JP7222404B2 - Cell-sheet-forming member, method for producing cell-sheet-forming member, and method for producing cell-sheet - Google Patents

Description

TECHNICAL FIELD The present disclosure relates to a cell sheet forming member for forming a cell sheet, a method for producing a cell sheet forming member, and a method for producing a cell sheet.

Various methods for culturing cells have been conventionally proposed. For example, in the method for culturing cells described in Patent Document 1, a plurality of spatial structures partitioned by fine side walls are provided in a cell culture substrate, and the direction in which the spatial structures communicate with each other and the direction in which cells extend are separated. Alignment controls the orientation of the cells in the cell sheet (see Patent Document 1).

JP 2006-191809 A

By the way, for example, the cell sheet described in Patent Literature 1 is fragile and may be damaged when peeled off from the cell culture substrate.
An object of the present disclosure is to provide a cell sheet-forming member, a method for manufacturing a cell-sheet-forming member, and a method for manufacturing a cell sheet that facilitates the detachment of a cell sheet.

A cell sheet-forming member according to an aspect of the present disclosure includes a surface for forming a cell sheet, the surface comprising a culturing unit for culturing cells, and a surface located outside the culturing unit, wherein the cell sheet and a peeling assisting portion that forms a peeling start site from the surface of the culture portion, the culture portion includes a plurality of first flat portions and a plurality of first uneven portions, each of the first flat portions It has a shape extending in a first direction, the first flat portions are arranged in a second direction intersecting the first direction, and the first concave and convex portions are adjacent to each other. a first stepped structure that fills the gap, the first stepped structure is composed of convex portions arranged at a first pitch, and the peeling assisting portion is composed of convex portions arranged at a second pitch It is composed of a second uneven portion or a second flat portion having a second stepped structure.

According to the above configuration, the cell sheet-forming member has the detachment assisting part outside the culture part, so that the cell sheet can be easily detached. That is, when the detachment assisting portion is composed of the second concave-convex portion having the second stepped structure, the contact area of the detachment assisting portion with the cell sheet is smaller than the contact area with the cell sheet of the culturing portion. , the adhesive strength also decreases. Therefore, the outer peripheral portion of the cell sheet is easily peeled off from the surface of the cell sheet-forming member. Further, when the peeling assisting portion is composed of the second flat portion, the strength of the outer peripheral portion of the cell sheet is increased, and the outer peripheral portion of the cell sheet is less likely to be damaged when peeled from the surface.

In the cell sheet-forming member, for example, the cells cultured in the cell sheet-forming member are at least one selected from the group consisting of myoblasts, fibroblasts, and cardiomyocytes. According to the above configuration, it is possible to easily produce an oriented cell sheet of myoblasts, fibroblasts, cardiomyocytes, and the like, and easily remove the cell sheet from the cell sheet forming member without damaging the cell sheet. can be peeled off.

In the above-described cell sheet-forming member, the peeling assisting portion is composed of a second uneven portion having a second stepped structure composed of convex portions arranged at a second pitch, and the second pitch is equal to the It is preferably equal to or greater than the first pitch. According to the above configuration, the contact area with the cell sheet in the detachment assisting section is smaller than the contact area with the cell sheet in the culturing section, and the adhesive strength is also low. Therefore, the outer peripheral portion of the cell sheet is easily peeled off from the surface of the cell sheet-forming member.

In the cell sheet-forming member, for example, it is preferable that the first pitch is 10 nm or more and 10 μm or less, and the second pitch is 100 nm or more and 10 μm or less.

In the cell sheet-forming member, it is preferable that the detachment assisting portion is a single annular portion surrounding the culture portion. According to the above configuration, the cell sheet can be easily peeled off from the peeling assisting portion composed of the single annular portion.

In the above-described cell sheet-forming member, the peeling assisting portion is composed of a second uneven portion having a second stepped structure composed of convex portions arranged at a second pitch, and the peeling assisting portion includes the Two or more annular parts surrounding the culture part, wherein the outer annular part has a concavo-convex structure with a pitch equal to or larger than that of the inner annular part. is preferred. According to the above configuration, the adhesive strength of the cell sheet can be weakened toward the outside. Thereby, the cell sheet can be easily peeled from the outside in the peeling assisting part.

In the cell-sheet-forming member, the surface is preferably coated with a temperature-responsive polymer. According to the above configuration, the cell sheet can be easily peeled off and collected.

In the above-mentioned cell sheet-forming member, the detachment assisting portion is composed of a second uneven portion having a second stepped structure composed of convex portions arranged at a second pitch, and is parallel to the surface. It is preferable that a contact area per unit area between the virtual plane and the peeling assisting portion is smaller than a contact area per unit area between the virtual plane and the first concave-convex portion. According to the above configuration, the area per unit area of contact between the virtual plane parallel to the surface and the peeling assisting portion is smaller than the area per unit area of contact between the virtual plane and the first uneven portion. Therefore, the adhesive strength is also low. Therefore, the outer peripheral portion of the cell sheet is easily peeled off from the surface of the cell sheet-forming member.

In the cell sheet-forming member, the detachment assisting portion is composed of a second flat portion, and the area per unit area of contact between the imaginary plane parallel to the surface and the detachment assisting portion is equal to the imaginary It is preferable that the contact area between the flat surface and the first concave-convex portion is larger than the contact area per unit area. According to the above configuration, the area per unit area of contact between the virtual plane parallel to the surface and the peeling assisting portion is larger than the area per unit area of contact between the virtual plane and the first concave-convex portion. , the strength of the outer peripheral portion of the cell sheet increases, and the outer peripheral portion of the cell sheet is less likely to be damaged when peeled off from the surface.

A cell sheet-forming member according to another aspect of the present disclosure includes a surface for forming a cell sheet, the surface comprising a culturing unit for culturing cells, and the cell sheet forming member located at the edge of the surface. a peeling assisting portion that forms a peel initiation site from the surface; the culture portion includes a plurality of first flat portions; and a plurality of first uneven portions; The first flat portion has a shape extending in a direction, and the first flat portion is arranged in a second direction that intersects with the first direction, and a virtual plane parallel to the surface and the peeling assisting portion are formed per unit area The contact area is smaller than the contact area per unit area between the virtual plane and the first concave-convex portion.

According to the above configuration, the area per unit area of contact between the virtual plane parallel to the surface and the peeling assisting portion is smaller than the area per unit area of contact between the virtual plane and the first uneven portion. Therefore, the adhesive strength is also low. Therefore, the outer peripheral portion of the cell sheet is easily peeled off from the surface of the cell sheet-forming member.

A cell sheet-forming member according to still another aspect of the present disclosure includes a surface for forming a cell sheet, the surface comprising a culture section for culturing cells, and a cell sheet-forming member located at the edge of the surface. and a peeling assisting portion that forms a peeling start site from the surface of the culture portion, the culture portion includes a plurality of first flat portions and a plurality of first uneven portions, each of the first flat portions The first flat portion has a shape extending in a first direction, and the first flat portion is arranged in a second direction that intersects with the first direction, and a virtual plane parallel to the surface and the peeling assisting portion have a unit area The contact area is larger than the contact area per unit area between the virtual plane and the first concave-convex portion.

According to the above configuration, the area per unit area of contact between the virtual plane parallel to the surface and the peeling assisting portion is larger than the area per unit area of contact between the virtual plane and the first concave-convex portion. , the strength of the outer peripheral portion of the cell sheet increases, and the outer peripheral portion of the cell sheet is less likely to be damaged when peeled off from the surface.

A method for manufacturing a cell sheet-forming member for solving the above problems includes forming an intaglio, and forming a surface of the cell sheet-forming member for forming the cell sheet by transferring the intaglio, The surface includes a culture portion for culturing cells, and a peeling assisting portion located outside the culture portion and forming a peeling start site of the cell sheet from the surface, wherein the culture portion includes a plurality of A first flat portion and a plurality of first uneven portions, each of the first flat portions having a shape extending in a first direction, and the first flat portions intersecting the first direction and each of the first uneven portions includes a first stepped structure filling between the adjacent first flat portions, and the first stepped structures are arranged at a first pitch. The peeling assisting portion is composed of a second uneven portion having a second stepped structure composed of projections arranged at a second pitch, or a second flat portion. , the intaglio includes a first flat molding portion for molding the first flat portion, a first uneven molding portion for molding the first uneven portion, and a second uneven portion for molding the second uneven portion or the second flat portion. and forming the intaglio by using a photolithography method, a colloidal lithography method, an anodization method, a photolithography method, a colloidal lithography method, an anodization method, and forming using at least one interference exposure method.

