bambanker cell cryopreservation solution (R) (NIPPON Genetics, CS-02-001)

[0194] -4% Paraformaldehyde (PFA; Wako Pure Chemical Industries, Ltd., Japan, 163-20146)

[0195] - VersaLyse Lysing Solution (Beckman Coulter, Inc., A09777) - Hemolysis reagent

[0196] -Fetal bovine serum (FBS) (Biosera, FB-1365)

[0197] -Phosphate-buffered saline (PBS)

[0198] -Xylene (Muto Chemical, 4313)

[0199] -100% ethanol (Muto Chemical, 4026)

[0200] - Mordant (Muto Chemical, 4006-1)

[0201] -Second mordant (Muto Chemical, 8141-1)

[0202] -Weigert's iron hematoxylin staining solution 1 (Muto Chemical, 4034-1)

[0203] -Weigert's iron hematoxylin staining solution 2 (Muto Chemical, 4035-1)

[0204] -0.75% Orange G (Muto Chemical, 4023-1)

[0205] -1% acetic acid (Muto Chemical, 4017-1)

[0206] -Masson staining solution B (Muto Chemical, 4025-1)

[0207] -2.5% phosphotungstic acid solution (Muto Chemical, 4018-1)

[0208] -Aniline blue dye (Muto Chemical, 4020-1)

[0209] -Maeda's improved resorcinol magenta solution (Muto Chemical, 4032-1)

[0210] -VAN GIESON staining solution A (Muto Chemical, 4036-1)

[0211] -VAN GIESON staining solution B (Muto Chemical, 4037-1)

[0212] -1% Sirius Red Staining Solution (Mutoh Chemical, 3306-1)

[0213] - Citrate buffer (Sigma, C9999-1000ML)

[0214] - Bovine serum albumin (BSA) (Wako Pure Pharmaceutical Co., Ltd., Japan, 013-25773)

[0215] <First Anti>

[0216] - Anti-CD90 antibody (abcam, ab123511)

[0217] - Anti-CD105 antibody (abcam, ab156756)

[0218] - Anti-SSEA4 antibody (abcam, ab16287)

[0219] - Anti-VEGFA antibody (abcam, ab1316)

[0220] - Anti-HGF antibody (abcam, ab83760)

[0221] - Anti-SSEA3 antibody (Bioss, bs-3575R)

[0222] - Anti-von Willebrand factor (vWF) antibody (abcam, ab6994)

[0223] - Anti-vimentin antibody (abcam, ab8069)

[0224] Secondary antibody (fluorescently labeled)

[0225] -Goat anti-mouse IgG H&L (Alexa Fluor) (R) 488)(abcam,ab150113)

[0226] -Goat anti-rabbit IgG H&L (Alexa Fluor) (R) 594)(abcam,ab150080)

[0227] -ProLong TM Gold quenching-resistant sealing tablets (containing DAPI (4',6-diamidinyl-2-phenylindole)) (Thermo Fisher Scientific, P36931)

[0228] - Cell Staining Buffer (BioLegend, 420201)

[0229] -FITC-labeled anti-CD90 antibody (BioLegend, 328107)

[0230] - Anti-FITC microbeads (Miltenyi Biotec, 130-048-700)

[0231] - Goat anti-rabbit IgG microbeads (Miltenyi Biotec, 130-048-600)

[0232] -MACS buffer (autoMACS) (R) Rinse solution; Miltenyi Biotec, 130-091-222)

[0233] -MACS separation column (MS chromatography column; Miltenyi Biotec, 130-042-201)

[0234] - Fluorescence microscope (Nikon, ECLIPSE Ti-U)

[0235] - Polarizing microscope (Nikon, ECLIPSE E1000)

[0236] [Example 1] Fabrication of tissue structures using in vivo tissue formation technology (iBTA)

[0237] After anesthetizing the dog, five incisions were made in the skin from the right abdomen to the back using an electric scalpel. Subcutaneous tissue was then inserted and implanted at each site. Figure 4 D and Figure 10 The wound was sutured using a tissue structure manufacturing instrument of shape A. Three weeks later, the dog was anesthetized, the skin was incised, and the subcutaneous tissue structure manufacturing instrument was removed. The tissue structure manufacturing instrument used had six cylindrical columnar components 13, each 1 mm in diameter and 60 mm long, fixed at both ends and in the middle by annular supports with a diameter of 11 mm. The opening width W was 4.0 mm and the depth D was 4.6 mm.

[0238] The instrument removed from the subcutaneous tissue was largely covered by soft tissue. Observation of the cross-section revealed that the hollow portion (hollow space) within the instrument was filled with tissue up to the center. It can be considered that the hollow portion of the instrument, used to manufacture tissue structures implanted and placed within a living organism, contains aggregated cells, thus forming tissue. The location of implantation has almost no effect on tissue formation.

[0239] Figure 10 The photographs exemplarily show the instrument used (A), the instrument removed after 3 weeks in the body (B), and a cross-section of the resulting tissue structure (C).

[0240] This demonstrates that tissue structures can be fabricated by implanting the tissue structure manufacturing device of the present invention within a living organism.

[0241] The instruments used to manufacture tissue structures removed from the subcutaneous tissue are placed into a tissue preservation solution, HypoThermosol. (R) Place the FRS in 15 mL centrifuge tubes and place on ice. Store at 4°C and proceed with subsequent processing within 24 hours.

