JP7180698B2 - Cell-sheet-forming member, base material, and method for producing cell-sheet-forming member - Google Patents

Description

The present disclosure relates to a cell-sheet-forming member for forming a cell sheet in which cell clusters are formed, a base material, and a method for manufacturing the cell-sheet-forming member.

In the field of drug discovery and development, cultured cells are used for drug screening, drug screening, drug safety evaluation, toxicity testing, regenerative medicine, etc. In these fields, three-dimensional tissues similar to in vivo tissues are used. Sometimes used. (See Patent Document 1).

WO2007/097120

By the way, tests using stem cells such as iPS cells are being introduced also in the fields of drug screening and toxicity testing. However, a test method using a three-dimensional tissue such as a cell mass requires a complicated method of three-dimensional organization.

An object of the present disclosure is to provide a cell-sheet-forming member, a base material, and a method for producing a cell-sheet-forming member that enable easy formation of a cell sheet containing cell clusters.

A cell sheet-forming member according to an aspect of the present disclosure includes a surface for forming a cell sheet, and the surface includes a flat portion where cell adhesion is dominant and a cell adhesion is inferior to the flat portion. The flat portion has a circular shape or a polygonal shape when viewed from the direction facing the surface, and is configured so that cell clusters are formed at least on the flat portion.

According to the above configuration, the cell mass is formed on the flat portion, and at least a part of the cell mass formed by aggregating the cells on the uneven portion adheres to the flat portion, making it difficult for the cell mass to separate. . Therefore, identification of cell clusters and measurement of movement such as size and pulsation of cell clusters are facilitated. In addition, analysis such as image processing is facilitated.

In the cell sheet-forming member, the pitch of the protrusions in the protrusions and recesses is preferably 10 nm or more and 10 μm or less. According to the above configuration, the cells in the concave-convex portion have reduced adhesiveness, can aggregate, and easily form cell clusters.

In the cell sheet-forming member, the area of the flat portion is preferably 100 μm ² or more and 10000 μm ² or less. According to the above configuration, the cell mass is reliably adhered to the flat portion.

A substrate according to an aspect of the present disclosure has cell clusters formed on the surface of the cell cluster forming member as described above. According to the above configuration, it becomes easy to identify the cell mass adhered to the flat portion while being fixed to the substrate, and to measure the size of the cell mass and movement such as pulsation. In addition, analysis such as image processing is facilitated.

A method for producing a cell sheet-forming member according to an aspect of the present disclosure includes forming an intaglio, and forming a surface of the cell sheet-forming member for forming a cell sheet by transferring the intaglio, The surface has a flat portion where cell adhesion is dominant and an uneven portion where the cell adhesion is inferior to the flat portion, and the intaglio includes a flat molding portion for molding the flat portion and the uneven portion. and forming the intaglio is performed by subjecting the flat molding portion and the uneven molding portion to at least one of a photolithography method, a colloidal lithography method, an anodization method, and an interference exposure method. including forming using

A cell sheet-forming member according to another aspect of the present disclosure includes a surface for forming a cell sheet, the surface comprising an orientation control section for controlling orientation of cells, and a cell mass forming portion for forming masses, wherein the orientation control portion has a shape extending in a first direction and is arranged in a second direction intersecting the first direction; and a first flat portion. and a first concave-convex portion filling between the first flat portions adjacent to each other, wherein the cell cluster forming portion includes a second flat portion adjacent to the orientation control portion and adjacent to the orientation control portion. and a second uneven portion connected to the second flat portion so that at least a part of the cell mass is formed on the second flat portion.

According to the above configuration, the orientation control unit can align the extension direction of the cells with the first direction. As a result, it is possible to improve the orientation of the cells in the cell sheet that spreads in two dimensions. Also, in the cell cluster forming portion, the cells located on the second uneven portion are aggregated on the second flat portion, and at least a part of the cell cluster adheres to the second flat portion. As a result, the cell mass is formed on the second flat portion, and the cell mass adheres to the second flat portion, making it difficult to separate. Therefore, identification of cell clusters and measurement of movement such as size and pulsation of cell clusters are facilitated. In addition, analysis such as image processing is facilitated.

