Patent Application
20230235291
The present invention relates to a somatic stem cell-accumulated tissue construct and a device for producing the same.
The self-defense reaction to eliminate foreign substances causes macrophages and the like to be accumulated around foreign substances that have invaded into a living body. Macrophages adsorb to the surfaces of the foreign substances to degrade foreign substances, and promote collagen production in fibroblasts through TGF-β production by monocytes. As a result, the foreign substances that have invaded into a living body are covered by connective tissues comprising fibroblasts and collagen to be isolated in a living body (i.e., encapsulation). As a technology for forming living body-derived tissue on the basis of such phenomena, a technique of forming connective tissue by implanting a base material, which is a foreign substance, in a living body is reported (e.g., Patent Documents 1 to 6). For example, Patent Document 5 discloses that a shell member formed helically along the circumference of the rod-like structural member is implanted in a living body, thereby producing a connective tissue body formed on the surface of the rod-like structural member and including the shell member embedded therein. Patent Document 6 discloses that an assembly comprising a rod-like body and a stent fitted on the circumferential surface of the rod-like body is implanted in a living body to form a connective tissue body on the circumferential surface of the assembly, the resultant is isolated from the living body, and the rod-like body is removed therefrom, thereby obtaining a stent covered by the connective tissue body. However, the tissue formed on the surface of the base material via encapsulation generally has a thickness of only approximately several tens to several hundreds of micrometers, and most of the cells included therein are fibroblasts.
In recent years, regenerative therapies by implanting cells cultured ex vivo or tissue constructed therefrom in a living body have drawn attention as a technology for reproducing functions of tissues or organs impaired by diseases or injuries. Stem cells are important materials for production of cells of interest or construction of tissues in regenerative therapies. Meanwhile, the basis for the use in regenerative therapies of embryonic stem cells that are required to be ethically considered, or of induced pluripotent stem cells of which safety is required to be assured has not been yet established. Accordingly, a technique of efficiently collecting somatic stem cells that are safer and less problematic in terms of ethics has been awaited.
An object of the present invention is to provide a technique of efficiently collecting somatic stem cells.
The present inventors have conducted concentrated studies in order to solve the above problem. As a result, they found that a tissue construct comprising somatic stem cells accumulated therein could be produced. This has led to the completion of the present invention.
Specifically, the present invention includes the following.
[1] A tissue construct comprising a core portion having a recess and composed of fibrous connective tissue, and loose fibrous, somatic stem cell-accumulated tissue comprising type III collagen and somatic stem cells which is formed in the recess.
[2] The tissue construct according to [1] above, wherein the core portion has one, or two or more recesses.
[3] The tissue construct according to [1] or [2] above, wherein the core portion has at least three recesses.
[4] The tissue construct according to any of [1] to [3] above, wherein the recess has an opening width of at least 2.5 mm.
[5] The tissue construct according to any of [1] to [4] above, wherein the core portion has a diametrical dimension of at least 2.5 mm.
[6] The tissue construct according to any of [1] to [5] above, wherein the somatic stem cells comprise at least either mesenchymal stem cells or pluripotent stem cells.
[7] The tissue construct according to any of [1] to [6] above, wherein the tissue construct has a rod-like shape, and a plurality of the recesses are located along the circumferential direction.
[8] The tissue construct according to any of [1] to [7] above, wherein the tissue construct has a rod-like shape, and a plurality of the recesses are located along the axial direction.
[9] The tissue construct according to any of [1] to [6] above, wherein the tissue construct has a generally polyhedral shape, and a plurality of the recesses are located on different planes thereof.
[10] The tissue construct according to any of [1] to [9] above, wherein the tissue construct has a non-tubular and non-sheet shape.
[11] The tissue construct according to any of [1] to [10] above, which is formed by placing a device with a hollow portion for producing a tissue construct having a hollow portion in an environment comprising body tissue materials,
wherein the device for producing a tissue construct has a frame body that forms the hollow portion,
the frame body has an opening that communicates from the hollow portion to an external space of the device for producing a tissue construct, and
the frame body defines the shape of the tissue construct formed in the hollow portion.
[12] The tissue construct according to [11] above, wherein the tissue construct fills the hollow portion in the device for producing a tissue construct and the core portion is adhered to the surface of the frame body.
[13] A method for collecting somatic stem cells comprising separating somatic stem cell-accumulated tissue or somatic stem cells from the tissue construct according to any of [1] to [12] above.
[14] A device for producing a tissue construct comprising:
a hollow portion; and
a frame body that forms the hollow portion,
wherein the frame body has an opening that communicates from the hollow portion to an external space of the device for producing a tissue construct, and
the tissue construct comprises a core portion having a recess and composed of fibrous connective tissue, and loose fibrous, somatic stem cell-accumulated tissue comprising type III collagen and somatic stem cells which is formed in the recess.
[15] The device for producing a tissue construct according to [14] above, which is used to form a tissue construct comprising a core portion having a recess and composed of fibrous connective tissue, and loose fibrous, somatic stem cell-accumulated tissue comprising type III collagen and somatic stem cells which is formed in the recess by placing the device in an environment comprising body tissue materials,
wherein the device for producing a tissue construct comprises a hollow portion, and a frame body that forms the hollow portion,
the frame body has an opening that communicates from the hollow portion to an external space of the device for producing a tissue construct,
the frame body defines the shape of the tissue construct formed in the hollow portion, and
when the device is placed in the environment comprising body tissue materials, fibrous connective tissue is formed to stretch from the surface of the frame body toward the inside of the hollow portion, thereby causing the core portion having a recess recessed from the opening toward the hollow portion to be formed, and loose fibrous tissue where somatic stem cells are accumulated is formed in the recess.
[16] The device for producing a tissue construct according to [14] or [15] above, wherein the opening has an opening width of at least 2.5 mm.
[17] The device for producing a tissue construct according to any of [14] to [16] above, wherein the opening has an opening configuration that enables a circle with a diameter of 2.5 mm or larger to be inscribed therein.
[18] The device for producing a tissue construct according to any of [14] to [17] above, wherein the frame body is composed of a columnar member and a supporting member, and the columnar member is fixed by the supporting member to maintain the shape of the frame body.
[19] The device for producing a tissue construct according to [18] above, wherein a plurality of the columnar members are fixed to the supporting members at the both ends of the columnar members and arranged in a circumferential direction on a hypothetical cylindrical plane sandwiched between the supporting members.
[20] The device for producing a tissue construct according to [18] or [19] above, wherein the opening is formed by the columnar member and the supporting member, or a plurality of the columnar members.
[21] The device for producing a tissue construct according to any of [14] to [20] above, wherein the opening has a polygonal, rectangular, trapezoidal, round, circular, or elliptic opening edge.
[22] The device for producing a tissue construct according to any of [18] to [21] above, wherein the supporting member further has an opening that communicates from the hollow portion to an external space of the device.
[23] A method for producing a tissue construct comprising a core portion having a recess and composed of fibrous connective tissue, and loose fibrous somatic stem cell-accumulated tissue comprising type III collagen and somatic stem cells which is formed in the recess,
wherein the method comprises placing the device for producing a tissue construct according to any of [14] to [22] above in an environment comprising body tissue materials, such as in a living body or in an isolated body tissue.
This description includes the contents as disclosed in Japanese Patent Application No. 2020-103747 from which the present application claims priority.
According to the present invention, somatic stem cells can be collected efficiently.
Hereafter, the present invention is described in detail.
The present invention relates to a device for producing a tissue construct capable of efficiently accumulating somatic stem cells from body tissue, and a tissue construct that is rich in somatic stem cells and can be prepared using such device.
For example, the present invention relates to a device for producing a tissue construct comprising:
a hollow portion; and
a frame body that forms the hollow portion,
wherein the frame body has an opening, preferably, an opening that communicates from the hollow portion to an external space of the device for producing a tissue construct, and
the tissue construct comprises a core portion having a recess and composed of fibrous connective tissue, and loose fibrous somatic stem cell-accumulated tissue comprising type III collagen and somatic stem cells which is formed in the recess. The present invention also relates to a tissue construct produced with the use of such device for producing a tissue construct.