In the method for producing a cell sheet according to an aspect of the present disclosure, the cell sheet-forming member as described above is used, and the adhesion to one of the first flat portion and the first uneven portion is superior to the adhesion to the other. forming a cell sheet on the surface of the cell sheet-forming member by causing certain cells to adhere to the surface of the cell sheet-forming member; and and peeling a sheet from said surface.

(a) is a perspective view showing the structure of the cell-sheet-forming member according to the present disclosure together with a Petri dish, (b) is a plan view showing the entire surface of the cell-sheet-forming member in FIG. FIG. 1(b) is an enlarged perspective view showing a part of the cultured part surface of the cell sheet-forming member, and (d) is an enlarged part of the cultured part surface of the cell sheet-forming member of FIG. 1(b). , (e) is a partial cross-sectional view showing an enlarged part of the culture portion of the cell sheet-forming member of FIG. 1 (b), and (f) is the cell sheet-forming member of FIG. An image taken by a scanning electron microscope of the surface of (a) is a plan view of a first cell-sheet-forming member, and (b) is a cross-sectional view of a main part of the first cell-sheet-forming member of FIG. 2(a). (a) is a plan view of a second cell-sheet-forming member, and (b) is a cross-sectional view of the second cell-sheet-forming member of FIG. 3(a). (a) is a plan view of a third cell-sheet-forming member, and (b) is a cross-sectional view of the main parts of the third cell-sheet-forming member of FIG. 4(a). (a) is a plan view of a fourth cell-sheet-forming member, and (b) is a cross-sectional view of a main part of the fourth cell-sheet-forming member in FIG. 5(a). Process drawing for demonstrating an example of the manufacturing method of the cell-sheet formation member shown to Fig.2 (a) and FIG.2(b). (a) to (c) are schematic diagrams for explaining the manufacturing process of a cell sheet. Fluorescent staining images of myoblasts cultured using the cell sheet forming member according to the present disclosure. A fluorescent staining image of myoblasts cultured using a cell culture petri dish as a reference example. (a) to (c) are schematic diagrams for explaining the manufacturing process of a cell sheet. (a) to (c) are schematic diagrams for explaining the manufacturing process of a cell sheet. (a) is a diagram showing cultured cells, (b) is a diagram showing a state in which the cultured cells are coated with an adhesive membrane, and (c) is a diagram showing the state in which the cultured cells are three-dimensionally organized. (d) is a fluorescence-stained image of myoblasts cultured using the cell sheet-forming member.

Embodiments of a cell sheet-forming member, a method for producing a cell sheet-forming member, and a method for producing a cell sheet will be described below. First, the structure of the cell-sheet-forming member will be described, and then the method for manufacturing the cell-sheet-forming member and the method for manufacturing the cell sheet will be described.

[Cell sheet forming member]
As shown in FIG. 1(a), the cell sheet-forming member 100 is, for example, a sheet material placed on a culture dish 110 of a Petri dish. The cell sheet-forming member 100 may be placed on the culture dish 110, or may be provided by directly processing a petri dish. When the cell-sheet-forming member 100 is provided by directly processing a petri dish, the cell-sheet-forming member 100 is formed, for example, by injection-molding the petri dish. The petri dish holds a cell suspension in a space surrounded by the culture dish 110 and the lid 120 . Cells contained in the cell suspension are, for example, at least one selected from the group consisting of myoblasts, fibroblasts, and cardiomyocytes, but are not limited thereto.

As shown in FIG. 1(b), the surface 111 of the cell sheet-forming member 100 includes a culturing portion 101 for culturing cells and a detachment assisting portion 102 that facilitates detachment of the cell sheet from the surface 111. As shown in FIG. The exfoliation assisting portion 102 is configured as an annular portion surrounding the culture portion 101 outside the culture portion 101 . Further, the peeling assisting portion 102 is positioned at the edge of the surface 111 . In the detachment assisting portion 102, as an example, the adhesive force of the cell sheet to the detachment assisting portion 102 is set to be lower than the adhesive force of the cell sheet to the culture portion 101, and the outer peripheral portion of the cell sheet serves as the detachment starting site. It is configured to be easily peeled off from the surface 111 . Further, in the detachment assisting portion 102, as another example, the adhesive force of the cell sheet to the detachment assisting portion 102 is made higher than the adhesive force of the cell sheet to the culture portion 101 to increase the strength of the outer peripheral portion of the cell sheet, The peripheral part of the cell sheet is made less likely to be damaged when peeled off from the surface 111. - 特許庁

[Culturing department]
As shown in FIG. 1C, the culture section 101 includes a plurality of first flat portions 130 and a plurality of first uneven portions 140. As shown in FIG. Each first concave-convex portion 140 has a stepped structure (first stepped structure), and a plurality of stepped structures fill spaces between adjacent first flat portions 130 . The stepped structure is a protrusion or a recess. The stepped structure in the present embodiment is the convex portion 141, and the first concave/convex portion 140 includes a concave portion sandwiched between adjacent first flat portions 130 and a plurality of convex portions 141 located on the bottom surface of the concave portion. and

As shown in FIG. 1(d), each first flat portion 130 is a flat surface extending in a first direction (vertical direction in FIG. 1(d)), which is one direction. The first flat portions 130 are arranged along the entire surface 111 in a second direction (horizontal direction in FIG. 1D) orthogonal to the first direction. Each first uneven portion 140 also extends in the first direction, and the first uneven portions 140 are aligned in the second direction across the surface 111 . This is also clear from the image of the surface of the cell sheet-forming member 100 taken with a scanning electron microscope, as shown in FIG. 1(f).

Each convex portion 141 forming the first concave-convex portion 140 is positioned, for example, at each vertex of a triangular lattice when viewed from the direction facing the surface 111 . Each first concave-convex portion 140 repeats such an arrangement of convex portions 141 in the first direction and the second direction. In the case of the first concave-convex portion 140 in which the convex portions 141 are positioned at the vertices of the triangular lattice, the original disk for forming the convex portions 141 is used as a mask suitable for forming a minute repeated structure, such as a monoparticle film. can be formed by an etching method using as a mask.

Each projection 141 has, for example, a circular shape when viewed from the direction facing the surface 111 . The mode of the distance between the centers of the convex portions 141 adjacent to each other is the pitch of the convex portions 141 . Further, the maximum width of the convex portion 141 in plan view is the diameter of the convex portion 141 .

A configuration in which the first pitch, which is the pitch of the convex portions 141, satisfies the following (A) and (B) is suitable for animal cells such as human and mouse, especially the above-described myoblasts, fibroblasts, and cardiomyocytes. in the first direction. That is, the configuration in which the pitch (first pitch) of the projections 141 satisfies the following (A) and (B) has superiority in adhesion to animal cells, particularly myoblasts, fibroblasts, and cardiomyocytes. , the first flat portion 130 and the first uneven portion 140 are clearly divided.

(A) First pitch of protrusions 141: 10 nm or more and 10 μm or less (preferably 100 nm or more and 10 μm or less)
In addition, if the pitch of the protrusions 141 is, for example, 300 nm or less, the visible light transmitted through the cell sheet is less likely to be interfered by the protrusions 141 . The color displayed by the interference of the protrusions 141 complicates observation of the cell sheet using a microscope. From the viewpoint of such visibility, the pitch of the protrusions 141 is preferably 10 nm or more and 300 nm or less. On the other hand, the larger the pitch of the projections 141, the more accurately the fine structure of the projections can be formed on the resin surface by injection molding or the like. For example, if the pitch of the protrusions 141 is 500 nm or more, it is easy to form a fine structure on the resin surface. From the viewpoint of workability, the pitch of the protrusions 141 is preferably 500 nm or more and 10 μm or less.

(B) Diameter of convex portion 141: 50% or more and 100% or less of the pitch of convex portion 141 is. Also, the length in the second direction (short side direction) between the flat portions 130 adjacent to each other is the width of the first uneven portion 140 .