[0242] Inside the laminar flow hood, the tissue fabrication instrument containing the formed tissue structures was removed from the tissue preservation solution, gently washed with a centrifuge tube containing PBS, and excess tissue around the instrument was removed. After removal, the instrument was photographed and stored in DMEM.

[0243] [Example 2] Histological analysis of tissue structures

[0244] The tissue structure extracted from the organism in Example 1 was treated with 4% PFA at 4°C for 24 hours to fix the tissue. The instrument was then removed to create a paraffin-embedded block of the tissue structure.

[0245] Paraffin-embedded blocks were prepared into sections 3–5 μm thick using a microtome in the width direction. The sections were stained with hematoxylin and eosin (HE), Masson's trichrome (MT), Elastica van Gieson (EVG), and Sirius red (SR) and observed under a microscope.

[0246] The staining results showed that the entire tissue structure stained pink after HE staining. Figure 11 A), stained blue by MT staining ( Figure 11 B), thus indicating that the obtained tissue structure is rich in collagen fibers. Furthermore, the entire tissue structure stained red by SR staining, indicating that it is rich in collagen (B). Figure 11 D). In the tissue structure, particularly the region from around the columnar components of the tissue structure manufacturing instrument to the center is heavily stained, indicating a dense presence of collagen. This suggests that fibrous connective tissue forms around and in the center of the tissue structure. In contrast, in the area between the columnar components of the tissue structure, there are areas that are lightly stained by HE, MT, and SR staining, where collagen fibers are present only very sparsely. In the obtained tissue structure, the connective tissue formed around and in the center of the columnar components has generally curved concave areas between the columnar components, indicating that soft tissue (sparse fibrous tissue) corresponding to the lightly stained areas (pocket areas) is formed in these concave areas.

[0247] It should be noted that since there are no areas that would stain black with EVG, this indicates that the tissue structure contains almost no elastic fibers. Figure 11 C).

[0248] To examine the types of collagen present in tissue structures, sections stained with Sirius red (SR) were observed using a polarizing microscope. Under polarizing light, type I collagen appeared yellow to orange, while type III collagen appeared green.

[0249] The results showed that the tightly packed collagen fibers in the areas stained with Sirius red (around and in the center of the instrument column) appeared yellow or orange, indicating the presence of predominantly type I collagen. Figure 12 A and Figure 12 B). On the other hand, the lightly stained areas between the columnar components were observed to be green, indicating the presence of more type III collagen compared to the area around and in the center of the instrument's columnar components. Figure 12C). The lightly stained areas (pocket sections) between columnar components contain fewer fibers and are randomly oriented. Figure 12 C).

[0250] [Example 3] Immunohistochemical staining

[0251] To investigate the cell types and distribution within the tissue structures, immunohistochemical staining was performed. For 3–5 μm thick sections of the tissue structures, immunohistochemical staining with corresponding primary and secondary antibodies detected CD90 and CD105 as markers of mesenchymal stem cells, SSEA4 and SSEA3 as markers of pluripotent stem cells, and vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) as growth factors. Additionally, DAPI (4',6-diamidindo-2-phenylindole) was used for staining of cell nuclei. The results are shown below. Figures 13-15 .

[0252] In the detection of mesenchymal stem cell markers CD90 and CD105, most cells in the pocket region were stained. Positive cells could also be identified around the aforementioned columnar component, in the region near the pocket region. Figure 13 In the diagram, arrows indicate representative staining sites. On the other hand, positive cells are completely absent in the central part of the tissue structure.

[0253] In the detection of pluripotent stem cell markers SSEA4 and SSEA3, as with mesenchymal stem cell markers, most cells in the pocket region were stained. Figure 14 In A and 14B, arrows indicate representative staining sites. On the other hand, positive cells are absent in the central part of the tissue structure.

[0254] Regarding the expression of growth factors within tissue structures, VEGF is highly expressed in relatively lateral regions of the tissue structure, particularly in round cells within pockets, where VEGF expression is stronger. Figure 15 HGF-expressing cells were more frequently seen in the pocket region, similar to VEGF-expressing cells, while round cells highly expressed HGF. Figure 15 It should be noted that round cells are generally highly undifferentiated cells. On the other hand, VEGF expression is weaker in fibroblasts.

[0255] To investigate the nature of stem cells, double staining was performed on the stem cell marker SSEA3 and other markers (CD90 and SSEA3, CD105 and SSEA3, SSEA4 and SSEA3, VEGF and SSEA3).

[0256] The double staining results showed that, in addition to double-positive cells (white arrows), single-positive cells for each marker were also observed (horizontal stripe arrows, dotted arrows). Figure 16 The horizontal striped arrows indicate green staining positive for CD90, CD105, SSEA4, or VEGF. The dotted arrows indicate red staining positive for SSEA4. This indicates that the cells constituting the pocket tissue are a hierarchical population of undifferentiated cells. Additionally, some cells simultaneously express the pluripotent stem cell markers SSEA4 and SSEA3. Figure 16 C), which is consistent with the characteristics of ES cells and other more undifferentiated pluripotent stem cells (Henderson JK, et al., Stem Cells, 2002; 20: 329-337). Furthermore, the particularly high expression of VEGF in SSEA3-positive cells suggests... Figure 16 D) The highly undifferentiated stem cells present in the pocket tissue possess a high angiogenic capacity. In fact, immunohistochemical staining with anti-vWF antibody detected strong expression of the vascular endothelial cell marker vWF in the region centered at the boundary between the pocket and the core, indicating the presence of vascular endothelial cells near this boundary and in the pocket. It should be noted that immunohistochemical staining with anti-vimentin antibody also detected the fibroblast marker vimentin in the pocket.