According to the present invention, the placement of the device for producing a tissue construct as described above in an environment comprising body tissue materials (e.g., in a living body/in vivo) enables cells to be the accumulated in the device for producing a tissue construct, and formation of a tissue construct comprising somatic stem cells accumulated therein to be induced in the device for producing a tissue construct.
Through the opening of the device for producing a tissue construct of the present invention placed in an environment comprising body tissue materials, cells and the like from body tissue materials enter a hollow portion (which is open to the external space of the device) of the device, and fibrous connective tissue is formed in the hollow portion. A recess of the fibrous connective tissue recessed from the opening toward the hollow portion of the device is formed directly below the opening of the device for producing a tissue construct, and the core portion having the recess and composed of fibrous connective tissue is formed from the surface of the device facing the hollow portion of the device toward the inside of the hollow portion of the device, and many somatic stem cells are accumulated in the recess, thereby forming the somatic stem cell-accumulated tissue. The placement of the device for producing a tissue construct in an environment comprising body tissue materials for a duration enough to form tissue (comprising fibrous connective tissue and somatic stem cell-accumulated tissue in the recess(es) thereof) to fill the hollow portion in the device for producing a tissue construct results in formulation of a tissue construct in the device. It is possible to easily and efficiently collect somatic stem cells from the somatic stem cell-accumulated tissue in the resulting tissue construct. The present invention is based on the findings by the present inventors as described above.
More specifically, the device for producing a tissue construct of the present invention comprises a frame body that forms a hollow portion of the device (e.g., composed of the frame body), and the frame body has an opening that communicates from the hollow portion to the external space of the device for producing a tissue construct. The device for producing a tissue construct is, preferably, configured to allow fibrous connective tissue to be formed to stretch from the surface of the frame body toward the inside of the hollow portion, thereby causing the core portion having a recess recessed from the opening toward the hollow portion to be formed, and to allow loose fibrous tissue where somatic stem cells are accumulated to be formed in the recess, when the device for producing a tissue construct is placed in an environment comprising body tissue materials (e.g., in a living body/in vivo). When a tissue construct is formed in the device for producing a tissue construct to fill the hollow portion formed by the frame body, the frame body defines the shape of a tissue construct (e.g., the shape of the whole tissue construct, the shape of the core portion, and the positions and the depths of the somatic stem cell-accumulated tissue/the recesses) formed in the hollow portion.
In an embodiment, the frame body may comprise a columnar member and a supporting member, and, for example, it may be composed of, a columnar member and a supporting member. The supporting member fixes the columnar member to maintain the shape of the frame body, thereby maintaining the shape of the hollow portion. A plurality of the columnar members, for example, may be fixed to the supporting member at the both ends of the columnar members. A plurality of the columnar members may further be fixed to a supporting member at the intermediate parts of the columnar members. The frame body preferably comprises a plurality of the columnar members. For example, the frame body may comprise 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 columnar members, although the number of columnar members is not limited thereto. The columnar member may have any shape such as, circular cylinder, elliptic column, prism, frustum, twist, or screw. The supporting member may be in any shape of, for example, a ring, disk, oval ring, oval disk, rectangular frame, or plate (e.g., a polygonal, trapezoidal, or atypical plate). The frame body may comprise 1, 2, 3, 4, or more supporting members. The frame body may comprise 2 or more structures tiered in the extending direction of the columnar members, each of the structures comprises a plurality of the columnar members fixed to a supporting member at the both ends of the columnar members and optionally at the intermediate parts (e.g., at 1, or 2 or more sites on the intermediate parts) of the columnar members. In such a case, for example, 3 to 20, 3 to 15, 3 to 10, 4 to 15, or 5 to 15 columnar members may be fixed to one supporting member, although the number of the columnar members to be fixed thereto is not limited thereto.
In an embodiment, in the frame body, a plurality of the columnar members may be fixed to a supporting member at the both ends of the columnar members and optionally at the intermediate parts of the columnar members and may be arranged in a circumferential direction on a hypothetical cylindrical plane (e.g., on a hypothetical circular cylindrical, oval cylindrical, or square cylindrical plane) sandwiched between the supporting members. In such a case, the supporting member may be in the shape of a ring, disk, oval ring, oval disk, rectangular frame, or plate (e.g., a polygonal, trapezoidal, or atypical plate), although the shape is not limited thereto. The plurality of the columnar members may be arranged at equal or different intervals on a hypothetical cylindrical plane sandwiched between the supporting members. The frame body may comprise 2 or more structures tiered in the extending direction of the columnar members, each of the structures comprises a plurality of the columnar members that are fixed to a supporting member at the both ends of the columnar members and optionally at the intermediate parts of the columnar members and arranged in the circumferential direction on a hypothetical cylindrical plane sandwiched between the supporting members. In such a case, for example, 3 to 20, 3 to 15, 3 to 10, 4 to 15, or 5 to 15 columnar members may be fixed to one supporting member, although the number of the columnar members to be fixed is not limited thereto.
In another embodiment, the frame body may comprise a plurality of the columnar members. For example, the frame body may be composed of a plurality of the columnar members, and the columnar members may be fixed to each other to maintain the shape of the frame body.
A columnar member being a component of the frame body may have a sectional width of at least 0.5 mm, or 0.5 mm to 5 mm, for example, 0.7 mm to 3 mm, or 0.7 mm to 1.5 mm, although the sectional width is not limited thereto.
A supporting member being a component of the frame body may have a sectional width of 5 mm to 30 mm, for example, 5 mm to 25 mm or 6 mm to 20 mm, although the sectional width is not limited thereto.
In another embodiment, the frame body may comprise a mesh or coiled linear member, and for example, may be composed of a mesh linear member. The linear member may have a sectional width of 0.5 mm to 5 mm, for example, 0.7 mm to 3 mm or 0.7 mm to 1.5 mm, although the sectional width is not limited thereto. The frame body may comprise a linear member and a supporting member, and, for example, the frame body may be composed of a linear member and a supporting member. Such supporting member fixes the linear member to maintain the shape of the frame body.
In the present invention, the term “sectional width” shall be defined as the longest distance between 2 parallel tangential lines that sandwich a section (in general, a transverse cross section) of a member or region of interest from the both sides, unless otherwise specified. The form of the cross section is not limited to a circular or elliptic form, and it may be, for example, a polygonal, trapezoidal, or atypical form.
The length in the longitudinal direction of the device for producing a tissue construct, the frame body, and the columnar member being a component of the device and the frame body may be 5 mm to 100 mm, for example, 15 mm to 90 mm, 20 mm to 80 mm, 15 mm to 70 mm, or 20 mm to 60 mm, although the length is not limited thereto.
The device for producing a tissue construct, the frame body, and the components thereof such as the columnar member, the supporting member, and the linear member, preferably comprise or are made of a biocompatible material. Examples of the biocompatible material include, but are not limited to, resin materials, such as acrylic resin or silicone resin, ceramics, metal materials such as stainless steel, cobalt-chromium alloy, titanium, titanium alloy materials (e.g., nickel-titanium alloy), platinum, and gold. The biocompatible material used in this context of the present invention preferably has rigidity to the extent that the biocompatible material would not be deformed in a living body at least within a period for production of the tissue construct.
The frame body may have 1, 2, 3, or 4 or more (e.g., 4, 5, 6, 7, 8, 9, or 10 or more) openings that communicates from the hollow portion of the device to an external space of the device for producing a tissue construct, in which a recess of the fibrous connective tissue being formed directly below the opening. For example, the number of the openings may be 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 opening may be preferably formed by a columnar member and a supporting member, or by a plurality of columnar members. Alternatively, the opening may be formed by a linear member, or by a linear member and a supporting member.
The supporting member itself of the frame body may have an opening that communicates from the hollow portion of the device to an external space of the device. For example, when the supporting member is in the shape of a ring, oval ring, rectangular frame, or the like, the supporting member may have the opening.