The width of the first flat portion 130 and the width of the first uneven portion 140 are, for example, 1/10 times or more and 10 times or less the size of cells to be cultured (5 μm or more and 100 μm or less). A configuration in which the width of the first flat portion 130 and the width of the first uneven portion 140 satisfy the following (C) and (D) is suitable for animal cells, particularly the above-described myoblasts, fibroblasts, and cardiomyocytes. This is preferable from the viewpoint of facilitating alignment of the extending direction with the first direction.

(C) Width of first flat portion 130: 10 μm or more and 50 μm or less (D) Width of first uneven portion 140: 10 μm or more and 50 μm or less As shown in FIG. and a concave portion 142 between the first flat portion 130 and the convex portion 141 adjacent thereto. Since the plurality of protrusions 141 are scattered on the first uneven portion 140 , the recesses 142 , which are spaces between the protrusions 141 , are connected in the first direction and the second direction in the first uneven portion 140 .

The length between the bottom surface of recess 142 and first flat portion 130 in the thickness direction of cell-sheet-forming member 100 is the height of first flat portion 130 . In addition, in the thickness direction of the cell-sheet-forming member 100, the height difference between the tip surface of each convex portion 141 and the first flat portion 130 is a boundary step. The height difference between the bottom surface of the concave portion 142 and the tip surface of each convex portion 141 is the height of the convex portion 141 . In a configuration in which the tip surface of each convex portion 141 and the flat portion 130 are flush with each other, the height of the first flat portion 130 and the height of the convex portion 141 are equal to each other. The ratio of the pitch of the protrusions 141 to the height of the protrusions 141 is the aspect ratio of the protrusions 141 .

A configuration in which the boundary step satisfies the following (E) is preferable from the viewpoint of enhancing the flatness of the cell sheet. The configuration in which the height of the convex portion 141 satisfies the following (F) and the configuration in which the aspect ratio of the convex portion 141 satisfies the following (G) are from the viewpoint of enhancing the structural stability of the first uneven portion 140. Moreover, it is preferable from the viewpoint of facilitating the formation of the first concave-convex portion 140 .

(E) Boundary step: 0.5 μm or less (preferably 0.3 μm or less)
(F) Height of convex portion 141: 5 nm or more and 5 μm or less (preferably 50 nm or more and 5 μm or less (G) Aspect ratio of convex portion 141: 0.1 or more and 10 or less And above (A) and (B) ), the cell preferentially adheres to one structure regardless of whether the cell preferentially adheres to the first flat portion 130 or the cell preferentially adheres to the first uneven portion 140. Combined with the inferiority of adhesion to the other structure, the extending direction of the cells is aligned with the first direction, which is the extending direction of both structures.As a result, the cells spread in two-dimensional directions along the surface 111. In the cell sheet, it is possible to align the extension direction of the cells in a one-dimensional direction, that is, to improve the orientation of the cells. It is also possible to form a three-dimensional structure by piling up in the thickness direction while having elasticity.

In addition, in a configuration that satisfies the above (E), in particular, a configuration in which the tip surface of each convex portion 141 and the first flat portion 130 are flush with each other, the first uneven portion 140 and the first flat portion 130 are covered. It is possible to increase the flatness of the formed cell sheet. Furthermore, the configuration that satisfies (F) above can further improve the flatness of the cell sheet.

In addition, since the surface 111 of the cell sheet-forming member 100 includes the first flat portion 130 and the first uneven portion 140, the cells that preferentially adhere to the first flat portion 130 and the first uneven portion 140 adhere to each other. It also becomes possible to apply a common cell sheet forming member 100 to both dominant cells. That is, it also becomes possible to enhance the versatility of the cell sheet-forming member 100 .

A stimuli-responsive material may be applied in order to facilitate the detachment and recovery of the cell sheet after cell sheet formation. The stimuli-responsive material is preferably a temperature-responsive polymer whose water affinity changes with temperature change. Specifically, poly-N-isopropylacrylamide (PIPAAm) is preferred. The stimulus-responsive material may be applied to the substrate using conventional application methods. In order to facilitate the detachment and recovery of the cell sheet, the cell sheet forming member having the cell sheet formed thereon may be subjected to ultrasonic treatment.

[Peeling auxiliary part]
[First cell sheet forming member]
As shown in FIGS. 2(a) and 2(b), the detachment assisting portion 102 of the first cell sheet-forming member, which is an embodiment of the cell sheet-forming member 100, is configured as an annular portion surrounding the culture portion 101. The second concave-convex portion 121 is provided. The second uneven portion 121 is located on the outer peripheral portion of the cell sheet-forming member 100 . The second uneven portion 121 has a single step structure (second step structure) surrounding the culture portion 101 . The stepped structure in the peeling assisting portion 102 is a convex portion 122, and the second uneven portion 121 includes a concave portion adjacent to the first flat portion 130 and a plurality of convex portions 122 located on the bottom surface of the concave portion.

Each convex portion 122 that constitutes the second uneven portion 121 is positioned, for example, at each vertex of a triangular lattice when viewed from the direction facing the surface 111 . For the second uneven portion 121, if the convex portion 122 is positioned at each vertex of the triangular lattice, the original disk for forming the convex portion 122 is used as a mask suitable for forming a minute repeated structure, for example, It can be formed by an etching method using a monoparticle film as a mask. Also, the second uneven portion 121 may be formed at the same time as the first uneven portion 140 . The width of the second uneven portion 121 is preferably 0.5 mm or more and 20 mm or less.

Each projection 122 has, for example, a circular shape when viewed from the direction facing the surface 111 . The mode of the distance between the centers of the convex portions 122 adjacent to each other is the pitch of the convex portions 122 . Also, the maximum width of the convex portion 122 in plan view is the diameter of the convex portion 122 .

The configuration in which the second pitch, which is the pitch of the projections 122, satisfies the following (H) is from the viewpoint of enhancing the detachability of animal cells such as human and mouse, particularly the myoblasts, fibroblasts, and cardiomyocytes described above. preferred.

(H) Second pitch of protrusions 122: 100 nm or more and 10 μm or less (preferably 300 nm or more and 5 μm or less, more preferably 500 nm or more and 1 μm or less)
Preferably, the second pitch is the same as or greater than the first pitch. That is, it is preferable that the second pitch has a size equal to or larger than the first pitch. In other words, the contact area per unit area between the virtual plane parallel to the surface 111 and the second uneven portion 121 of the peeling assisting portion 102 is the contact area per unit area between the virtual plane and the first uneven portion 140 . smaller than the area to be As a result, the adhesive strength of the cell sheet to the exfoliation assisting section 102 becomes smaller than the adhesive strength to the culture section 101 . Therefore, the cell sheet is easily detached from the outer edge (detachment start site) of the surface 111 and is less likely to be damaged. Further, even when the second pitch is the same as the first pitch, when the second pitch is adjacent to the outer peripheral side of the first flat portion 130, the contact area of the second uneven portion 121 with the cell sheet is the same as that of the first flat portion 130. When the contact area with the cell sheet is smaller than the contact area of the cell sheet, the adhesive strength is weakened and the peeling from the outer peripheral edge of the surface 111 is facilitated.

In the thickness direction of the cell-sheet-forming member 100 , the length between the bottom surface of the concave portion and the convex portion 122 is the height of the convex portion 122 . In a configuration in which the tip surface of each convex portion 122 is flush with the convex portion 141 and the first flat portion 130, the flatness of the cell sheet can be enhanced.

[Second cell sheet forming member]
As shown in FIGS. 3( a ) and 3 ( b ), the second cell-sheet-forming member, which is another embodiment of the cell-sheet-forming member 100 , has a peeling assisting portion 102 with an inner annular It comprises a portion 123 and an outer annular portion 124 . That is, the second uneven portion 121 has a double stepped structure (second stepped structure) surrounding the culture portion 101, the inner annular portion 123 is positioned adjacent to the culture portion 101, and the outer annular portion 124 is located adjacent to the culture portion 101. , adjoins the inner annular portion 123 on the inner side, and constitutes the outer peripheral edge of the cell sheet-forming member 100 on the outer side.

Each stepped structure of the inner annular portion 123 and the outer annular portion 124 is a convex portion or a concave portion. The stepped structure of the inner annular portion 123 is a convex portion 123a, and the inner annular portion 123 includes a concave portion adjacent to the first flat portion 130 and a plurality of convex portions 123a positioned on the bottom surface of the concave portion. The stepped structure of the outer annular portion 124 is a convex portion 124a, and the outer annular portion 124 includes a plurality of convex portions 124a positioned on the bottom surface of the concave portion.