[0257] The above results indicate that stem cells aggregate and activate in the pocket region to promote tissue formation. That is, the tissue formed in the concave region (pocket region) can be termed tissue formed by the aggregation of adult stem cells (adult stem cell aggregation tissue). Fabricating tissue structures with this pocket region allows for the efficient acquisition and utilization of activated stem cells.

[0258] [Example 4] Cell isolation and analysis

[0259] 20 mL of DMEM (serum-free) was added to the type I collagenase (50 mg) in the vial to prepare a 0.25% collagenase solution. A tissue fabrication instrument containing internal tissue structures, taken from an organism in Example 1, was placed in a 15 mL centrifuge tube containing 6 mL of the prepared collagenase solution and shaken at 37°C. After shaking for 1.5 hours, the instrument and any undigested tissue were removed, and the tube was centrifuged at 1000 rpm for 5 minutes at 4°C, removing the supernatant. 6 mL of DMEM was added, and the cells (4 vials, 24 mL) were recovered in a 50 mL centrifuge tube. The recovered fraction was centrifuged at 1000 rpm for 5 minutes at 4°C, removing the supernatant.

[0260] To induce hemolysis of red blood cells, 1 mL of VersaLyse Lysing Solution was added to the recovered cells, which were then pipetted and incubated at room temperature for 10 minutes. 10 mL of DMEM containing 10% FBS was added, and the cells were centrifuged at 1000 rpm for 5 minutes at 4°C, removing the supernatant. 5 mL of BAMBANKER cell cryopreservation medium was added. (R) Resuspension was performed, and 10 μL of the cell suspension was separated. An equal volume of trypan blue staining solution was added for staining, and the cell count was measured. The cells were then aliquoted into 1 mL aliquots and stored in liquid nitrogen.

[0261] 5mL BAMBANKER (R) The cell concentration of the suspension was 6.25 × 10⁻⁶. 6 Cells / mL, with a cell viability of 98.4%. Therefore, approximately 4 × 10⁶ cells / mL can be recovered from each tissue structure. 6 Cells. It should be noted that approximately the same number of cells were also recovered in other independently conducted experiments. Under the treatment conditions of this embodiment, while the general whole of the adult stem cell aggregate and its surrounding connective tissue (peripheral part of the tissue structure) were decomposed, the central part of the tissue structure (core part) was not decomposed.

[0262] To examine the proportion of cells present in the recovered cell population, the number of CD90-positive cells, SSEA3-positive cells, and SSEA4-positive cells were measured using flow cytometry.

[0263] The results showed that in the recovered cell population, 73% were CD90-positive, 23% were SSEA3-positive, and 18% were SSEA4-positive. Figure 17 ); Approximately 80% of the recovered cell population expressed stem cell markers. Based on marker expression, approximately 70% were mesenchymal stem cells (2.8 × 10⁻⁶). 6 (cells), approximately 20% are pluripotent stem cells (8.0 × 10⁻⁶). 5 cell).

[0264] During collagenase treatment, because tissue structures are broken down from the outside and cells are separated, the cells separated in the initial stage of collagenase treatment are almost all adult stem cells from adult stem cell aggregates. This indicates that the proportion of fibroblasts increases with the duration of collagenase treatment.

[0265] The common source of mesenchymal stem cells, bone marrow, contains only 10–100 cells / mL of mesenchymal stem cells, while adipose tissue contains only 5 × 10⁶ cells / mL. 3Mesenchymal stem cells per g. The cell structure involved in this invention contains a high proportion of stem cells, therefore, this invention can recover stem cells with higher efficiency compared to existing methods for stem cell recovery.

[0266] [Example 5] Concentration of CD90 or SSEA3 positive cells

[0267] The tissue structures were treated with collagenase in the same manner as in Example 4, and CD90 or SSEA3-positive stem cells were isolated and concentrated from the recovered cell population. The recovered cell population was labeled with anti-CD90 antibody (BioLegend, 328107) or anti-SSEA3 antibody (Bioss, bs-3575R), reacted with magnetic bead antibodies [anti-FITC microbeads (Miltenyi Biotec, 130-048-700) and goat anti-rabbit IgG microbeads (Miltenyi Biotec, 130-048-600)], and cells were separated by magnetic cell sorting (MACS).

[0268] As a result, the proportion of CD90-positive cells was concentrated from 62% before separation to 90% after separation. Figure 18 In addition, it was able to concentrate the proportion of SSEA3-positive cells from 29% before separation to 84% after separation. Figure 19 ).

[0269] The cell populations before and after MACS cell separation were compared at a concentration of 3000 cells / cm³. 2 Cells were seeded at a density of [missing information] into 6-well plates and cultured in DMEM containing 10% FBS. Cell morphology was observed. Results showed that fibroblast-like cells adhered to and proliferated on the bottom surface of the wells. Figure 20 Cells isolated using stem cell markers also exhibited a fibroblast-like morphology, but many cells were observed to be smaller than the pre-isolation cell population. Figure 20 ).