The opening of the frame body has an opening width that is wide enough to form the fibrous connective tissue/core portion, the recess of the fibrous connective tissue/core portion, and somatic stem cell-accumulated tissue in the recess. In the present invention, the term “opening width” of the opening of the frame body is defined as an opening width in the transverse direction of the opening. The opening of the frame body preferably has an opening width of at least 2.5 mm, for example, an opening width of 3.0 mm or larger. In an embodiment, the opening may 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 body is also referred to as the “opening width W” herein.
In the present invention, the terms “longitudinal (direction)” and “transverse (direction)” are used for any form or an object with any form. For example, such terms are used for various shapes or objects such as, in addition to a rectangle or cuboid, other polygons, circle, oval, circular cylinder, prism, cone, polygonal pyramid, frustum, and so on, and frame bodies having such shapes. In some shapes or objects, such as a circle, square, or cube, the length in the “longitudinal direction” may be the same as the length in the “transverse direction.”
The opening width of the opening of the frame body can be determined at the opening edge that is defined by points positioned on the component(s) (e.g., columnar member(s), supporting member(s), and/or linear member(s)) forming the opening, and in the same plane where the opening has the smallest opening area (see, for example,
Alternatively, or in addition to the above, the opening of the frame body preferably has an opening configuration (a shape and size) that enables a circle with a diameter of 2.5 mm or larger (e.g., a diameter of 3.0 mm or larger) to be inscribed in the opening. The opening of the frame body may have an opening configuration that enables a circle with a diameter of, 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, to be inscribed in the opening. The opening of the frame body having such opening configuration is particularly suitable to form a recess of the fibrous connective tissue and somatic stem cell-accumulated tissue in the recess. The opening of the frame body having “an opening configuration that enables a circle to be inscribed in the opening” used herein means an opening where a circle having a diameter of a given size can fit inside the opening configuration (e.g., a rectangular or polygonal configuration) of the opening, in contact with the opening at one or more points. For example, an opening with a rectangular configuration (longer side: 20 mm; shorter side: 2.5 mm) enables a circle with a diameter of up to 2.5 mm to be inscribed in the opening. In the present invention, an opening configuration of the opening of the frame body can be defined at positions on the opening where the opening has the smallest opening area. For example, such configuration can be defined on any plane (e.g., a plane) including the opening edge.
In an embodiment, the opening of the frame body may have an opening edge of any configuration, for example, having a polygonal, rectangular, trapezoidal, round, circular, or elliptic shape.
In at least one opening of the frame body of the device for producing a tissue construct according to the present invention, opposite sides of the opening edge may provide opening widths varying in the extending direction of the sides. When the device for producing a tissue construct has such configuration, a single opening can have varying opening widths.
The hollow portion formed by the frame body preferably has a sufficient depth to form the core portion. In the frame body, the depth of the hollow portion from the opening may be at least 2 mm. The depth of the hollow portion from the opening may be, for example, 2 mm to 30 mm, 2 mm to 26 mm, or 2 mm to 12 mm. The depth of the hollow portion from the opening in the frame body (referred to as the “depth D” herein) is determined, on a transverse cross section plane of the frame body, as the distance from the midpoint of the straight line connecting points located on the opening edge of a single opening, to the deepest point of the hollow portion (
Concerning the device for producing a tissue construct of the present invention, at least one the openings of the frame body is required to be configured as described above, and preferably all the openings of the frame body be configured as described above.
In the frame body of the device for producing a tissue construct according to the present invention, a plurality of the openings may be positioned adjacent to each other while sandwiching a columnar member. A plurality of the openings may be arranged along the circumferential direction of the frame body. Alternatively, a plurality of the openings may be arranged along the axial direction of the frame body. Alternatively, a plurality of the openings may be arranged along the circumferential direction and along the axial direction of the frame body. Such opening configuration is capable of increasing the density of the recesses to be formed on the outer part of the tissue construct, which are intended to accumulate somatic stem cells therein. In a preferred embodiment, accordingly, the opening configuration also enables the efficiency of somatic stem cell accumulation to be increased, and, in turn, the number of somatic stem cells collected with the device to be increased. When such device for producing a tissue construct is used, the core portion of the resulting tissue construct may comprise a plurality of the recesses separated by recess peripheries. In that case, each recess is adjacent to another recess across at least one recess edge.
It is preferred that the device for producing a tissue construct according to the present invention do not comprise other members such as rod-like members in the hollow portion formed by the frame body. It is preferred that the device would be configured not to be placed in an environment comprising body tissue materials in a state where other members such as rod-like members are set in the hollow portion of the device.
In an embodiment, the device for producing a tissue construct according to the present invention is one as illustrated in
One first supporting member 11A and one second supporting member 11B fix 3 circular cylindrical columnar members 13. The 3 columnar members 13 are arranged at equal intervals in the circumferential direction Ci on each circular cylindrical plane S, and 3 openings 10H are defined and formed by the 3 columnar members 13 together with the first supporting member 11A and the second supporting member 11B. The columnar members 13, the 2 first supporting members 11A, and the second supporting member 11B constitute the frame body of the device 10. Before the device 10 is implanted in an environment comprising body tissue materials, such as in body tissue in a living body, a space surrounded by the columnar members 13, the first supporting member 11A, and the second supporting member 11B is the hollow portion 10S, and each opening 10H communicates from the hollow portion 10S of the device to the external space of the device. When the device 10 is implanted in an environment comprising body tissue materials, such as in body tissue in a living body, cells and the like including fibroblasts can flow into the device from the body tissue toward the hollow portion 10S of the device through the through-holes 11H.
The distance between the center (center point) Ct on the cylindrical plane S on the cross section illustrated in
In the device 10 for producing a tissue construct implanted in an environment comprising body tissue materials, such as in body tissue in a living body, tissue formation is considered to occur as summarized in
As shown in
In another embodiment of the device 10 for producing a tissue construct, as shown in
In a further embodiment of the device for producing a tissue construct, as shown in
In a further embodiment of the device for producing a tissue construct, as shown in
In a further embodiment of the device 10 for producing a tissue construct, as shown in
In a further embodiment of the device for producing a tissue construct, as shown in
In a further embodiment of the device for producing a tissue construct, as shown in
In a further embodiment of the device for producing a tissue construct, as shown in
The device 10 as illustrated in
As shown in
The device 10 as illustrated in
As shown in
The device 10 as illustrated in
The positions of the openings 10H are not limited to positions aligned in the circumferential direction and/or the axial direction Ax of the device 10, and they may be helically and periodically positioned in the longitudinal direction of the device 10. The positions of the openings 10H are not necessarily configured to provide a repeat of single opening 10 in a given direction. For example, the positions of the openings 10H may be configured to form a group of openings composed of a plurality of openings 10H, and to provide a repeat of the group of openings in a given direction.
In a further embodiment of the device for producing a tissue construct, the shape of the device 10 or the frame body is not particularly limited, and it may be a rod-like shape, a generally polyhedral shape (e.g., a cube or cuboid), or a generally spherical or hemispherical shape. The shape of the device 10 or the frame body is not limited to a linear rod-like shape, and it may be a curved shape.
In a further embodiment of the device for producing a tissue construct, as shown in
In a further embodiment of the device for producing a tissue construct, for example as shown in
The frame body of the device 10 may have a configuration in which a plurality of the cubic or cuboid structures, for example, as illustrated in
In a further embodiment of the device for producing a tissue construct, the device 10 may be in the form of a stent. The device 10 in the form of a stent as illustrated in
The present invention also relates to a method for producing a tissue construct comprising somatic stem cells accumulated therein, with the use of the device for producing a tissue construct according to the present invention. The method for producing a tissue construct comprising somatic stem cells accumulated therein with the use of the device for producing a tissue construct comprises placing the device for producing a tissue construct in an environment comprising body tissue materials, such as in a living body or in body tissue. More specifically, the method comprises positioning (e.g., implanting) the device for producing a tissue construct in an environment comprising body tissue materials, such as in a living body or in body tissue, and placing the device therein for a given period of time. The environment comprising body tissue materials, such as body tissue in a living body or isolated body tissue, may comprise fibroblasts. The body tissue may be ectodermal tissue, mesodermal tissue, or endodermal tissue. The body tissue in which the device for producing a tissue construct is to be placed may be a tissue in healthy conditions in which somatic stem cells are present, or a tissue such as a damaged or lost tissue in which somatic stem cells necessary for tissue repair are present. A preferred example of the body tissue comprising fibroblasts is subcutaneous tissue.