Each of the convex portions 123a and 124a is positioned at each vertex of a triangular lattice, for example, when viewed from the direction facing the surface 111 . If each of the annular portions 123 and 124 has the convex portions 123a and 124a positioned at the respective vertices of the triangular lattice, the original plate for forming the convex portions 123a and 124a is formed into a fine repeating structure. It can be formed by an etching method using a suitable mask, for example, a monoparticle film as a mask. Also, the annular portions 123 and 124 can be formed at the same time as the first uneven portion 140 .

A configuration in which the inner second pitch, which is the pitch of the projections 123a, and the outer second pitch, which is the pitch of the projections 124a, satisfy the following (I) and (J) are suitable for animal cells such as human and mouse, particularly myoblasts as described above. It is suitable from the viewpoint of increasing the detachability of cells, fibroblasts, and cardiomyocytes. Furthermore, this configuration is preferable from the viewpoint that the outer annular portion 124 makes it easier to peel off the cell sheet than the inner annular portion 123 .

(I) Inner second pitch of protrusions 123a in inner annular portion 123: 100 nm or more and 10 μm or less (preferably 300 nm or more and 1 μm or less)
(J) Outer second pitch of protrusions 124a in outer annular portion 124: 500 nm or more and 10 μm or less (preferably 700 nm or more and 2 μm or less)
With such a configuration, the contact area per unit area between the virtual plane parallel to the surface 111 and the annular portions 123 and 124 of the peeling assisting portion 102 is the unit area between the virtual plane and the first uneven portion 140. It is smaller than the area in contact with each other. As a result, the adhesive strength of the cell sheet to the exfoliation assisting section 102 becomes smaller than the adhesive strength to the culture section 101 . Therefore, the cell sheet is easily detached from the outer edge (detachment start site) of the surface 111 and is less likely to be damaged.

More specifically, the inner second pitch is preferably the same as or greater than the first pitch. The contact area of the cell sheet with respect to the exfoliation assisting section 102 is smaller than the contact area per unit area with respect to the culture section 101, thereby weakening the contact force. Even if the inner second pitch is the same as the first pitch of the first uneven portion 140, when it is adjacent to the outer peripheral side of the first flat portion 130, the contact area of the inner annular portion 123 with the cell sheet is the first pitch. When the contact area of the 1 flat portion 130 with respect to the cell sheet becomes smaller, the adhesive strength is weakened. As a result, the inner annular portion 123 has less adhesion to the cell sheet than the first flat portion 130, making it easier to peel off. The inner second pitch of the projections 123 a of the inner annular portion 123 is smaller than the outer second pitch of the projections 124 a of the outer annular portion 124 . That is, the outer second pitch is greater than the inner second pitch. As a result, the contact area per unit area of the outer annular portion 124 with respect to the cell sheet is smaller than that of the inner annular portion 123, and the adhesive strength of the cell sheet is weakened, making it easier to peel off.

In the thickness direction of the cell-sheet-forming member 100, the length between the bottom surface of the recess and the protrusions 123a and 124a is the height of the protrusions 123a and 124a. In a configuration in which the tip surfaces of the convex portions 123a and 124a are flush with the convex portion 141 and the first flat portion 130, the flatness of the cell sheet can be enhanced.

[Third cell sheet forming member]
As shown in FIGS. 4( a ) and 4 ( b ), the detachment assisting portion 102 of the third cell sheet-forming member, which is still another embodiment of the cell sheet-forming member 100 , has the second uneven portion 121 , It comprises an inner annular portion 125 and an outer annular portion 126 . That is, the second uneven portion 121 has a double stepped structure surrounding the culture portion 101, the inner annular portion 125 is positioned adjacent to the culture portion 101, and the outer annular portion 126 is located inside the inner annular portion. It is adjacent to the portion 125 and constitutes the outer peripheral edge of the cell sheet-forming member 100 on the outside.

Each stepped structure of the inner annular portion 125 and the outer annular portion 126 is a convex portion or a concave portion. The stepped structure (second stepped structure) of the inner annular portion 125 is a convex portion 125a having a truncated cone shape. and a plurality of positioned convex portions 125a. The stepped structure of the outer annular portion 126 is a convex portion 126a having a conical shape, and the outer annular portion 126 has a plurality of convex portions 126a positioned on the bottom surface of the concave portion.

Each convex part 125a, 126a is located at each vertex of a triangular lattice, for example, when viewed from the direction facing the surface 111 . If each of the annular portions 125 and 126 has the convex portions 125a and 126a located at the vertices of the triangular lattice, the original plate for forming the convex portions 125a and 126a is formed into a fine repeating structure. It can be formed by an etching method using a suitable mask, for example, a monoparticle film as a mask. Also, the annular portions 125 and 126 can be formed at the same time as the first uneven portion 140 .

The inner second pitch, which is the pitch of the convex portions 125a, and the outer second pitch, which is the pitch of the convex portions 126a, satisfy the following (K): It is suitable from the viewpoint of enhancing the detachability of cells.

(K) Inner second pitch and outer second pitch of convex portions 125a and 126a in annular portions 125 and 126: 100 nm or more and 10 μm or less (preferably 300 nm or more and 1 μm or less)
With such a configuration, the contact area per unit area between the virtual plane parallel to the surface 111 and the annular portions 125 and 126 of the peeling assisting portion 102 is the unit area between the virtual plane and the first uneven portion 140. It is smaller than the area in contact with each other. As a result, the adhesive strength of the cell sheet to the exfoliation assisting section 102 becomes smaller than the adhesive strength to the culture section 101 . Therefore, the cell sheet is easily detached from the outer edge (detachment start site) of the surface 111 and is less likely to be damaged.

More specifically, the inner second pitch is preferably the same as or greater than the first pitch. The contact area of the cell sheet with respect to the exfoliation assisting section 102 is smaller than the contact area per unit area with respect to the culture section 101, thereby weakening the contact force. Even if the inner second pitch is the same as the first pitch, when the inner annular portion 125 is adjacent to the outer peripheral side of the first flat portion 130, the contact area of the inner annular portion 125 with the cell sheet is the same as that of the cells of the first flat portion 130. Adhesive strength is weakened by making the area smaller than the contact area with the sheet. As a result, the inner annular portion 125 has a weaker adhesive force to the cell sheet than the first flat portion 130, making it easier to peel off. In the outer annular portion 126, each convex portion 126a has a conical shape, and in the inner annular portion 125, each convex portion 125a has a truncated cone shape. Therefore, the inner annular portion 125 has a larger contact area per unit area than the outer annular portion 124 . As a result, the outer annular portion 126 is easier to peel off the cell sheet than the inner annular portion 125 .

In the thickness direction of the cell-sheet-forming member 100, the length between the bottom surface of the recess and the protrusions 125a and 126a is the height of the protrusions 125a and 126a. In a configuration in which the tip surfaces of the convex portions 125a and 126a are flush with the convex portion 141 and the first flat portion 130, the flatness of the cell sheet can be enhanced.

[Fourth cell sheet forming member]
As shown in FIGS. 5( a ) and 5 ( b ), the separation assisting portion 102 of the fourth cell sheet-forming member, which is still another embodiment of the cell sheet-forming member 100 , has a second flat portion 127 . ing. The second flat portion 127 is a flat surface. The length between the bottom surface of recess 142 and second flat portion 127 in the thickness direction of cell-sheet-forming member 100 is the height of second flat portion 127 . The height difference between the bottom surface of the concave portion 142 and the tip surface of the second flat portion 127 is the height of the second flat portion 127 . A configuration in which the tip surface of each convex portion 141, the first flat portion 130, and the second flat portion 127 are flush with each other is preferable. Such a surface 111 can be formed by an etching method using a mask suitable for forming a minute repeated structure, for example, a monoparticle film, as a mask for forming the projections 122. . Also, the second flat portion 127 may be formed at the same time as the first uneven portion 140 . The width of the second flat portion 127 is preferably 0.5 mm or more and 20 mm or less.

With such a configuration, the contact area per unit area between the virtual plane parallel to the surface 111 and the second flat portion 127 of the peeling assisting portion 102 is the unit area between the virtual plane and the first uneven portion 140. It becomes larger than the area that touches the hit. As a result, in the second flat portion 127 , for example, the adhesive force of the cell sheet to the detachment assisting portion 102 is higher than the adhesive force of the cell sheet to the culture portion 101 . Therefore, the strength of the outer peripheral portion of the cell sheet is increased, and the outer peripheral portion of the cell sheet is less likely to be damaged when peeled off from the surface 111 .