In the cell sheet-forming member, it is preferable that the second flat portion is positioned sandwiched between the first uneven portions. According to the above configuration, it is possible to form a cell cluster near the oriented cells.

In the cell-sheet-forming member, it is preferable that the second concave-convex portion is sandwiched between the first concave-convex portions. According to the above configuration, it is possible to form a cell cluster with cells near the oriented cells.

In the cell sheet-forming member, the second flat portion preferably has a length in the second direction of 50 μm or more and 200 μm or less and a length in the first direction of 100 μm or more. According to the above configuration, the cells located on the second concave-convex portion aggregate on the second flat portion, and at least part of the cell mass can adhere to the second flat portion.

In the above cell sheet-forming member, the length of the portion sandwiched between the adjacent orientation control portions in the second uneven portion in the second direction is 50 μm or more and 1 mm or less, and the convex portion in the second uneven portion The pitch is preferably 10 nm or more and 10 μm or less. According to the above configuration, the cells positioned on the second concave-convex portion easily aggregate.

In the cell sheet-forming member, the first uneven portion preferably has a length in the second direction of 10 μm or more and 50 μm or less and a length in the first direction of 100 μm or more. According to the above configuration, it is possible to improve the orientation of the cells in the cell sheet that spreads in the two-dimensional direction.

A substrate according to another aspect of the present disclosure has cell clusters formed on the surface of the cell cluster forming member as described above. According to the above configuration, it becomes easy to identify the cell mass adhered to the flat portion while being fixed to the substrate, and to measure the size of the cell mass and movement such as pulsation. In addition, analysis such as image processing is facilitated. In the base material, it is preferable that cells having orientation in the first direction are formed in the orientation control part.

A method for manufacturing a cell sheet-forming member according to another aspect of the present disclosure includes forming an intaglio, and forming a surface of the cell sheet-forming member for forming a cell sheet by transferring the intaglio, The surface includes an orientation control section for controlling the orientation of cells, and a cell cluster forming section for forming a cell cluster by aggregating the cells, and the orientation control section is arranged in a first direction. The cell mass includes first flat portions having an elongated shape and arranged in a second direction intersecting the first direction, and first uneven portions filling between the first flat portions adjacent to each other. The forming portion includes a second flat portion adjacent to the orientation control portion, and a second uneven portion adjacent to the orientation control portion and connected to the second flat portion. A first flat molding portion for molding a first flat portion, a first uneven molding portion for molding the first uneven portion, a second flat molding portion for molding the second flat portion, and a second uneven portion for molding. forming the intaglio plate by using a photolithography method, a colloidal lithography method, an anodization method, and an interference exposure method to form the first unevenness molding portion and the second unevenness molding portion; including forming using at least one of

The perspective view which shows a cell sheet formation member with a petri dish. (a) is a plan view of the cell sheet-forming member in the first embodiment, and (b) is a cross-sectional view of the range indicated by the arrow in FIG. 2(a). The figure for demonstrating an example of the manufacturing method of the cell sheet formation member in 1st Embodiment shown to Fig.2 (a). (a) is a plan view of the cell sheet-forming member in the second embodiment, (b) is a cross-sectional view of the range indicated by the arrow in FIG. 4(a), and (c) is the cell of FIG. 4(a). FIG. 4D is a perspective view of the orientation control portion of the sheet-forming member, and FIG. 4D is an image of the surface of the orientation control portion of the cell sheet-forming member of FIG. The figure for demonstrating an example of the manufacturing method of the cell sheet formation member in 2nd Embodiment shown to Fig.4 (a). (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) 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.

An embodiment 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.

[First embodiment]
[Cell sheet forming member]
As shown in FIG. 1, the cell sheet-forming member 100 is, for example, a sheet material arranged in 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 the petri dish, the cell sheet-forming member 100 is insert-molded, for example, when the petri dish is injection-molded. 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 not particularly limited as long as they are adhesive cells, but cells derived from various tissues in vivo, stem cells, various cells induced to differentiate from ES cells and iPS cells, etc. can be used.