[0270] By using the cell structure involved in this invention as a source, it is possible to efficiently concentrate target stem cells.

[0271] [Example 6]

[0272] A skin puncture was performed on the dog, and a balloon catheter with a collapsing support (29mm long x 20mm in diameter when dilated) was inserted subcutaneously. Figure 21 A, 21D). Subcutaneous water pressure is applied to inflate the balloon (…). Figure 21 B), increase the diameter of the support ( Figure 21 C).

[0273] The water pressure was released, causing the balloon to contract and be removed. The stent was then placed subcutaneously while the balloon was in a dilated state. Three weeks later, the dog was anesthetized, the skin was incised, and the subcutaneous stent was removed.

[0274] Inside the scaffold, the tissue filled the central portion ( Figure 21 E). Removal of tissue structures formed within the scaffold ( Figure 21 F). Fibrous connective tissue forms around and at the center of the scaffold's metal frame, and between adjacent metal frames, the connective tissue forms curved concave areas. Pockets containing aggregated stem cells (adult stem cell aggregation tissue) are formed within these concave areas. Figure 21 (G, arrow). Additionally, similar to the metal frame, recesses containing aggregated adult stem cell tissue were also identified at the through openings 12H at both ends of the scaffold. (Using...) Figure 9 The scaffold of the shape shown can also be used to manufacture the tissue structure involved in this invention.

[0275] [Example 7]

[0276] Tissue structures were manufactured using the tissue structure manufacturing apparatus of the present invention, which has various shapes. Preparations were made... Figure 4 A~ Figure 4 C Figure 5 A and Figure 5 The five types of tissue structure manufacturing instruments 10 shown in Figure B are all cylindrical in shape with a diameter of 1 mm. An incision was performed on a beagle under full anesthesia to implant the tissue structure manufacturing instruments subcutaneously in the abdomen, and the wound was sutured. At 3, 10, 2 weeks (14 days), 17 days, or 3 weeks (21 days) after instrument implantation, an incision was performed on the beagle to remove the tissue structure manufacturing instruments from the subcutaneous tissue of the abdomen.

[0277] The tissue structure manufacturing instrument, which was taken from under the skin, was almost entirely covered by soft tissue. The hollow part (hollow space) inside the instrument was filled with tissue, and tissue structures were observed to form in the same way as in the above embodiment.

[0278] The tissue structures obtained from the aforementioned instruments were subjected to fiber staining treatment using the same method as in Example 2 to stain the collagen fibers. As an example, [the following method will be used]. Figure 4 HE-stained images of sections of tissue structures located near the center of length in tissue structure fabrication instruments (aperture width W = 6.0 mm) produced by B are shown below. Figure 22The results were very similar to those of Example 2. Connective tissue was present from the periphery of each columnar component to the center of the tissue structure. Between the columnar components, the connective tissue formed recesses 22H, in which sparse fibrous adult stem cell aggregates (pockets) 23 were present. It should be noted that the main component of the connective tissue was dense collagen fibers.

[0279] The tissue analysis results show that fibrous connective tissue gradually forms from the surface of each columnar component of the instrument to the interior of the hollow part of the instrument. The connective tissue formed around adjacent columnar components is connected to each other, thereby forming a core part with a concave part on the outer surface that extends from the opening of the instrument to the hollow part of the instrument. At the same time, adult stem cells from the organism gather in the concave part to form an adult stem cell aggregation tissue.

[0280] In addition, immunohistochemical staining was performed in the same manner as in Example 3. Numerous cell nuclei stained with DAPI were confirmed in the connective tissue of the core region, while expression of the mesenchymal stem cell marker CD90 and the pluripotent stem cell marker SSEA-4 was negative. On the other hand, numerous CD90-positive and SSEA-4-positive adult stem cells were confirmed in the adult stem cell aggregate tissue (pocket region).

[0281] When the implantation period of the tissue structure manufacturing device was 3 days, although the formation of a core with a concave outer surface was confirmed, no CD90-positive cells or SSEA-4-positive cells were detected in the adult stem cell aggregate tissue or the core. When the implantation period was 10 days, 2 weeks, 17 days, and 3 weeks, the presence of CD90-positive cells and SSEA-4-positive cells was observed in the core.

[0282] like Figure 23 As shown, HE staining results confirmed that the adult stem cell aggregates contained a high amount of fibrin during a 10-day placement period. Conversely, during a 3-week placement period, the fibrin content was significantly lower compared to the 10-day placement. In contrast, the number of CD90-positive and SSEA-4-positive cells detected in the adult stem cell aggregates during a 3-week placement period was significantly higher than the 10-day placement period.

[0283] The number of CD90-positive cells in the adult stem cell aggregate tissue at 2 weeks of placement was approximately the midpoint between the 10-day and 3-week placement periods. In contrast, the number of SSEA-4-positive cells in the adult stem cell aggregate tissue at 2 weeks of placement was significantly higher than at 10 days of placement. This indicates a substantial increase in SSEA-4-positive cells in the adult stem cell aggregate tissue between 2 and 3 weeks after implantation.

[0284] The above results indicate that the number of adult stem cells in adult stem cell aggregates increases during in vivo placement of the tissue structure manufacturing device.