Note that the peeling assisting portion 102 may be configured such that the second flat portion 127 is a second uneven portion having a stepped structure. In this case, the pitch of the second uneven portion may be smaller than the pitch of the first uneven portion 140 so that the contact area with the cell sheet per unit area is larger than that of the culture portion 101 . With such a configuration, the strength of the cell sheet at the detachment assisting portion 102 can be increased. When the second concave-convex portion is composed of two or more multiple annular portions, the pitch of the concave-convex structure of the outer annular portion is smaller than that of the inner annular portion. With such a configuration, the strength of the cell sheet can be increased as it approaches the edge of the surface 111 .

[Method for producing cell sheet-forming member]
Next, an example of a method for producing a cell sheet-forming member will be described. In the following description, an example of forming the surface 111 of the cell sheet forming member by transferring the intaglio 150 using the nanoimprint method will be described. Here, as an example, a method for manufacturing the above-described first cell sheet-forming member (FIGS. 2(a) and 2(b)) will be described.

As shown in FIG. 6 , the method for manufacturing a cell sheet-forming member includes the steps of forming an intaglio 150 and forming the surface 111 of the cell sheet-forming member 100 by transferring the intaglio 150 .

The lower surface of the intaglio 150 has a shape extending in a first direction (direction orthogonal to the paper surface), and a plurality of first flat molded portions 151 arranged in a second direction (horizontal direction of the paper surface) intersecting the first direction. and a first concave-convex molded portion 152 having a plurality of stepped structures filling spaces between adjacent first flat molded portions 151 . The first flat molding portion 151 is a portion for forming the first flat portion 130 , and the first uneven molding portion 152 is a portion for forming the first uneven portion 140 . Further, the portion of the intaglio 150 outside the first flat forming portion 151 is a portion forming the peeling assisting portion 102, and in the example of FIG. 153 is provided. The concave-convex structure of the first concave-convex molded portion 152 has a pitch, height, etc. corresponding to the concave-convex structure of the first concave-convex portion 140 . Also, the uneven structure of the second uneven molding portion 153 has a pitch, height, etc. corresponding to the uneven structure of the second uneven portion 121 . Incidentally, when forming the second flat portion 127 as shown in FIG.

In the step of forming the intaglio 150, for example, the silicon substrate for forming the intaglio 150 is subjected to at least one of photolithography, colloidal lithography, anodic oxidation, and interference exposure to form unevenness. be done. Also, the intaglio 150 itself may be obtained by one or more transfers from the master. A shape corresponding to the surface shape of the intaglio 150 is formed on the master by using, for example, at least one of photolithography, colloidal lithography, anodization, and interference exposure on a silicon substrate.

Next, the lower surface of the intaglio 150 is made to face the surface 111 of the substrate 160 for forming the cell sheet-forming member 100 . The material forming the base material 160 is, for example, a thermoplastic resin or a photocurable resin. Then, the lower surface of the intaglio 150 is pressed against the surface 111 of the substrate 160 while the substrate 160 has fluidity. Next, the intaglio 150 is released from the surface 111 of the substrate 160 while the fluidity of the substrate 160 is suppressed. As a result, the first flat part 151, the first uneven part 152, and the second uneven part 153 of the intaglio 150 are transferred to the surface 111 of the substrate 160, and the first flat part 130 and the first uneven part 140 are transferred. , the second concave-convex portion 121 and the like are formed.

For example, laminin, collagen, gelatin, fibronectin, polylysine (PDL or PLL), hyaluronic acid, etc. are applied to the surface of the thermoplastic resin or photocurable resin forming material of the base material 160 for the purpose of increasing the adhesiveness of cells. Organic substances containing adhesion factors such as extracellular matrices, polymers, gels, etc. may be applied. As a material for forming the base material 160, biomaterials such as polysaccharides and proteins may be used.

[Method for producing cell sheet]
Next, a cell sheet manufactured using the cell sheet forming member 100 will be described.
As shown in FIG. 7(a), the cell suspension located on the surface 111 of the cell sheet forming member 100 contains cells S1 adhered to the first flat portion 130, for example. At this time, each first flat portion 130 extends in the longitudinal direction (first direction) of the first uneven portion 140, and the width of each first flat portion 130 is one to several times the size of a typical cell. degree. Therefore, as shown in FIG. 7B, the positions of the cells S1 are preferentially distributed within the range of the first flat portion 130, and the long axis direction of the cells S1 is arranged in the first direction. connected in a straight line. That is, the extending direction of the cells S1 is controlled so as to be aligned with the long side direction of the first flat portion 130 . FIG. 8 shows an example of myoblasts cultured using the cell sheet-forming member 100. In the example shown in the figure, the myoblasts are controlled to align in one direction.

As shown in FIG. 7(c), in the cell sheet forming substrate that does not satisfy (A) above, the orientation of the cells S1 is not controlled, so the long axis direction of the cells is arranged in a random direction. FIG. 9 shows an example of myoblasts cultured using a commercially available cell culture petri dish serving as a reference example. In the example shown in the figure, the myoblasts are randomly arranged in the extending direction.

As shown in FIG. 10( a ), the cell suspension located on the surface 111 of the cell sheet forming member 100 contains cells S2 adhered to the first uneven portion 140, for example. At this time, each first uneven portion 140 extends in the long side direction (first direction) of the first uneven portion 140, and the width of each first uneven portion 140 is one to several times the size of a general cell. degree. Therefore, as shown in FIG. 10(b), the cells S2 are preferentially distributed within the range of the first concave-convex portion 140, and the cells S2 are linearly arranged with the longitudinal direction of the cells arranged in the first direction. connected to That is, the extension direction of the cell S2 is controlled so as to be aligned with the long side direction of the first concave-convex portion 140 .

As shown in FIG. 10(c), in the cell sheet forming substrate that does not satisfy (A) above, the orientation of the cells S1 is not controlled, so the long axis direction of the cells exists in random directions.
On the other hand, in the cell sheet-forming substrate that satisfies the above (A), as shown in FIG. It is the cell S1 that preferentially adheres, and is also the cell S2 that is allowed to adhere to the first uneven portion 140, although it is inferior to the first flat portion . Alternatively, the cells in the cell suspension held on the cell sheet-forming member 100 are the cells S2 that preferentially adhere to the first uneven portion 140, and although they are inferior to the first uneven portion 140, they are flat. It is also the cell S1 allowed to adhere to the part 130 .

In this case, as shown in FIG. 11B, the first flat portions 130 and the first uneven portions 140 extend in the first direction and are alternately arranged in the second direction. Therefore, on the surface 111 of the cell-sheet-forming member, for example, the orientation of the cells S1 preferentially adhered to the flat portion 130 is determined by the structure of the flat portion 130 and the structure of the first uneven portion 140 that partitions it. controlled by

In the first uneven portion 140 sandwiched between the flat portions 130 adjacent to each other, although inferior to the flat portion 130, the cells S2 adhered to the first uneven portion 140 show the orientation due to the flat portion 130. control is reflected. As a result, cells S1 and S2 whose orientation is controlled in the first direction form a cell sheet SA that spreads over the entire surface 111, as shown in FIG. 11(c).

Alternatively, the orientation of the cells S2 preferentially adhered to the first uneven portion 140 is controlled by the structure of the first uneven portion 140 and the structure of the first flat portion 130 that partitions it. In the first flat portion 130 sandwiched between the first uneven portions 140 adjacent to each other, the cell S1 adhered to the first flat portion 130, although inferior to the first uneven portion 140, Orientation control by the uneven portion 140 is reflected. As a result, cells S1 and S2 whose orientation is controlled in the first direction form a cell sheet SA that spreads over the entire surface 111, as shown in FIG. 11(c).