As shown in FIG. 2( a ), the surface 111 of the cell sheet-forming member 100 in the first embodiment includes a plurality of flat portions 130 and a plurality of uneven portions 140 provided around the flat portions 130 . Seen from the direction facing the surface 111 , each flat portion 130 has, for example, a circular shape when seen from the side facing the surface 111 . Alternatively, it may have a polygonal shape such as a triangle, quadrangle, or hexagon. The concave-convex portion 140 includes a concave portion and a stepped structure of the convex portion 141, and the stepped structure fills between the flat portions 130, which are island-shaped regions. The flat portion 130 is superior in cell adhesion, and the uneven portion 140 is inferior in cell adhesion to the flat portion 130 . As shown in FIG. 2( b ), the cells seeded on the base material are cultured in a monolayer spread on the base material at the beginning of the culture, but after a certain period of time, the cells are naturally located on the uneven part 140 aggregate to form spherical or rod-shaped cell clusters 11 . At least a part of the cell mass 11 adheres to the flat portion 130 and does not detach from the surface 111 and does not separate. The cell mass 11 may be adhered almost entirely to the flat portion 130 in some cases.

Each convex portion 141 forming the 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 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 uneven portion 140 in which the convex portions 141 are positioned at the vertices of the triangular lattice, the original plate for forming the convex portions 141 is a mask suitable for forming a minute repeated structure, for example, a single particle film. can be formed by an etching method such as

The pitch of the protrusions 141 in the protrusions and recesses 140 preferably satisfies (A) below.
(A) Pitch of projections 141 in projections and depressions 140: 10 nm or more and 10 μm or less. Preferably, it is 100 nm or more and 5 μm or less. More preferably, it is 300 nm or more and 5 μm or less. Even more preferably, it is 500 nm or more and 2 μm or less.

Moreover, the area of the flat portion 130 preferably satisfies the following (B).
(B) Area of flat portion 130: 100 μm ² or more and 10000 μm ² or less.
By satisfying the condition (A), the protrusions 141 facilitate aggregation of the cells spread in a monolayer on the base material at the initial stage of culture, and the cell clusters 11 can be formed. Moreover, by satisfying the condition (B), the flat portion 130 adheres to the cell mass 11 and becomes difficult to separate. Since the cell mass 11 is not separated, it becomes easy to identify the cell mass 11 and to measure the size of the cell mass 11 and movement such as pulsation. In addition, analysis such as image processing is facilitated.

Under the condition (A), if the pitch of the projections 141 is, for example, 300 nm or less, visible light transmitted through the cell sheet is less likely to be interfered by the projections 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.

[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.

As shown in FIG. 3 , the method for manufacturing the 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 includes a flat molding portion 151 for molding the plurality of flat portions 130 and an uneven molding portion 152 for molding the uneven portions 140 . In the step of forming the intaglio 150, for example, a silicon substrate for forming the intaglio 150 is subjected to at least one of a photolithography method, a colloidal lithography method, an anodization method, and an interference exposure method. is formed. 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 molded portions 151 and 152 of the intaglio 150 are transferred to the surface 111 of the substrate 160, and the flat portion 130 and the uneven portion 140 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.
The cell suspension located on the surface 111 of the cell sheet-forming member 100 contains cells forming the cell mass 11, for example. At this time, the cells seeded on the surface 111 are cultured in a monolayer spreading state on the surface 111 at the initial stage of culture. Then, after a certain period of time has passed, the cells located on the uneven portion 140 naturally aggregate to form spherical or rod-shaped cell clusters 11 on at least the flat portion 130 . At least a part of the cell mass 11 should be adhered to the flat portion 130 so as not to separate. The cell mass 11 may be wholly adhered to the flat portion 130 or partially adhered to the flat portion 130 . In either case, the cell mass 11 does not detach from the surface 111 and is difficult to release.