[0285] [Example 8]

[0286] about Figures 4-6 The relationship between the structure of the tissue structure manufacturing instrument shown and the formation of the concave portion of connective tissue and the aggregated tissue of adult stem cells was examined by sectioning tissue structures manufactured during a 3-week placement period using the same method as in Example 7, after being treated with fiber staining (HE staining).

[0287] It was used in the manufacture of tissue structures. Figure 4 When manufacturing instruments for tissue structures of shape A, the manufactured tissue structures ( Figure 24 The device has a core portion 25 as described in all of the following (i) and (ii), and an adult stem cell aggregate tissue 23 formed in its recess, wherein: (i) a hollow portion 10S (upper section of the device) with an opening width W of 3.0 mm and a depth D of 2.0 mm between three columnar components 13 and (ii) a hollow portion 10S (lower section of the device) with an opening width W of 2.5 mm and a depth D of 2.2 mm between four columnar components 13; wherein, with respect to the core portion 25, fibrous connective tissue is present around and at the center of each columnar component 13, support portion 11A and 11B, and a recess 22H is formed from each opening surrounded (formed) by two adjacent columnar components 13, support portion 11A and support portion 11B into the hollow portion of the device. The fibrous connective tissue 22 constituting the core portion 25 extends into each through hole 11H of the two support portions 11A of the tissue structure manufacturing device. The tissue structure also has a recess 22H that extends from the through hole (support opening) 11H into the hollow part of the device, in which an aggregate of adult stem cell tissue 23 has been confirmed to have formed. The fibrous connective tissue formed in the hollow parts of the device in (i) and (ii) above are connected via the fibrous connective tissue formed in the through hole of the support 11B.

[0288] It was used in the manufacture of tissue structures. Figure 4When manufacturing instruments for tissue structures of shape B, the manufactured tissue structures ( Figure 25 The device has a core portion 25 as described in all of the following (i), (ii), and (iii), and an adult stem cell aggregation tissue 23 formed therein, wherein: (i) a hollow portion 10S (upper section of the device) with an opening width W of 4.0 mm and a depth D of 4.6 mm between 6 columnar components 13; (ii) a hollow portion 10S (middle section of the device) with an opening width W of 6.0 mm and a depth D of 4.0 mm between 4 columnar components 13; and (iii) 8 columnar components 13... The hollow portion 10S (lower section of the instrument) of the device has an opening width W of 3.0 mm and a depth D of 4.8 mm between the components 13. Regarding the core portion 25, fibrous connective tissue is present around and at the center of each columnar component 13, support portions 11A and 11B, and a recess 22H is formed from each opening surrounded (formed) by two adjacent columnar components 13 and two opposing support portions (support portions 11A and 11B, or two support portions 11B) into the hollow portion of the instrument. The fibrous connective tissue 22 constituting the core portion 25 extends into each through-hole 11H of the two support portions 11A of the tissue structure manufacturing instrument. The tissue structure also has a recess 22H from the through-hole (support portion opening) 11H into the hollow portion of the instrument, in which an aggregate of adult stem cell tissue 23 has also been confirmed to form. The fibrous connective tissues formed in the hollow portions of the instruments (i) to (iii) above are connected via the fibrous connective tissues formed in the through holes 11H of the two support portions 11B.

[0289] It was used in the manufacture of tissue structures. Figure 4 When manufacturing instruments for tissue structures of shape C, the manufactured tissue structures ( Figure 26The device has a core portion 25 as described in all of the following (i), (ii), and (iii), and an adult stem cell aggregation tissue 23 formed therein, wherein: (i) a hollow portion 10S (upper section of the device) with an opening width W of 5.0 mm and a depth D of 7.1 mm between 8 columnar components 13; (ii) a hollow portion 10S (middle section of the device) with an opening width W of 6.5 mm and a depth D of 6.8 mm between 6 columnar components 13; and (iii) 10 columnar components... The hollow portion 10S (lower section of the instrument) of the device has an opening width W of 3.5 mm and a depth D of 7.3 mm between the columnar components 13; wherein, regarding the core portion 25, fibrous connective tissue is present around and at the center of each columnar component 13, support portions 11A and 11B, and has recesses 22H that are recessed into the hollow portion of the instrument from the openings formed by the two adjacent columnar components 13 and the two opposing support portions (support portions 11A and support portions 11B, or two support portions 11B). The fibrous connective tissue 22 of the core portion 25 extends into the through holes 11H of the two support portions 11A of the tissue structure manufacturing instrument. The tissue structure also has recesses 22H that are recessed into the hollow portion of the instrument from the through holes (support portion openings) 11H, in which aggregates of adult stem cell tissue 23 have also been found. The fibrous connective tissues formed in the hollow portions of the instruments (i) to (iii) above are connected via the fibrous connective tissues formed in the through holes 11H of the two support portions 11B.