Examples of the cell-sheet-forming member, the cell-sheet-forming member manufacturing method, and the cell-sheet manufacturing method according to the above embodiments will be described below.
<Example 1>
<Preparation of cell sheet forming member>
The annular portions 123 and 124 of the first flat portion 130, the first uneven portion 140 and the second uneven portion 121 of the cell sheet-forming member 100 provided with the detachment assisting portion 102 shown in FIGS. A nickel intaglio plate was prepared for forming by transfer. Next, using a nickel intaglio as a stamper, a polystyrene sheet in which a temperature-responsive polymer (PIPAAm) is fixed to the surface of the substrate is subjected to nanoimprinting to form the ring shape of the first flat portion 130, the first uneven portion 140 and the second uneven portion 121. Parts 123 and 124 were processed, thereby producing the cell sheet-forming member 100 of Example 1. FIG. Each first flat portion 130 in the culture section 101 has a shape extending in the first direction, and is arranged in a second direction intersecting the first direction. length) was 10 μm. The first uneven portion 140 has a plurality of stepped structures that fill spaces between the flat portions 130 adjacent to each other. The pitch of the protrusions 141 in the protrusions and recesses 140 was 300 nm. The height of each protrusion in the first uneven portion 140 was measured using an AFM, and the average height from the bottom of the recess to the tip of the protrusion was 446 nm. Also, the average height from the bottom surface of the concave portion to the first flat portion 130 was 455 nm.

Further, in the second concave-convex portion 121, the width of the outer annular portion 124 located at the outer edge of the cell sheet-forming member 100 was 5 mm, and the outer second pitch of the convex portions 124a was 600 nm. The height of each protrusion in the outer annular portion 124 was measured using an AFM, and the average height from the bottom of the recess to the tip of the protrusion was 763 nm. The inner second pitch of the protrusions 123a in the inner annular portion 123 located inside the outer annular portion 124 and outside the culture portion 101 was 300 nm. The height of the protrusions 123a in the inner annular portion 123 was measured using an AFM, and the average height from the bottom of the recess to the tip of the protrusion was 446 nm.

Then, the temperature-responsive polymer was fixed to the cell-sheet-forming member 100 of Example 1, UV irradiation was performed, and after performing this sterilization treatment, the cell-sheet-forming member 100 was used in a cell culture test.
<Cell culture test>
First, mouse-derived myoblasts (C2C12 cells, manufactured by DS Pharma Biomedical) were cultured in a cell culture flask (25 cm ² ). Culture conditions are FBS (fetal bovine serum) 1
DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 0% was used at 37° C. under 5% CO ₂ atmosphere. Cells were collected using trypsin according to a standard method. The collected cells were counted using a hemacytometer.

Next, the cell sheet-forming member 100 of Example 1 cut into a circle with a diameter of 8.8 mm was placed on the bottom surface of a cell culture multiwell plate (48 holes), and 1 ml of myoblasts were seeded. After culturing for 24 hours or more in a _CO2 incubator, the medium was changed, and the medium was changed every other day. Seven days after the initiation of culture, the cultured cell sheets were collected.

The cell sheet was recovered by first removing the medium to the extent that the cells were submerged, and leaving the multiwell plate in an incubator at -20°C for 30 minutes. Next, a PDVF membrane was placed on the cell sheet as a cell support, and the cell sheet was adhered. With the cell sheet attached to the PDVF membrane, the PDVF membrane was lifted and moved onto the collagen gel. The medium was dropped and only the PDVF membrane was removed. It was confirmed by microscopic observation that the collected cell sheets were free of sheet breakage and cell dropout.

According to the above embodiment, the following effects can be obtained.
(1) The cell sheet-forming member 100 shown in FIGS. 2(a) to 4(b) is provided with a detachment assisting portion 102 composed of a second uneven portion 121 outside the culture portion 101. FIG. The detachment assisting portion 102 can weaken the adhesive force by making the contact area of the cell sheet smaller than that of the culture portion 101 . Thereby, the cell sheet can be easily peeled off at the peeling assisting portion 102 . The cell sheet-forming member 100 including the detachment assisting portion 102 composed of the second concave-convex portion 121 is suitable, for example, for culturing cells having relatively strong adhesiveness. That is, even when a cell sheet having relatively strong adhesiveness is to be peeled, the peeling assisting portion 102 can weaken the adhesive strength of the cell sheet, thereby facilitating the peeling of the cell sheet.

On the other hand, the cell sheet-forming member 100 shown in FIGS. 5(a) and 5(b) has a detachment assisting portion 102 consisting of a second flat portion 127 outside the culture portion 101. As shown in FIG. When the adhesive force of the cell sheet to the detachment assisting portion 102 is made higher than the adhesive force of the cell sheet to the culture portion 101 in this manner, the strength of the outer peripheral portion of the cell sheet is increased, and the outer peripheral portion of the cell sheet is made to be the surface. It is possible to make it difficult to damage when peeling off from 111 . The cell sheet-forming member 100 including the detachment assisting portion 102 consisting of the second flat portion 127 is suitable for culturing cells having relatively weak adhesiveness, for example. Cells with weak adhesiveness have the problem that during cell culture and during cell sheet recovery (detaching), cell detachment and cell sheet breakage tend to occur at unintended timings. In this regard, when the cell sheet-forming member 100 including the detachment assisting portion 102 composed of the second flat portion 127 is used, the second flat portion 127 of the detachment assisting portion 102 (peripheral portion) has relatively high adhesiveness to cells. Therefore, a cell sheet having high mechanical strength can be formed, and the cell sheet can be easily peeled off.

(2) It is possible to easily produce oriented cell sheets of myoblasts, fibroblasts, cardiomyocytes, etc., and to easily peel them off from the cell sheet-forming member 100 without damaging the cell sheets. can do.

(3) The second pitch of the stepped structure at the second uneven portion 121 in the peeling assisting portion 102 is the same as or larger than the first pitch of the stepped structure at the first uneven portion 140 . As a result, the contact area of the second uneven portion 121 with respect to the cell sheet becomes smaller than the contact area of the first uneven portion 140 with respect to the cell sheet, and the adhesive strength also decreases. Therefore, the outer peripheral portion of the cell sheet can be easily peeled off from the surface 111 .

(4) The exfoliation assisting portion 102 shown in FIG. 2( a ) is the second concave-convex portion 121 configured by a single annular portion surrounding the culture portion 101 . Therefore, the contact area of the cell sheet with respect to the culture section 101 is reduced in the second concave-convex portion 121 , thereby weakening the adhesive force. Thereby, the cell sheet can be easily peeled off at the peeling assisting portion 102 .

(5) The exfoliation assisting portion 102 shown in FIGS. 3(a) to 4(b) is a second uneven portion 121 configured by a double annular portion surrounding the culture portion 101. The pitch of the concave-convex structure of the portion is the same as the pitch of the concave-convex structure of the inner annular portion, or larger than the pitch of the concave-convex structure of the inner annular portion. As a result, even in the detachment assisting portion 102, the adhesive force of the cell sheet can be weakened toward the outside. Thereby, the cell sheet can be easily peeled off at the peeling assisting portion 102 .

(6) The surface of the substrate 160 is coated with a temperature-responsive polymer. This facilitates peeling and collection of the cell sheet.
It should be noted that the above embodiment may be modified as follows.

- It is also possible to form a cell sheet in which an oriented three-dimensional tissue is formed by the above-described extracellular matrix coating method. As shown in FIGS. 12(a) to 12(c), in this case, the cell suspension located on the surface 111 of the cell sheet-forming member 100 consists of a first solution containing the first cell adhesion component and a first It contains cultured cells 1 coated alternately with one component and a second solution containing a cell adhesion second component that interacts. That is, the cultured cells 1 shown in FIG. 12(a) are covered with the adhesive film 2 as shown in FIG. 12(b). The adhesive film 2 is composed of a first film 2a containing a first component and a second film 2b containing a second component (FIG. 12(b)). The combination of the first component and the second component is, for example, a combination of a macromolecule containing an arginine-glycine-aspartic acid (RGD) sequence to which integrin binds and a macromolecule that interacts with the macromolecule containing the RGD sequence. be.

The polymer containing the RGD sequence, which contains the first component, may be a protein having the RGD sequence, or a protein to which the RGD sequence is chemically bound. In addition, it may be a naturally-derived polymer other than protein, or a synthetic polymer, as long as it has an RGD sequence. Examples of macromolecules comprising protein and containing an RGD sequence include conventionally known adhesive proteins such as fibronectin (molecular weight of about 500,000), vitronectin, laminin, cadherin, and collagen.