The device for producing a tissue construct can be implanted and placed in body tissue in a living body. Alternatively, the device for producing a tissue construct may be placed in an artificial environment that mimics the body tissue in a living body; specifically, an in vitro culture system comprising isolated body tissue from a living body, or an environment constructed outside a living body to mimic the body tissue in a living body. Examples of the artificial environment that mimics the body tissue in a living body include an artificial tissue or artificial organ prepared by three-dimensional culture of isolated cells or tissue.
When implanting and placing the device for producing a tissue construct in body tissue in a living body, a living body and/or body tissue is incised, under anesthesia as appropriate, to form an insertion opening. Subsequently, the device for producing a tissue construct is inserted into the insertion opening. After the device for producing a tissue construct is implanted, the incisional wound is sutured.
Body tissue in which the device for producing a tissue construct is positioned (e.g., implanted) can be from any animal from which somatic stem cells may be obtained. The device for producing a tissue construct can be positioned (e.g., implanted) and placed in a living body of any animal. Examples of the animal include, but are not limited to, mammalian animals, including primates such as human, monkey, and chimpanzee, and dog, cat, cow, pig, horse, goat, sheep, rat, and mouse; birds, fish, and amphibians. In an embodiment, such animal may be human, or non-human animals, such as non-human mammalian animals. In an embodiment, the device for producing a tissue construct may be placed in body tissue in a living body or isolated body tissue from a living body of an animal individual in which somatic stem cells collected from the tissue construct to be produced with the device for producing a tissue construct or cells differentiated from the collected somatic stem cells are to be implanted (i.e., an animal subject, such as a human or non-human animal) or an individual that is genetically closely related to the animal individual. This enables the implantation of autologous somatic stem cells or somatic stem cells derived from a closely related individual. In the present invention, a part in the living body in which the device for producing a tissue construct is to be implanted is not particularly limited. For example, the device may be implanted in abdomen, chest, shoulder, back, limbs, or abdominal cavity.
The device for producing a tissue construct positioned in an environment comprising body tissue materials is placed therein until a core portion having a recess and composed of fibrous connective tissue and a tissue comprising somatic stem cells accumulated in the recess are formed in the hollow portion of the device.
The period of time for which the device for producing a tissue construct is placed in an environment comprising body tissue materials, such as in body tissue in a living body or the isolated body tissue, is preferably enough to enable somatic stem cells to be sufficiently accumulated without causing excessive inflammation in body tissue. The period of time can be adequately determined depending on, for example, the site of implantation, the organism species of the living body, and/or the size of the device. The period of time for placement may be, in general, 5 days to 4 months, and preferably 1 week (7 days) to 3 months, for example, 1 week to 2 months, 1 week to 5 weeks, 9 days to 5 weeks, 9 days to 4 weeks, 2 weeks to 5 weeks, 2 weeks to 4 weeks, 2 weeks to 3 weeks, or 3 weeks to 5 weeks.
After the period of time for placement, the device for producing a tissue construct is removed from the environment comprising body tissue materials. In the removed apparatus for producing a tissue construct, a tissue construct comprising a core portion comprising fibrous connective tissue, with a recess that comprises tissue comprising somatic stem cells accumulated therein is formed.
When removing the device 10 from the living body, the site of implantation of the device for producing a tissue construct is incised, under anesthesia as appropriate. After the device for producing a tissue construct is removed from the site of incision, the incisional wound is preferably sutured.
By removing the device for producing a tissue construct from the environment comprising body tissue materials, such as from body tissue in a living body as described above, the tissue construct can be isolated. The tissue construct may be separated from the removed device for producing a tissue construct to collect the tissue construct. With the use of the device for producing a tissue construct, as described above, a tissue construct can be produced.
The present invention also provides the tissue construct according to the present invention that can be produced with the use of the above-mentioned device for producing a tissue construct. More specifically, the present invention relates to a tissue construct comprising a core portion having a recess and composed of fibrous connective tissue, and a loose fibrous tissue containing type III collagen and somatic stem cells which is formed in the recess (somatic stem cell-accumulated tissue).
In the present invention, the fibrous connective tissue 22 composing the core portion 25 of the tissue construct is a rigid tissue with high fiber density that contains collagen fibers and fibroblasts as main components. In the present invention, the fibrous connective tissue composing the core portion can also be referred to as a “dense connective tissue,” and it is different from adipose tissue or loose connective tissue. In the present invention, it is preferred that the fibrous connective tissue composing the core portion comprise type I collagen, as a main collagen component. In the present invention, on the other hand, the content of type III collagen in the core portion according to the present invention is typically low, and, in particular, the content of type III collagen in the center part of the core portion is only very low. In a preferred embodiment of the present invention, the proportion of type I collagen to the total collagen in the core portion is approximately 65% to 90% by weight, for example, approximately 70% to 85% by weight; and the proportion of type III collagen to the total collagen in the core portion is approximately 5% to 30% by weight, for example, approximately 10% to 25% by weight (e.g.,
The somatic stem cell-accumulated tissue 23 formed in the recess (the pocket portion) is loose fibrous tissue which contains somatic stem cells, and particularly is enriched in somatic stem cells 23C. In the present invention, the somatic stem cell-accumulated tissue formed in the recess contains type III collagen, and the type III collagen content thereof is larger than the type III collagen content in the core portion. The somatic stem cell-accumulated tissue formed in the recess may also contain other cells, such as fibroblasts or vascular endothelial cells, and it may further contain collagen of other types or other substances. In a preferred embodiment of the present invention, the proportion of type I collagen to total collagen in the somatic stem cell-accumulated tissue is approximately 30% to 75% by weight, for example, approximately 35% to 65% by weight; and the proportion of type III collagen to total collagen is approximately 20% to 65% by weight, for example, approximately 30% to 60% by weight (e.g.,
In a preferable embodiment, the proportion of type III collagen to total collagen in the somatic stem cell-accumulated tissue formed in the recess (the pocket portion) of the tissue construct of the present invention is larger than the proportion of type III collagen to total collagen in the core portion of the tissue construct. In an embodiment, the former (i.e., the proportion of type III collagen in the pocket portion) may be 30% to 60% by weight, and the latter (i.e., the proportion of type III collagen in the core portion) may be 10% to 25% by weight.
In a preferred embodiment, the proportion of type I collagen to total collagen in the somatic stem cell-accumulated tissue formed in the recess (the pocket portion) of the tissue construct of the present invention is lower than the proportion of type I collagen to total collagen in the core portion of the tissue construct. In an embodiment, the former (i.e., the proportion of type I collagen in the pocket portion) may be 35% to 65% by weight, and the latter (i.e., the proportion of type I collagen in the core portion) may be 70% to 85% by weight.
The tissue construct according to the present invention, in particular, the core portion and the somatic stem cell-accumulated tissue, do not substantially comprise elastic fiber. The phrase “not substantially comprise elastic fiber” used in the context of the present invention refers to a situation in which no or substantially no elastic fibers are detectable by Elastica van Gieson (EVG) staining.
The tissue construct according to the present invention is formed in the hollow portion of the device for producing a tissue construct to fill the hollow portion of the device. Thus, the resulting tissue construct is shaped to fit the form of the hollow portion in the device. When the tissue construct is produced with the use of the device for producing a tissue construct as illustrated in
In the present invention, the “core portion” composed of the fibrous connective tissue is a core of the tissue construct which supports the somatic stem cell-accumulated tissue formed in the recess of the tissue construct. In a preferred embodiment of the present invention, the core portion continuously extends from the center part of the tissue construct toward the outermost surface of the tissue construct. In the present invention, the “center part” of the tissue construct, if it has a rod-like shape, for example, refers to the full-length of the central axis (the central axis in the longitudinal direction) or a part thereof of the tissue construct and/or a region in the vicinity thereof in the circumferential direction. The central axis is a line that connects center points (the geometric centers) of the transverse cross sections (cross sections orthogonal to the longitudinal direction) of the tissue construct. Alternatively, the “center part” of the tissue construct according to the present invention, if it has a generally polyhedral shape, for example, refers to the full-length of the central axis (the central axis in the longitudinal direction) or a part thereof of the tissue construct and/or a region in the vicinity thereof in the circumferential direction. The central axis is a line that connects center points (the geometric centers) of the transverse cross sections of the tissue construct.