[0290] It was used in the manufacture of tissue structures. Figure 5 When manufacturing an instrument with the tissue structure shown in A, a tissue structure having a core portion 25 and an adult stem cell aggregate tissue 23 formed in its recess is manufactured; wherein, regarding the core portion 25, fibrous connective tissue is present around and at the center of each columnar member 13 and support portion 11A, and a recess 22H is formed from each opening surrounded (formed) by two adjacent columnar members 13 and two opposing support portions 11A into the hollow portion of the instrument. Regarding each opening, a recess 22H is confirmed to be formed in the range where the opening width W between the columnar members 13 is 2.5 mm to 5.0 mm, and an adult stem cell aggregate tissue 23 is confirmed to be present in the recess 22H. No recess 22H is confirmed to be formed in the range where the opening width W is narrower than 2.5 mm. For example, Figure 27 A is an HE-stained photograph of a section located approximately at the center of the tissue structure along its length. A recess 22H was identified at an opening width W of 3.0 mm and a depth D of 4.8 mm, within which an aggregate of adult stem cells 23 was also identified. Figure 27B is an HE-stained photograph of a section located near the end of the tissue structure along its length. A recess 22H was identified at a location with an opening width W of 4.0 mm and a depth D of 4.6 mm, within which an aggregate of adult stem cells 23 was also confirmed. However, no recess 22H was identified at a location with an opening width W of 2.0 mm and a depth D of 4.9 mm. Figure 27 B). The fibrous connective tissue 22 of the core 25 extends into the through holes 11H of the two support portions 11A of the tissue structure manufacturing device. The tissue structure also has a recess 22H that extends from the through holes (support openings) 11H into the hollow part of the device, in which adult stem cell aggregates 23 have also been found to be formed.

[0291] It was used in the manufacture of tissue structures. Figure 5 When manufacturing the device with the tissue structure shown in B, a tissue structure having a core portion 25 and an adult stem cell aggregate tissue 23 formed in its recess is manufactured; wherein, regarding the core portion 25, fibrous connective tissue is present around and at the center of each columnar member 13 and support portion 11A, and a recess 22H is formed from each opening surrounded (formed) by two adjacent columnar members 13 and two opposing support portions 11A into the hollow portion of the device. Regarding each opening, a recess 22H is confirmed to be formed in the range of opening width W between columnar members 13 being 2.5 mm to 8.0 mm, and an adult stem cell aggregate tissue 23 is confirmed to be present in the recess 22H. In the range where the opening width W is narrower than 2.5 mm, no recess 22H is confirmed to be formed. For example, Figure 28 A is an HE-stained photograph of a section located approximately at the center of the tissue structure along its length. A recess 22H was identified at an opening width W of 4.0 mm and a depth D of 7.2 mm, within which an aggregate of adult stem cells 23 was also confirmed. Figure 28 B is an HE-stained photograph of a section located near the end of the tissue structure. A recess 22H was identified at a location with an opening width W of 7.0 mm and a depth D of 6.6 mm, within which an aggregate of adult stem cells 23 was also confirmed; however, no recess 22H was identified at a location with an opening width W of 2.0 mm and a depth D of 7.4 mm. Figure 28 B). The fibrous connective tissue 22 of the core 25 extends into the through holes 11H of the two support portions 11A of the tissue structure manufacturing device. The tissue structure also has a recess 22H that extends from the through holes (support openings) 11H into the hollow part of the device, in which adult stem cell aggregates 23 have also been found to be formed.

[0292] Figure 29 Is using Figure 5This is a magnified image of a fibrous-stained section of a tissue structure with a cross-section along the axial direction Ax, manufactured using a tissue structure manufacturing instrument of shape B. The image shows the structure of the tissue formed directly below an opening whose width W continuously varies from 2.0 mm to 5.0 mm. Figure 29 As shown, within the range of 2.0 mm to 5.0 mm for the opening width W, the larger the opening width W, the greater the depth of the recess 22H formed directly below it. Consequently, the size (volume) of the adult stem cell aggregate 23 formed in the recess 22H directly below it also increases with the opening width W. In particular, since the depth of the recess 22H increases sharply with an opening width W = 3.0 mm, the size of the adult stem cell aggregate 23 also increases sharply when the opening width W = 3.0 mm or more.

[0293] It was used in the manufacture of tissue structures. Figure 6 When manufacturing the device for the tissue structure shown in Figure A, a tissue structure having a core portion 25 and an adult stem cell aggregate tissue 23 formed in its recess is manufactured; wherein, regarding the core portion 25, fibrous connective tissue is present around and at the center of each columnar component 13, the large rectangular frame 11C, and the small rectangular frame 11D, and a recess 22H is formed from each opening enclosed by two adjacent columnar components 13, the large rectangular frame 11C, and the small rectangular frame 11D into the hollow part of the device. Furthermore, the tissue structure is manufactured using... Figure 6 When manufacturing an instrument using a tissue structure of shape B, a tissue structure having a core portion 25 and an adult stem cell aggregate 23 formed in its recess is manufactured; wherein, regarding the core portion 25, fibrous connective tissue is present around and at the center of each columnar member 13, large rectangular frame 11C, small rectangular frame 11D and plate-like body 26, and has: (i) an opening formed by two adjacent columnar members 13 and large rectangular frame 11C, small rectangular frame 11D, and (ii) recesses 22H that are recessed into the hollow portion of the instrument from the opening formed by one columnar member 13, large rectangular frame 11C, small rectangular frame 11D and plate-like body 26. No recesses are formed in the opening of the slit 26S (opening width 1 mm) of the plate-like body 26. Regarding each opening, a recess 22H was found to be formed within an opening width W of 3.0 mm to 20.0 mm between the columnar components 13, and an aggregate of adult stem cell tissue 23 was found to be present in this recess 22H. It should be noted that... Figure 6 The depth D of the instrument used to manufacture the tissue structure of shape A ranges from 2.0 mm to 10.5 mm. Additionally, Figure 6The depth D of the tissue structure manufacturing instrument shown in B ranges from 2.0 mm to 20.5 mm because the bottom of the instrument is closed and the deepest point of the hollow part is located at the bottom. The fibrous connective tissue 22 of the core 25 extends into the frames of the large rectangular frame 11C and the small rectangular frame 11D of the tissue structure manufacturing instrument. The tissue structure also has recesses 22H that are recessed into the hollow part of the instrument from the openings of the large rectangular frame 11C and the small rectangular frame 11D (both of which are support parts), and adult stem cell aggregates 23 have also been found to form in these recesses.