As the polymer that interacts with the polymer containing the RGD sequence containing the second component, both substances can, for example, bind, adhere, adsorb, and transfer electrons by interacting with the polymer containing the RGD sequence. There is no particular limitation as long as it is a substance that can be in close proximity, but it may be any one of proteins, naturally occurring polymers, and synthetic polymers that interact with a polymer containing an RGD sequence. Proteins that interact with macromolecules containing RGD sequences include, for example, water-soluble proteins, and specific examples include collagen, gelatin (eg, molecular weight of 100,000), proteoglycan, integrin, enzyme, antibody, and the like. be. Examples of naturally-derived polymers that interact with polymers containing RGD sequences include water-soluble polypeptides, low-molecular-weight peptides, polyamino acids such as α-polylysine and ε-polylysine (molecular weight 5,000), heparin and heparan sulfate, Sugars such as dextran sulfate (molecular weight: 500,000) and hyaluronic acid (molecular weight: 1,000,000-).

Combinations of the first component and the second component are, for example, fibronectin and gelatin, fibronectin and heparin, fibronectin and dextran sulfate, and laminin and collagen.

Next, a method for producing a cell sheet having a three-dimensional tissue will be described.
First, cultured cells 1 are prepared, and the entire surface thereof is coated with an adhesive film 2 to produce coated cells. Specifically, the prepared cultured cells 1 are placed in a test tube.

The first component is then brought into contact with the cultured cells 1 . As a method of contacting the cultured cells with the first component, for example, a method of directly adding the first component, a method of immersing the cells in a liquid containing the first component, a method of dropping the liquid containing the first component onto the cultured cells 1, or There are other methods such as spraying. Immersion is preferred because it is easy to operate. The contact conditions can be appropriately determined depending on the contact method, the concentration of the contained liquid used, and the like. Specifically, the contact time is, for example, 15 seconds to 60 minutes, preferably 15 seconds to 15 minutes, more preferably 15 seconds to 5 minutes, and even more preferably 15 seconds to 1 minute. The contact temperature is not particularly limited, but is, for example, 4 to 60°C, preferably 20 to 40°C, more preferably 30 to 37°C, and still more preferably 37°C.

When preparing the liquid containing the first component, the solvent includes aqueous solvents such as water and buffers, and the buffers include, for example, Tris buffers such as Tris-HCl buffer, phosphate buffers, HEPES buffer, citrate-phosphate buffer, glycylglycine-sodium hydroxide buffer, Britton-Robinson buffer, GTA buffer and the like.

After the cultured cells 1 are thus coated with the first film 2a composed of the first component, the free first component is removed. As a method for removing the first component, centrifugation is performed using the above solvent to separate the cultured cells 1 from the liquid containing the first component, and the supernatant is removed to remove the free first component. A method of removing is mentioned. Thereby, the cultured cells 1 coated with the first film 2a made of the first component can be obtained.

When forming a laminated film consisting of a first film 2a of the first component and a second film 2b of the second component as the adhesive film 2, the cultured cells 1 coated with the first component are brought into contact with the second component. As a result, it is coated with the second film 2b made of the second component. The method of contacting with the second component can be performed in the same manner as in the first component, and when contacting with the liquid containing the second component, the method of preparing the liquid containing the first component is the same. A liquid containing the second component can be prepared. As a result, the cultured cells 1 coated with the adhesive film 2 consisting of the first film 2a composed of the first component and the second film 2b composed of the second component are obtained. A laminated film composed of the first film 2a of the first component and the second film 2b of the second component can be repeatedly coated to form a plurality of coating film layers. The non-peritoneal layer is, for example, 1 to 20 layers, preferably 1 to 10 layers, more preferably 1 to 5 layers. A cell suspension containing the cultured cells 1 is dropped onto the surface 111 of the cell sheet forming member 100, and the cultured cells 1 are seeded. After that, the cultured cells 1 are cultured.

Culture conditions for completing the three-dimensional cell sheet of cultured cells 1 are appropriately determined according to the cells to be cultured. For example, the culture temperature is, for example, 4 to 60°C, preferably 20 to 40°C, more preferably 30 to 37°C, and the culture time is, for example, 1 to 168 hours, preferably 3 to 24 hours. hours, more preferably 3 to 12 hours. As a result, the cell sheet 10 having a three-dimensional structure can be manufactured by bonding the cultured cells 1 to each other or by bonding the proliferated cultured cells 1 to each other via the adhesive film 2 (FIG. 12 ( c) see).

In the cell sheet 10, other cultured cells extending in the one-dimensional direction are cultured so as to overlap the cultured cells extending in the one-dimensional direction, so the size and shape of the cultured cells extending in the one-dimensional direction are , not only in the first direction, but also in the second direction, and in the direction orthogonal to the first and second directions. As a result, in the three-dimensional tissue, the spaces between the cultured cells extending in the first direction and adjacent in the second direction are filled with other cells that are smaller than the relevant cell and extend in the first direction. Therefore, the three-dimensional tissue has a layered structure in the first layer constituting the first surface adjacent to the surface 111 and in a position close thereto. 1 surface) and approaching the second surface opposite to the first surface, the layer structure collapses, and another cultured cell 1 is piled up so as to enter the gap between the cultured cells 1 . The oriented three-dimensional tissue allows cells to be cultured in a state close to that of a living body. Further, as shown in FIG. 12(d), the orientation has an inclination in the second direction with respect to the first direction in which the elongation direction of the cultured cells 1 is the specific direction. In a three-dimensional tissue having such an orientation, cells are cultured in a state close to that of a living body, so drug responsiveness and the like can be made close to that of a living body.

The surface 111 of the cell sheet-forming member 100 is formed of, for example, laminin, collagen, gelatin, fibronectin, polylysine (PDL or PLL), extracellular matrix such as hyaluronic acid, polymer, or gel for the purpose of enhancing cell adhesiveness. It may be coated with an organic substance containing an adhesion factor such as the like, or it may be a surface composed of metal. In addition, the surface 111 of the cell sheet-forming member 100 may be hydrophilic or hydrophobic for the purpose of enhancing the adhesion of cells and the flatness of the cell sheet.

- An extracellular matrix production promoting factor may be added to the cell suspension. Examples of extracellular matrix production-promoting factors include TGF-β1, TGF-β3, ascorbic acid, ascorbic acid diphosphate, derivatives thereof, and salts thereof. From the viewpoint of collagen production, ascorbic acid, ascorbic acid diphosphate, derivatives thereof, and salts thereof (eg, sodium salts, magnesium salts, potassium salts, etc.) are preferred. Ascorbic acid is preferably L-form.

[Culturing part of cell sheet-forming member]
The shape of the convex portion 141 can be any one of a conical shape such as a cone or a pyramid, a columnar shape such as a cylinder or a prism, a truncated cone such as a truncated cone or a truncated pyramid, or a hemispherical shape. be.

The positions of the protrusions 141 can be irregular at each lattice point on the square lattice, at each lattice point on the hexagonal lattice, and further at the first concave-convex portion 140 .
- The shape of the first concave-convex portion 140 is not limited to a linear shape extending in the first direction, and may be changed to a polygonal shape extending in the first direction or a curved shape extending in the first direction.

- It is also possible to change the bottom surface of the first uneven portion 140 and the flat portion 130 to be flush with each other, that is, change the base end portion of the convex portion 141 and the first flat portion 130 to be flush with each other. In addition, as described above, the configuration in which the tip surface of the first concave-convex portion 140 and the first flat portion 130 are flush with each other is preferable from the viewpoint of improving the flatness of the cell sheet.

- It is also possible to change the stepped structure that constitutes the first concave-convex portion 140 to a concave portion, and it is also possible to change it to both a concave portion and a convex portion. For example, the first concave-convex portion 140 may have one side surface continuous with the first flat portion 130, and may have a structure in which a plurality of concave portions are formed in the side surface.

- The width of one first uneven portion 140 and the width of the other first uneven portion 140 may be different from each other, or may be equal to each other. Note that if the width of one first uneven portion 140 and the width of the other first uneven portion 140 are equal to each other, the uniformity of the characteristics of the cell sheet in the second direction can be improved. becomes possible.

- The width of one first flat portion 130 and the width of the other first flat portion 130 may be different from each other, or may be equal to each other. If the width of one first flat portion 130 and the width of the other first flat portion 130 are equal to each other, the uniformity of the properties of the cell sheet in the second direction can be improved. becomes possible.