The core portion of the tissue construct according to the present invention preferably has a sectional width of at least 2.5 mm. The “sectional width” of the core portion is as defined above, and it is determined as the longest distance between 2 parallel tangential lines that sandwich a transverse cross section of the core portion. Typically, the sectional width of the core portion may be the longest distance between the edges 24E of two recess peripheries 24 (
In an embodiment, the tissue construct or the core portion thereof according to the present invention may or may not be solid. The “solid” use in the context of the tissue construct or the core portion thereof means that the tissue construct and the core portion thereof are filled with tissue to the center part thereof. Concerning the tissue construct removed from the device for producing a tissue construct that had been placed in an environment comprising body tissue materials, a thin tissue membrane may also be formed outside of columnar members. However, the presence of such a thin tissue membrane does not indicate that the tissue construct according to the present invention or the core portion thereof is not solid.
The tissue construct according to the present invention has, on the core portion, at least one recess in which the somatic stem cell-accumulated tissue has been formed, and the tissue construct may have preferably 2 or more, and more preferably 3 or more such recesses. The tissue construct according to the present invention may have, on the core portion, for example, 4, 5, 6, 7, 8, 9, or 10 or more recesses in which the somatic stem cell-accumulated tissue is formed. The core portion of the tissue construct according to the present invention may have, 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 recesses in which the somatic stem cell-accumulated tissue is formed, per tissue construct. The recess is formed of a fibrous connective tissue. The inner surface of each recess on the core portion is typically configured to have a generally curved surface (e.g., in the form of a mortar).
In the tissue construct according to the present invention, the recess in which the somatic stem cell-accumulated tissue is formed preferably has an opening width WW of at least 2.5 mm, although the opening width is not limited thereto. For calculation, the opening width WW of the recess can be regarded as the same as the opening width W of the opening of the frame body of the device for producing a tissue construct used for the production of the tissue construct. Alternatively, the opening width WW of the recess of the tissue construct according to the present invention can be determined as the shortest distance between the edges (corresponding to the opening edge of the opening of the frame body) on the surface of the frame body (e.g., the columnar member, supporting member, or linear member) of the recess periphery 24 formed at the periphery of the frame body (e.g., the columnar member, supporting member, or linear member) of the device for producing a tissue construct, on the transverse cross section of the tissue construct after the removal of the device for producing a tissue construct. In the tissue construct according to the present invention, the recess on the core portion preferably has an opening configuration with the opening width WW of 2.5 mm or larger, 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, or in addition to the above, the recess in which the somatic stem cell-accumulated tissue is formed, of the core portion of the tissue construct according to the present invention preferably has an opening configuration (a shape and size) that enables a circle with a diameter of 2.5 mm or larger (e.g., a diameter of 3.0 mm or larger) to be inscribed in the opening. The opening of the recess may have a shape and a size that enable a circle with a diameter of, 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 to be inscribed in the opening. The opening of the recess having such shape and size corresponds to the opening configuration of the opening of the frame body that is particularly suitable to form a recess of the fibrous connective tissue and somatic stem cell-accumulated tissue in the recess. The opening of the recess on the core portion of the tissue construct according to the present invention having “an opening configuration that enables a circle to be inscribed in the opening” means an opening where a circle having a given diameter can fit inside the opening configuration (e.g., a rectangular or polygonal configuration) of the opening, in contact with the opening at one or more points. For example, an opening with a rectangular configuration having a longer edge of 20 mm and a shorter edge of 2.5 mm enables a circle with a diameter of up to 2.5 mm to be inscribed therein.
In an embodiment, a fibrous connective tissue or a core portion of the tissue construct according to the present invention may comprise a plurality of the recesses separated by recess peripheries.
In an embodiment, the outer surface of the fibrous connective tissue or the core portion of the tissue construct according to the present invention may be composed of a plurality of the recesses and a plurality of the recess peripheries.
The tissue construct according to the present invention may have any shape such as a rod-like (e.g., a generally cylindrical shape, or generally polygonal prism-like), generally polygonal, generally frustum-like, or generally spherical. In an embodiment, the tissue construct of the present invention may have a rod-like shape, and a plurality of the recesses may be located along the circumferential direction thereof. In another embodiment of the present invention, the tissue construct of the present invention may have a rod-like shape, and a plurality of the recesses may be located along the axial direction thereof. In another embodiment, the tissue construct of the present invention may have a rod-like shape, and a plurality of the recesses may be located along the axial direction and the circumferential direction thereof.
In a further embodiment, the tissue construct of the present invention may have a generally polyhedral shape, and a plurality of the recesses may be located on different faces of the polyhedron.
The tissue construct according to the present invention preferably has a non-tubular shape, and more preferably a non-sheet shape.
The tissue construct according to the present invention has a loose fibrous tissue formed in the recess of the core portion. In the tissue formed in the recess of the tissue construct according to the present invention, somatic stem cells are accumulated (somatic stem cell-accumulated tissue). Somatic stem cells contained in the somatic stem cell-accumulated tissue preferably comprise at least either mesenchymal stem cells or pluripotent stem cells, and more preferably comprise both the mesenchymal stem cells and the pluripotent stem cells. The somatic stem cell-accumulated tissue may comprise stem cells expressing stem cell markers, such as pluripotent stem cells expressing at least 1 pluripotent stem cell marker and/or mesenchymal stem cells expressing at least 1 mesenchymal stem cell marker. The somatic stem cell-accumulated tissue preferably comprises pluripotent stem cells expressing both the pluripotent stem cell markers SSEA4 and SSEA3. The somatic stem cell-accumulated tissue may also comprise stem cells, such as pluripotent stem cells, expressing at least either SSEA3 or SSEA4. Also, the somatic stem cell-accumulated tissue may also comprise mesenchymal stem cells expressing either or both the mesenchymal stem cell markers CD90 and CD105. Also, the somatic stem cell-accumulated tissue may comprise stem cells expressing the mesenchymal stem cell marker CD90, or both CD90 and SSEA3. Also, the somatic stem cell-accumulated tissue may comprise stem cells expressing the mesenchymal stem cell marker CD105, or both CD105 and SSEA3. Also, the somatic stem cell-accumulated tissue may comprise stem cells expressing the growth factor marker VEGF, or both VEGF and SSEA3. Such stem cells have a high angiogenic capacity. The somatic stem cell-accumulated tissue may also comprise stem cells expressing the growth factor marker HGF. The somatic stem cell-accumulated tissue in the tissue construct according to the present invention may comprise somatic stem cells (such as pluripotent stem cells or mesenchymal stem cells) expressing other markers, in addition to or instead of the markers indicated above.
In a preferred embodiment, the tissue construct of the present invention may comprise somatic stem cells (including mesenchymal stem cells and pluripotent stem cells) in a proportion of 1% or more, 5% or more, 10% or more, or 30% or more; and 60% or less, 50% or less, 40% or less, or 30% or less, for example, 1% to 60%, 5% to 40%, 5% to 30%, or 5% to 20%, relative to the total number of cells contained in the tissue construct (the proportion of stem cells based on the number of cells).
In a preferred embodiment, the somatic stem cell-accumulated tissue of the present invention may comprise somatic stem cells (including mesenchymal stem cells and pluripotent stem cells) in a proportion of 20% or more, 30% or more, or 50% or more; and 90% or less, 80% or less, or 70% or less; for example, 20% to 90%, 30% to 90%, 30% to 80%, or 50% to 90%, relative to the total number of cells contained in the somatic stem cell-accumulated tissue (the proportion of stem cells based on the number of cells).