[0294] It was used in the manufacture of tissue structures. Figure 6 When manufacturing the tissue structure of shape C, a tissue structure having a core portion 25 and an adult stem cell aggregate 23 formed in its recess was manufactured. Figure 30 Regarding the core portion 25, fibrous connective tissue is present around and at the center of each columnar component 13, the large rectangular frame 11C, the medium rectangular frame 11E, and the small rectangular frame 11D, and it has: (i) an opening formed by two adjacent columnar components 13, the large rectangular frame 11C, and the medium rectangular frame 11E, and (ii) recesses 22H that are recessed into the hollow portion of the instrument from the openings formed by the two columnar components 13, the medium rectangular frame 11E, and the small rectangular frame 11D. Regarding each opening, a recess 22H is confirmed to be formed within an opening width W of 3.0 mm to 20.0 mm between the columnar components 13, and an aggregate of adult stem cell tissue 23 is confirmed to be present in this recess 22H. It should be noted that... Figure 6 The depth D of the tissue structure manufacturing instrument shown in shape C ranges from 2.0 mm to 10.5 mm. The fibrous connective tissue 22 of the core 25 extends into the frames of the large rectangular frame 11C, the medium rectangular frame 11E, and the small rectangular frame 11D of the tissue structure manufacturing instrument. The tissue structure also has recesses 22H that are recessed into the hollow part of the instrument from the openings of the large rectangular frame 11C and the small rectangular frame 11D (both of which are support parts), and adult stem cell aggregates 23 have been found to form in these recesses.

[0295] Will use Figure 6 The tissue structure of shape C is obtained by manufacturing an instrument for a tissue structure. Figure 30 HE staining results of the sections are shown in Figure 31 .exist Figure 30 The tissue structure was cut at the location shown. Figure 31 A is a stained image of the longitudinal section (a-a') of the adult stem cell aggregate 23 formed directly below the opening of the large rectangular frame 11C. Figure 31B is a stained image of a longitudinal section (width direction; b-b') of the adult stem cell aggregate 23 formed directly below the opening of the small rectangular frame 11D. At any opening, a recess 22H indenting into the hollow portion of the instrument was observed, and adult stem cell aggregate 23 was formed within the recess. Collagen fibers are densely present in the fibrous connective tissue surrounding the large rectangular frame 11C or the small rectangular frame 11D.

[0296] In the above embodiments, it was confirmed that a recess 22H was formed on the tissue structure when the opening width W of the tissue structure manufacturing instrument was 2.5 mm or more. On the other hand, it was not confirmed that a recess 22H was formed directly below the opening where the opening width W was less than 2.5 mm. When the depth D, or the maximum value of the depth D, was at least 2 mm or more, it was not confirmed that it affected the formation of the recess 22H.

[0297] The above demonstrates that, using the tissue structure manufacturing apparatus 10 of the present invention, it is possible to manufacture tissue structures that aggregate and enrich somatic stem cells 23C in the recess 22H even with a short placement period, such as less than one month, or even about two to three weeks.

[0298] All publications, patents and patent applications referenced in this specification are incorporated herein by direct reference.

[0299] Symbol Explanation

[0300] Ax…Axial direction; Ci…Circumferential direction; Ct…Center point; D…Depth; S…Cylindrical surface; W…Opening width; 10…Tissue structure manufacturing instrument; 10F…Opening edge; 10H…Opening portion; 10S…Hollow portion; 11…Support portion; 11A…First support portion; 11B…Second support portion; 11H…Through hole; 12H…Through opening portion; 13…Columnar component; 16…Linear component; 21…Biological tissue; 22…Fibrotic connective tissue; 22C…Fibroblasts; 22H…Concave portion; 23…Adult stem cell aggregation tissue; 23C…Adult stem cells; 24…Peripheral part of concave portion; 25…Core portion.

Claims

1. A tissue structure comprising a core and a sparsely fibrous aggregate of adult stem cells, wherein, The core portion has a recess and is composed of fibrous connective tissue, and the sparsely fibrous adult stem cell aggregate is formed in the recess and contains type III collagen and adult stem cells. The recess has an opening width of at least 2.5 mm. The adult stem cells are selected from at least one of mesenchymal stem cells and pluripotent stem cells. The mesenchymal stem cells express at least one of the mesenchymal stem cell markers CD90 and CD105, and the pluripotent stem cells express at least one of the pluripotent stem cell markers SSEA4 and SSEA3.