- The width of the first flat portion 130 and the width of the first concave-convex portion 140 may be different from each other, or may be equal to each other. For example, when cell adhesion is predominant in the first flat portion 130, the width of the first flat portion 130 is within a range in which the orientation can be controlled and is larger than the width of the first uneven portion 140. preferred. Further, when cell adhesion is predominant in the first uneven portion 140, the width of the first uneven portion 140 is within a range in which the orientation can be controlled and is larger than the width of the first flat portion 130. preferred.

The second direction in which the first flat portions 130 and the first concave-convex portions 140 are alternately arranged is not limited to the direction perpendicular to the first direction. Directions forming an angle of 45° are also possible.

- The cell sheet-forming member is not limited to a transfer body using an intaglio plate, but may be a transfer body using a letterpress plate, and may be formed by injection molding. That is, it is also possible to manufacture a cell sheet molded member using injection molding.

[Exfoliation Auxiliary Portion of Cell Sheet Forming Member]
- In the second concave-convex portion 121, the height of the convex portion is preferably 50 nm or more and 5 μm or less. Also, the aspect ratio of the convex portion is preferably 0.1 or more and 10 or less. Such a configuration is preferable from the viewpoint of enhancing the structural stability of the second uneven portion 121 and from the viewpoint of facilitating the formation of the second uneven portion 121 .

- In the second uneven portion 121, the shape of the convex portion is any one of a conical shape such as a cone and a pyramid, a columnar shape such as a cylinder and a prism, a truncated cone shape such as a truncated cone and a truncated pyramid, and a hemispherical shape. It is possible to

In the second uneven portion 121 , the positions of the convex portions may be irregular at each lattice point on the square lattice, at each lattice point on the hexagonal lattice, and further at the first uneven portion 140 .
- The height of the second uneven portion 121 may be the same as, higher, or lower than the height of the first flat portion 130 and/or the first uneven portion 140 . When a height difference is formed between the culture portion 101 and the detachment assisting portion 102, a gap is likely to be formed at and around the position where the height difference is formed, weakening the adhesive force between the cell sheet and the surface 111. becomes possible.

- The second concave-convex portion 121 in the peeling assisting portion 102 may be composed of three or more annular portions. In this case, it is preferable to increase the pitch of the stepped structure toward the annular portion of the outer periphery so as to weaken the adhesive force. In addition, in the stepped structure in the adjacent annular portion, the pitch may be the same and there may be portions.

[others]
- The cell sheet-forming member can be applied to multiwell plates, petri dishes, flasks, chamber slides, etc., as long as they can hold a cell suspension.

DESCRIPTION OF SYMBOLS 1... Cultured cell, 2... Adhesive membrane, 2a... First membrane, 2b... Second membrane, 10... Cell sheet, 100... Cell sheet forming member, 101... Culture part, 102... Peeling auxiliary part, 110... Culture plate, DESCRIPTION OF SYMBOLS 111... Surface 120... Lid 121... Second uneven part 122... Convex part 123... Inner annular part 123a... Convex part 124... Outer annular part 124a... Convex part 125... Inner annular part 125a... Projection 126 Outer annular portion 126a Projection 127 Second flat portion 130 First flat portion 140 First projection 141 Projection 142 Recess 150 Intaglio 151 First flat molded portion 152 First uneven molded portion 153 Second uneven molded portion 160 Base material.

Claims (9)

having a surface for forming a cell sheet,
The surface comprises a culture section for culturing cells, and a detachment assisting section located outside the culture section and forming a detachment starting site of the cell sheet from the surface,
The culture section includes a plurality of first flat portions and a plurality of first uneven portions,
each of the first flat portions has a shape extending in a first direction, and the first flat portions are arranged in a second direction intersecting the first direction;
Each of the first concave-convex portions has a first stepped structure that fills a gap between the first flat portions adjacent to each other, and the first stepped structure is composed of convex portions arranged at a first pitch. ,
The peeling assisting portion is composed of a second uneven portion having a second stepped structure composed of convex portions arranged at a second pitch, or a second flat portion ,
The first pitch is 10 nm or more and 10 μm or less,
The second pitch is 100 nm or more and 10 μm or less.
Cell sheet forming member. The cell sheet forming member according to claim 1, wherein the cells cultured in the cell sheet forming member are at least one selected from the group consisting of myoblasts, fibroblasts, and cardiomyocytes. The cell sheet-forming member according to claim 1 or 2 , wherein the detachment assisting portion is a single annular portion surrounding the culturing portion. The peeling assisting portion is composed of a second uneven portion having a second stepped structure composed of convex portions arranged at a second pitch,
The exfoliation assisting part is a double or more annular part surrounding the culture part,
3. The cell sheet-forming member according to claim 1 , wherein the outer annular portion has a concavo-convex structure having a pitch equal to or greater than that of the inner annular portion. The cell sheet-forming member according to any one of claims 1 to 4 , wherein the surface is coated with a temperature-responsive polymer. having a surface for forming a cell sheet,
The surface comprises a culturing portion for culturing cells, and a detachment assisting portion located at the edge of the surface and forming a detachment starting site of the cell sheet from the surface,
The culture section includes a plurality of first flat portions and a plurality of first uneven portions,
each of the first flat portions has a shape extending in a first direction, and the first flat portions are arranged in a second direction intersecting the first direction;
Each of the first concave-convex portions has a first stepped structure that fills a gap between the first flat portions adjacent to each other, and the first stepped structure is composed of convex portions arranged at a first pitch. ,
The peeling assisting portion is composed of a second uneven portion having a second stepped structure composed of convex portions arranged at a second pitch, or a second flat portion,
The first pitch is 10 nm or more and 10 μm or less,
The second pitch is 100 nm or more and 10 μm or less,
The area per unit area of contact between the virtual plane parallel to the surface and the detachment assisting portion is smaller than the area per unit area of contact between the virtual plane and the first concave-convex portion. . having a surface for forming a cell sheet,
The surface comprises a culturing portion for culturing cells, and a detachment assisting portion located at the edge of the surface and forming a detachment starting site of the cell sheet from the surface,
The culture section includes a plurality of first flat portions and a plurality of first uneven portions,
each of the first flat portions has a shape extending in a first direction, and the first flat portions are arranged in a second direction intersecting the first direction;
Each of the first concave-convex portions has a first stepped structure that fills a gap between the first flat portions adjacent to each other, and the first stepped structure is composed of convex portions arranged at a first pitch. ,
The peeling assisting portion is composed of a second uneven portion having a second stepped structure composed of convex portions arranged at a second pitch, or a second flat portion,
The first pitch is 10 nm or more and 10 μm or less,
The second pitch is 100 nm or more and 10 μm or less,
The area per unit area of contact between the virtual plane parallel to the surface and the detachment assisting portion is larger than the area per unit area of contact between the virtual plane and the first concave-convex portion. . forming an intaglio;
forming a surface of a cell sheet forming member for forming a cell sheet by transferring the intaglio,
The surface comprises a culture section for culturing cells, and a detachment assisting section located outside the culture section and forming a detachment starting site of the cell sheet from the surface,
The culture section includes a plurality of first flat portions and a plurality of first uneven portions,
each of the first flat portions has a shape extending in a first direction, and the first flat portions are arranged in a second direction intersecting the first direction;
Each of the first concave-convex portions has a first stepped structure that fills a gap between the first flat portions adjacent to each other, and the first stepped structure is composed of convex portions arranged at a first pitch. ,
The peeling assisting portion is composed of a second uneven portion having a second stepped structure composed of convex portions arranged at a second pitch, or a second flat portion,
The intaglio includes a first flat molding portion that molds the first flat portion;
a first concave-convex forming part that forms the first concave-convex part;
A second molding portion that molds the second uneven portion or the second flat portion,
The first pitch is 10 nm or more and 10 μm or less,
The second pitch is 100 nm or more and 10 μm or less,
Forming the intaglio includes forming the first flat molding portion, the first concave-convex molding portion, and the second molding portion by at least a photolithography method, a colloidal lithography method, an anodization method, and an interference exposure method. comprising forming with one
A method for producing a cell sheet-forming member. The cell sheet-forming member according to any one of claims 1 to 7 is used, and the cell having adhesion to one of the first flat portion and the first uneven portion is more dominant than the adhesion to the other. Forming a cell sheet on the surface of the cell sheet forming member by adhering it to the surface of the cell sheet forming member;
A method for producing a cell sheet, comprising peeling the cell sheet from the surface from the peeling start site on the peeling assisting portion of the cell sheet forming member.

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