In a preferred embodiment, when the tissue construct and/or the somatic stem cell-accumulated tissue of the present invention are subjected to a treatment in a 0.25% collagenase type I solution at 37° C. for 1.5 hours, as described in Example 4, the cells collected from the tissue degraded as a result of such treatment (i.e., the somatic stem cell-accumulated tissue and its vicinity) may comprise somatic stem cells (including mesenchymal stem cells and pluripotent stem cells) in a proportion of 20% or more, 30% or more, or 50% or more; and 90% or less, 80% or less, or 70% or less; for example, 20% to 90%, 30% to 90%, 30°/o to 80%, or 50% to 90%, relative to the total number of the collected cells (the proportion of stem cells based on the number of cells).
The above-mentioned proportion of stem cells can be calculated with the use of the number of cells expressing at least one stem cell marker (e.g., at least one marker selected from the group consisting of CD90, CD105, SSEA3, and SSEA4, or at least one marker selected from the group consisting of CD90, SSEA3, and SSEA4, although the marker is not limited thereto) as the number of stem cells (that is, the number of somatic stem cells).
In an embodiment, the tissue construct and the somatic stem cell-accumulated tissue of the present invention can comprise mesenchymal stem cells and pluripotent stem cells at a ratio of mesenchymal stem cells to pluripotent stem cells of 60 to 95:5 to 40; 60 to 80:10 to 30; or 65 to 75:15 to 25, although the ratio is not limited thereto. The ratio of mesenchymal stem cells to pluripotent stem cells can be calculated with the use of the number of cells expressing at least one mesenchymal stem cell marker (e.g., CD90 or CD105, although the marker is not limited thereto) as the number of mesenchymal stem cells, and the number of cells expressing at least one pluripotent stem cell marker (e.g., SSEA3 or SSEA4, although the marker is not limited thereto) as the number of pluripotent stem cells.
The somatic stem cell-accumulated tissue formed in the recess in the tissue construct according to the present invention comprises, in addition to the somatic stem cells as described above, type III collagen. The somatic stem cell-accumulated tissue may further comprise other cells, such as fibroblasts or vascular endothelial cells, collagen of other types, or other substances. The presence of vascular endothelial cells can be examined by detecting the expression of, for example, the vascular endothelial cell marker vWF. The presence of fibroblasts can be examined by detecting the expression of, for example, the fibroblast marker vimentin.
In the tissue construct according to the present invention, a high-level expression of VEGF can be observed particularly in a region on the relatively outer side. On the other hand, in fibroblasts that are contained at a high level in the center part of the tissue construct according to the present invention, an expression level of VEGF is typically low.
The tissue construct according to the present invention may be one formed by placing the device for producing a tissue construct in an environment comprising body tissue materials, such as in body tissue in a living body.
The tissue construct according to the present invention may be in a state as it remains in the device after the production of the tissue construct in an environment comprising body tissue materials with the use of the device for producing a tissue construct. In that case, it is preferred that the tissue construct fill the hollow portion in the device for producing a tissue construct and the core portion be adhered to the surface of the frame body (e.g., the columnar member, the supporting member, and/or the linear member) of the device. After the tissue construct according to the present invention is formed, alternatively, it may be separated from the device for producing a tissue construct.
The somatic stem cell-accumulated tissue formed in the recess of the fibrous connective tissue (the core portion) of the tissue construct according to the present invention can be easily collected separately from the fibrous connective tissue. For example, the somatic stem cell-accumulated tissue 23 can be easily scraped from the recess 22H of the fibrous connective tissue 22 (the core portion 25) of the tissue construct according to the present invention. Alternatively, the somatic stem cell-accumulated tissue 23 or the somatic stem cells 23C can be collected from the device 10 removed from the environment comprising body tissue materials, such as body tissue in a living body, by scraping, from the recess 22H, the somatic stem cell-accumulated tissue 23 comprising the accumulated somatic stem cells 23C or enzymatic degradation of the somatic stem cell-accumulated tissue 23. The device 10 removed from the environment comprising body tissue materials may be directly subjected to enzymatic treatment with a degrading enzyme, such as collagenase, elastase, trypsin, TrypLE™ (Thermo Fisher Scientific), Accutase® (Innovative Cell Technologies, Inc.), or Dispase, to separate the somatic stem cells 23C from the somatic stem cell-accumulated tissue 23 formed in the recess in the hollow portion of the device. Alternatively, the somatic stem cell-accumulated tissue 23 scraped from the recess 22H may be subjected to enzymatic treatment with a degrading enzyme, such as collagenase, elastase, trypsin, TrypLE™ (Thermo Fisher Scientific), Accutase® (Innovative Cell Technologies, Inc.), or Dispase, to separate the somatic stem cells 23C from the somatic stem cell-accumulated tissue 23. While the duration of enzymatic treatment depends on the amount of samples to be treated, typically, it may be approximately 0.5 to 2 hours. The cells survived after the enzymatic treatment may be separated from the fibrous connective tissue 22, and the somatic stem cells 23C may be collected therefrom.
A further step of isolation and purification may be performed to enhance the purification level of the somatic stem cells 23C. For example, a filtration treatment may be performed with the use of a membrane filter or a mesh filter to separate cells comprising the somatic stem cells 23C. The somatic stem cells 23C can be isolated and/or purified with the use of markers specifically expressed on the surfaces of somatic stem cells, such as stem cell markers (e.g., the pluripotent stem cell markers and/or the mesenchymal stem cell markers). For example, either or both the pluripotent stem cell markers SSEA4 and SSEA3, or either or both the mesenchymal stem cell markers CD105 and CD105 can be used as stem cell markers. Somatic stem cells can be easily separated and/or concentrated from the somatic stem cell-accumulated tissue, in accordance with a conventional technique including a cell sorting technique, such as magnetic cell sorting (MACS) or fluorescence-activated cell sorting (FACS).
The present invention also relates to a method for collecting somatic stem cells 23C comprising separating somatic stem cell-accumulated tissue or somatic stem cells from the tissue construct according to the present invention.
The somatic stem cells isolated from the somatic stem cell-accumulated tissue in the tissue construct according to the present invention may or may not be cultured and proliferated as appropriate. Alternatively, a cell population comprising the somatic stem cell-accumulated tissue or somatic stem cells partially purified from the somatic stem cell-accumulated tissue may or may not be cultured and proliferated as appropriate. The somatic stem cell-accumulated tissue, the cell population comprising the somatic stem cells, or the somatic stem cells can be used for tissue construction or cell differentiation in vitro or ex vivo. The somatic stem cell-accumulated tissue, the cell population comprising the somatic stem cells, or the somatic stem cells can be used, by administration to a subject (e.g., a patient), for regenerative medicine, such as tissue repair (tissue regeneration), prevention or treatment (e.g., repairment or improvement) of impairment (e.g., congenital or acquired defect and congenital or acquired dysfunction, malfunction, or damage) or functional deterioration of tissue or organs; or functional improvement of tissue or organs, by administering them to a target (e.g., a patient) and used for tissue engineering. Specifically, a cell-based medicine comprising the somatic stem cell-accumulated tissue, a cell population comprising the somatic stem cells, or the somatic stem cells obtained in the present invention may be used for regenerative therapies, such as tissue repair (tissue regeneration), prevention or treatment (e.g., repairment or improvement) of impairment or functional deterioration of tissue or organs, or functional improvement of tissue or organs. The present invention also provides a cell-based medicine (or a pharmaceutical composition) comprising the somatic stem cell-accumulated tissue obtained from the tissue construct according to the present invention, the somatic stem cells derived from the somatic stem cell-accumulated tissue, or a cell population comprising the somatic stem cells. The present invention also provides a method for producing a cell-based medicine (or a pharmaceutical composition) comprising preparing a cell-based medicine (or a pharmaceutical composition) using the somatic stem cell-accumulated tissue obtained from the tissue construct according to the present invention, the somatic stem cells derived from the somatic stem cell-accumulated tissue, or the cell population comprising the somatic stem cells. The cell-based medicine or pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable additive which includes, but are not limited to, carrier, solvent, excipient, moistening agent, stabilizing agent, tonicity agent, buffer, preservative, coloring agent, and cryoprotective agent.