2. The tissue structure according to claim 1, wherein, The core portion has one or more of the aforementioned recesses.

3. The tissue structure according to claim 1 or 2, wherein, The core portion has at least three of the recesses.

4. The tissue structure according to claim 1, wherein, The core portion has a diameter of at least 2.5 mm.

5. The tissue structure according to claim 1, wherein, The structure is rod-shaped, with more than one of the recesses arranged circumferentially.

6. The tissue structure according to claim 1, wherein, The tissue structure is rod-shaped, with more than one of the recesses arranged axially.

7. The tissue structure according to claim 1, wherein, The structure is polyhedral in shape, with more than one of the recesses located on different faces.

8. The tissue structure according to claim 1, wherein, The tissue structure is neither tubular nor sheet-like in shape.

9. An organizational structure, which is the organizational structure according to claim 1, wherein, The tissue structure is formed by placing a tissue structure manufacturing instrument with a hollow portion in an environment where biological tissue material is present. The tissue structure manufacturing apparatus has a frame that forms the hollow portion. The frame has four or more openings that connect the hollow portion to the external space of the tissue structure manufacturing instrument, and these openings have an opening width of at least 2.5 mm. The depth of the hollow portion from the opening is at least 2 mm. The frame defines the shape of the tissue structure formed in the hollow portion.

10. The tissue structure according to claim 9, wherein, The tissue structure is in a state where it fills the hollow portion within the tissue structure manufacturing instrument, and the core portion is in close contact with the surface of the frame.

11. A method for collecting adult stem cells, the method comprising: Isolate adult stem cell aggregates or adult stem cells from the tissue structure according to any one of claims 1 to 10.

12. A tissue structure manufacturing apparatus, wherein the tissue structure manufacturing apparatus comprises: Hollow portion, and frame forming said hollow portion, The frame has four or more openings that connect the hollow portion to the external space of the tissue structure manufacturing instrument, and these openings have an opening width of at least 2.5 mm. The depth of the hollow portion from the opening is at least 2 mm. The tissue structure has a core and a sparsely fibrous aggregate of adult stem cells. The core has a concave portion and is composed of fibrous connective tissue. The sparsely fibrous aggregate of adult stem cells is formed in the concave portion and contains type III collagen and adult stem cells. The adult stem cells are selected from at least one of mesenchymal stem cells and pluripotent stem cells. The mesenchymal stem cells express at least one of the mesenchymal stem cell markers CD90 and CD105, and the pluripotent stem cells express at least one of the pluripotent stem cell markers SSEA4 and SSEA3.

13. The tissue structure manufacturing apparatus according to claim 12, used to form a tissue structure having a core and sparsely fibrous adult stem cell aggregate by placement in an environment containing biological tissue material, said core having a recess and composed of fibrous connective tissue, said sparsely fibrous adult stem cell aggregate formed in said recess and containing type III collagen and adult stem cells. The tissue structure manufacturing instrument has a hollow portion and a frame forming the hollow portion. The frame has an opening that connects the hollow portion to the external space of the tissue structure manufacturing instrument. The frame defines the shape of the tissue structure formed in the hollow portion. The tissue structure manufacturing apparatus is configured to, when placed in an environment in which biological tissue material is present, form fibrous connective tissue in a manner that extends from the surface of the frame toward the interior of the hollow portion, thereby generating a core portion having a recessed portion extending from the opening toward the hollow portion, and forming sparse fibrous tissue in the recessed portion in which adult stem cells are aggregated.

14. The tissue structure manufacturing apparatus according to claim 12, wherein, The frame contains a biocompatible metallic material.

15. The tissue structure manufacturing apparatus according to claim 12, wherein, The opening has an opening shape that can accommodate a circle with a diameter of 2.5 mm or more.

16. The tissue structure manufacturing apparatus according to claim 12, wherein, The frame is composed of columnar components and a support portion, the support portion fixing the columnar components to maintain the shape of the frame.

17. The tissue structure manufacturing apparatus according to claim 16, wherein, More than one columnar component is fixed at both ends by supports and is circumferentially arranged on the virtual cylindrical surface between the supports.

18. The tissue structure manufacturing apparatus according to claim 16, wherein, The opening is formed by a columnar component and a support, or by more than one columnar component.

19. The tissue structure manufacturing apparatus according to claim 12, wherein, The opening has polygonal, rectangular, trapezoidal, spherical, circular, or elliptical opening edges.

20. The tissue structure manufacturing apparatus according to claim 18, wherein, The support portion also has an opening that connects the hollow portion to the external space of the device.

21. A method for manufacturing an organizational structure, the method comprising: The tissue structure manufacturing apparatus according to any one of claims 12 to 20 is placed in an environment in the presence of biological tissue material, wherein the tissue structure has a core and a sparsely fibrous aggregate of adult stem cells, the core having a concave portion and being composed of fibrous connective tissue, and the sparsely fibrous aggregate of adult stem cells being formed in the concave portion and containing type III collagen and adult stem cells. The recess has an opening width of at least 2.5 mm. The adult stem cells are selected from at least one of mesenchymal stem cells and pluripotent stem cells. The mesenchymal stem cells express at least one of the mesenchymal stem cell markers CD90 and CD105, and the pluripotent stem cells express at least one of the pluripotent stem cell markers SSEA4 and SSEA3.

Images