The somatic stem cell-accumulated tissue obtained in the present invention, the cell population comprising the somatic stem cells, or the somatic stem cells may be differentiated into cells of interest by induction outside body tissue, followed by administration to a subject (e.g., a patient); or may be administered to an appropriate body tissue (e.g., a site of tissue or an organ with impairment or functional deterioration) without differentiation induction. The somatic stem cell-accumulated tissue obtained in the present invention, the cell population comprising the somatic stem cells, or the somatic stem cells can be cryopreserved in accordance with a conventional technique and can be administered when necessary. Differentiation induction may be performed in accordance with a conventional technique for cell differentiation induction or any known method. The present invention also provides a cell-based medicine (or a pharmaceutical composition) comprising the somatic stem cell-accumulated tissue of the present invention, the somatic stem cells derived from the somatic stem cell-accumulated tissue, or a cell population comprising the somatic stem cells, which have been subjected to the differentiation induction treatment; such as cells differentiated from the somatic stem cells derived from the somatic stem cell-accumulated tissue according to the present invention. The present invention also provides a method for producing a cell-based medicine (or a pharmaceutical composition) comprising preparing a cell-based medicine (or a pharmaceutical composition) using the somatic stem cell-accumulated tissue of the present invention, the somatic stem cells derived from the somatic stem cell-accumulated tissue, or the cell population comprising the somatic stem cells, which have been subjected to the differentiation induction treatment. The cell-based medicine or pharmaceutical composition may further comprise a pharmaceutically acceptable additive which includes, but are not limited to, carrier, solvent, excipient, moistening agent, stabilizing agent, tonicity agent, buffer, preservative, coloring agent, and cryoprotective agent.
The present invention also relates to a method for providing a regenerative therapy to a subject (e.g., a patient), a method of prevention or treatment (e.g., repairment or improvement) of impairment or functional deterioration of tissue or organ, or a method of functional improvement of tissue or organs, the method comprising administering the somatic stem cell-accumulated tissue according to the present invention, the somatic stem cells derived from the somatic stem cell-accumulated tissue, the cell population comprising the somatic stem cells, or cells differentiated from the somatic stem cells to the subject. Examples of the subject (e.g., patient) include, but are not limited to, mammalian animals, including primate, such as human, monkey, and chimpanzee, and dog, cat, cow, pig, horse, goat, sheep, rat, and mouse; birds, fish, and amphibians. In an embodiment, the subject (e.g., patient) may be human, or non-human animals, such as non-human mammalian animals. In an embodiment, the subject (e.g., patient) may be in need of a regenerative therapy, such as tissue repair (tissue regeneration), prevention or treatment (e.g., repairment or improvement) of impairment or functional deterioration of tissue or organs, or functional improvement of tissue or organs. The subject (e.g., patient) may have impairment or functional deterioration of tissue or organs, which can be expected to be cured via implantation of somatic stem cells or cells differentiated from somatic stem cells.
According to the present invention, a tissue construct comprising somatic stem cells accumulated therein can be produced, somatic stem cells can be efficiently collected, and somatic stem cells become easily obtained and used.
Hereafter, the present invention is described in greater detail with reference to the following Examples, although the technical scope of the present invention is not limited to these Examples.
In the Examples of this description, the test animal, the reagents, and the devices indicated below were used.
A beagle dog was anesthetized, the skin was incised with an electrosurgical knife at 5 sites from the right lateroabdominal region to the back, one device for producing a tissue construct having the configuration illustrated in
The device removed from the subcutaneous region was almost entirely covered with soft tissue. The cross section of the device was observed and the device was found to be filled with tissue to the center of the hollow portion (hollow space) of the device. It was considered that cells were accumulated in the hollow portion of the device for producing a tissue construct that had been implanted and placed in the living body, and tissue formation occurred as a result. A difference in the site of implantation did not substantially affect tissue formation.
It was shown that a tissue construct could be produced by implanting and placing the device for producing a tissue construct according to the present invention in vivo.
The device for producing a tissue construct removed from the subcutaneous region was transferred into a 15-ml centrifuge tube containing a tissue preservation medium HypoThermosol® FRS, placed on ice, stored at 4° C., and subjected to the subsequent treatment within 24 hours thereafter.
In a clean bench, the device for producing a tissue construct having the tissue construct formed therein was removed from the tissue preservation medium, the device was gently washed in a centrifuge tube containing PBS, and excess tissue around the device was trimmed away. Thereafter, the device was photographed and stored in DMEM.
The device for producing a tissue construct removed from the living body in Example a was treated with 4% PFA at 4° C. for 24 hours to fix tissue, and the device was then detached from the tissue construct and a paraffin-embedded block of the tissue construct was prepared.
The paraffin-embedded block was transversely cut with a microtome to prepare sections with thickness of 3 to 5 μm. The sections were subjected to hematoxylin-eosin (HE) staining, Masson trichrome (MT) staining, Elastica van Gieson (EVG) staining, and Sirius Red (SR) staining, and then observed under the microscope.
As a result of staining, the tissue construct was stained generally pink via HE staining (
No region was stained black with EVG staining. This indicates that the tissue construct comprises substantially no elastic fibers (
In order to determine the type of collagen present in the tissue construct, the sections subjected to Sirius Red (SR) staining were observed under a polarizing microscope. In polarizing microscopy, type I collagen is observed as yellow to orange, and type III collagen is observed as green.
As a result of observation, densely aligned collagen fibers were observed as yellow or orange in a region deeply stained with Sirius Red (i.e., around the columnar members of the device and the center part of the tissue construct). This indicates that type I collagen is mainly present in such region (
In order to determine the types of cells present in the tissue construct and the distribution thereof, immunohistochemical staining of the tissue construct was performed. Sections of the tissue construct with thickness of 3 to 5 μm were subjected to immunohistochemical staining to detect the mesenchymal stem cell markers CD90 and CD105, the pluripotent stem cell markers SSEA4 and SSEA3, the vascular endothelial growth factor (VEGF), and the hepatic cell growth factor (HGF) with the use of primary antibodies and secondary antibodies against them. In addition, cell nuclear staining was performed by the treatment with DAPI (4′,6-diamidino-2-phenylindole). The results are shown in
In the staining for detecting the mesenchymal stem cell markers CD90 and CD105, many cells were stained in the pocket portions. Even around the columnar members, positive cells were observed near the pocket portions. In
In the staining for detecting the pluripotent stem cell markers SSEA4 and SSEA3, many cells were stained in the pocket portions, as in the case of the mesenchymal stem cell markers. In
Regarding the expression inside the tissue construct, the VEGF expression level was high in a region on the relatively outer side of the entire tissue construct, and the VEGF expression level was particularly high in round cells in the pocket portions (
In order to examine properties of the stem cells, dual staining for the stem cell marker SSEA3 and other markers (CD90 and SSEA3; CD105 and SSEA3; SSEA4 and SSEA3; and VEGF and SSEA3) was performed.
As a result of dual staining, double positive cells (indicated by white arrowheads) as well as cells positive for a single marker (indicated by horizontal-striped arrowheads and dotted arrowheads) were observed (
These results mentioned above demonstrate that stem cells are accumulated and activated in the pocket portions, so as to promote tissue formation. That is, the tissue (pocket portion) formed in the recess is a tissue in which somatic stem cells are accumulated (“somatic stem cell-accumulated tissue”). The production of a tissue construct comprising such pocket portion enables activated stem cells to be efficiently corrected and available.
DMEM (serum-free; 20 ml) was added to 50 mg of collagenase, type I in a vial to prepare a 0.25% collagenase solution. The device for producing a tissue construct comprising the tissue construct formed therein, which was removed from the living body in Example 1, was transferred into a 15-ml centrifuge tube containing 6 ml of the collagenase solution prepared, and the tube was incubated at 37° C. with shaking in an incubator. After the incubation with shaking for 1.5 hours, the device and the tissue undegraded with collagenase were removed away. The centrifuge tube was centrifuged at 4° C. and 1,000 rpm for 5 minutes and the supernatant was removed. DMEM (6 ml) was added, pipetted, and then cells were collected into 50-ml centrifuge tubes (4 tubes, 24 ml). The collected fraction was centrifuged at 4° C. and 1,000 rpm for 5 minutes and the supernatant was removed.
In order to hemolyze the erythrocytes, 1 ml of the VersaLyse Lysing Solution was added to the collected cells, pipetted, and the cells were incubated at room temperature for 10 minutes. DMEM supplemented with 10% FBS (10 ml) was added, the resultant was centrifuged at 4° C. and 1,000 rpm for 5 minutes, and the supernatant was removed. The cell cryopreservation medium Bambanker® (5 ml) was added thereto to prepare a suspension, 10 μl of the cell suspension was sampled, an equal volume of a trypan blue stain solution was added for staining, and the number of stained cells was counted. The cell suspension was divided into 1 ml aliquots in cryotubes and stored in a liquid nitrogen tank.
The cell concentration of the 5 ml suspension in the Bambanker® was 6.25×106 cells/ml, and the cell viability was 98.4%. Thus, approximately 4×106 cells were collected per tissue construct. Also in the other experiments performed independently, comparable number of cells were collected. Under the treatment conditions of this Example, substantially the whole somatic stem cell-accumulated tissue and the connective tissue in the vicinity thereof (i.e., the peripheral region of the tissue construct) were degraded; however, the center part of the tissue construct (in particular, that of the core portion) was not degraded.
In order to determine the cell abundance ratio in the collected cell population, the CD90-positive cells, the SSEA3-positive cells, and the SSEA4-positive cells were counted using a flow cytometer.
As a result, the percentage of the CD90-positive cells was 73%, that of the SSEA3-positive cells was 23%, and that of the SSEA4-positive cells was 18% in the collected cell population (
Upon collagenase treatment, degradation of the tissue construct begins from the outer side and cells are separated therefrom. Accordingly, most cells separated at the early stage of collagenase treatment were somatic stem cells derived from the somatic stem cell-accumulated tissue. It was shown that the proportion of fibroblasts would increase depending on the duration of collagenase treatment.
The amount of the mesenchymal stem cells contained in bone marrow is as small as 10 to 100 cells/ml, and that in adipose tissue is as small as 5×103 cells/g, both of which are common sources of mesenchymal stem cells. Since the cells from the tissue construct according to the present invention contain stem cells at a high proportion, the present invention enables stem cells to be collected with higher efficiency, compared with a conventional method for stem cell collection.
From the cell population collected from the tissue construct subjected to collagenase treatment in the same way as in Example 4, CD90- or SSEA3-positive stem cells were separated and concentrated. Specifically, the collected cell population was labeled with anti-CD90 antibody (BioLegend, 328107) or anti-SSEA3 antibody (Bioss, bs-3575R), reacted with magnetic bead-conjugated antibodies; anti-FITC MicroBeads (Miltenyi Biotec, 130-048-700) and goat anti-rabbit IgG MicroBeads (Miltenyi Biotec, 130-048-600), and then subjected to the cell separation via magnetic cell sorting (MACS).
As a result, CD90-positive cells were concentrated from 62% (before separation) to 90% (after separation) in the cell proportion (
The cell population before cell separation via MACS (control) and the cell population after the cell separation were seeded on a 6-well plate at a density of 3,000 cells/cm2, cultured in DMEM supplemented with 10% FBS, and the cell morphology was observed. As a result, fibroblast-like cells were found to have adhered to the bottom of the plate and have proliferated (
As such, with the use of the tissue construct according to the present invention as a cell source, stem cells of interest could be concentrated efficiently.
The skin of a beagle dog was punctured and then a balloon catheter with a closed stent (having a length of 29 mm and a diameter of 20 mm, in the expanded state) mounted thereon was inserted thereinto subcutaneously (
The hydraulic pressure was released, the balloon was deflated and the catheter was withdrawn, thereby subcutaneously placing the stent in the expanded state. The beagle dog was anesthetized 3 weeks later, the skin was incised, and the stent placed subcutaneously was isolated.
The stent was found to be filled with tissue to the center part (
The devices for producing a tissue construct according to the present invention with various configurations were used to produce tissue constructs. Five types of devices for producing a tissue construct 10 having the configurations illustrated in
The device for producing a tissue construct removed from the subcutaneous region was almost entirely covered with soft tissue, and the hollow portion (hollow space) of the device was filled with tissue. As with the above-mentioned Examples, formation of the tissue construct was observed.
The tissue construct removed from the device was subjected to fiber staining to stain collagen fibers in the same way as in Example 2. As one example thereof,
On the basis of the result of tissue analysis of the tissue constructs, it was found that the formation of fibrous connective tissue progresses so as to stretch from the surface of each columnar member of the device for producing a tissue construct toward the inside of the hollow portion of the device, and the connective tissues formed around columnar members adjacent to each other become joined to each other, thereby causing a core portion having, on its outer surface, a recess that is recessed from the opening of the device toward the hollow portion of the device to be formed; and somatic stem cells derived from the living body are accumulated in the recess to form a somatic stem cell-accumulated tissue.
In addition, immunohistochemical staining was performed in the same way as in Example 3. In the connective tissue of the core portion, while many DAPI-stained cell nuclei were detected, mesenchymal stem cell marker CD90 expression was negative, and pluripotent stem cell marker SSEA-4 expression was also negative. In contrast, many CD90-positive somatic stem cells and many SSEA-4-positive somatic stem cells were detected in the somatic stem cell-accumulated tissue (the pocket portion).
When the device for producing a tissue construct was placed for 3 days after implantation in a living body, formation of the core portion having a recess on its outer surface was shown, but none of CD90-positive cells and SSEA-4-positive cells were detected in the somatic stem cell-accumulated tissue and in the core portion. When the device was placed for 10 days, 2 weeks, 17 days, or 3 weeks, CD90-positive cells and SSEA4-positive cells were shown in the core portion.
As shown in
The number of CD90-positive cells in the somatic stem cell-accumulated tissue detected when the device was placed for 2 weeks was approximately intermediate between the number of the cells detected when the device was placed for 10 days and the number of cells detected when the device was placed for 3 weeks. The number of SSEA-4-positive cells in the somatic stem cell-accumulated tissue detected when the device was placed for 2 weeks was only somewhat higher than that detected when the device was placed for 10 days. This indicates that SSEA-4-positive cells in the somatic stem cell-accumulated tissue largely increased between 2 weeks and 3 weeks after the implantation in a living body.
The results above indicate that the number of somatic stem cells in the somatic stem cell-accumulated tissue increases depending on the duration during which the device for producing a tissue construct is placed in a living body.
Concerning the association between the structures of the devices for producing a tissue construct illustrated in
When the device for producing a tissue construct of the configuration illustrated in
When the device for producing a tissue construct of the configuration illustrated in
When the device for producing a tissue construct of the configuration illustrated in
When the device for producing a tissue construct of the configuration illustrated in
When the device for producing a tissue construct of the configuration illustrated in
When the device for producing a tissue construct of the configuration illustrated in
When the device for producing a tissue construct of the configuration illustrated in
The Examples above show that formation of recess 22H was formed in the tissue construct within a range of the opening width W of 2.5 mm or larger of the device for producing a tissue construct. In contrast, formation of recess 22H was not observed directly below an opening with the opening width W of smaller than 2.5 mm. At the least, when the depth D or the maximal depth D was 2 mm or more, formation of the recess 22H was not affected.
It was shown that the invention using the device 10 for producing a tissue construct according to the present invention enables a tissue construct comprising the somatic stem cells 23C accumulated in the recess 22H and enriched in the somatic stem cells 23C to be produced in a shorter period of placement in a living body, such as within one month or even in approximately 2 to 3 weeks.
All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.
Ax: axial direction; Ci: circumferential direction; Ct: center point, D: depth; S: circular cylindrical plane; W: opening width; 10: device for producing a tissue construct; 10F: opening edge; 10H: opening; 10S: hollow portion; 11: supporting member; 11A: first supporting member; 11B: second supporting member; 11H: through-hole; 12H: pass-through opening; 13: columnar member; 16: linear member; 21: body tissue; 22: fibrous connective tissue; 22C: fibroblast; 22H: recess; 23: somatic stem cell-accumulated tissue; 23C: somatic stem cell; 24: recess periphery; 25: core portion.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2020-103747 | Jun 2020 | JP | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2021/020347 | 5/28/2021 | WO |