<Example 1>
Example 1 of the cell-sheet-forming member, the method for producing the cell-sheet-forming member, and the method for producing the cell-sheet according to the above embodiments will be described below.

<Preparation of cell sheet forming member>
As shown in FIGS. 2(a) and 2(b), a nickel intaglio plate was prepared for forming the cell sheet-forming member 100 having a plurality of flat portions 130 inside the uneven portion 140 by transfer. Next, using a nickel intaglio as a stamper, a polystyrene sheet in which a temperature-responsive polymer (PIPAAm) was fixed to the surface of the base material was processed by nanoimprinting to form uneven portions 140 and flat portions 130. A cell sheet-forming member 100 was produced. The uneven portion 140 in the cell-sheet-forming member 100 of Example 1 had a plurality of stepped structures, and the pitch of the convex portions 141 in the uneven portion 140 was 300 nm. The height of each convex portion 141 in the uneven portion 140 was measured using an AFM, and the average height from the bottom surface of the concave portion 142 to the tip of the convex portion 141 was 446 nm. Also, the average height from the bottom surface of the concave portion 142 to the flat portion 130 was 455 nm. The flat portion 130 had a circular shape with a diameter of 12 μm, and the shortest distance between adjacent flat portions 130 was 50 μm. Then, the cell sheet-forming member 100 of Example 1 was cut into a circle with a diameter of 8.8 mm, and was used for a cell culture test after UV irradiation as a sterilization treatment.

<Cell culture test>
First, mouse-derived cardiomyocytes (manufactured by Cosmo Bio) were cultured in a cell culture flask (25 cm ² ). The culture conditions were 37° C., 5% CO ₂ atmosphere, using a dedicated medium. 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). Cardiomyocytes adjusted to a concentration of 4×10 ⁵ cells/ml were seeded in 0.2 ml portions. After culturing in a CO ₂ incubator for 24 hours, the medium was changed, and the shape of the cells was observed after changing the medium every other day.

When the culture was started on day 0, the cells proliferated in a monolayer to a confluent state after 2 days of culture, and after 4 days, cell aggregates were formed in which the cells aggregated into spherical shapes. The cell mass adhered to the flat portion 130 and was not separated from the substrate by medium exchange. Next, the cell culture multi-well plate on which the cell sheet-forming member 100 was placed was left in an incubator at 20°C for 30 minutes. After standing, the medium in the well was pipetted, and the released cell aggregates were collected together with the medium in a 15 ml centrifuge tube.

According to the said 1st Embodiment, the effect enumerated below is acquired.
(1-1) Cells positioned on the uneven portion 140 are aggregated on the flat portion 130 and at least part of the cells adhere to the flat portion 130 . As a result, the cell mass 11 is formed on the flat portion 130, and the cell mass 11 adheres to the flat portion 130, making it difficult to separate. Therefore, in a state where the cell mass 11 is adhered to the flat portion 130, identification of the cell mass 11 and measurement of the size of the cell mass 11 and movement such as pulsation are facilitated. In addition, analysis such as image processing is facilitated.

(1-2) The pitch of the protrusions 141 in the protrusions 140 is 10 nm or more and 10 μm or less. As a result, the cells of the uneven portion 140 are less adhesive and can aggregate, facilitating the formation of the cell mass 11 .

(1-3) Since the area of the flat part 130 is 100 μm ² or more and 10000 μm ² or less, the cell mass 11 is reliably adhered to the flat part 130 .
Note that the above-described first embodiment may be implemented with the following modifications.

- The area of each flat portion 130 is not limited to 100 μm ² or more and 10000 μm ² or less. That is, each flat portion 130 should have an area to which at least a part of the cell mass 11 adheres to the extent that the cell mass 11 does not separate.

- The pitch of the protrusions 141 in the protrusions and recesses 140 is not limited to 10 nm or more and 10 μm or less as long as the cells can be held on the surface 111 .
In the uneven portion 140, the shape of the convex portion 141 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

The positions of the convex portions 141 in the uneven portion 140 can be irregular in each lattice point on the square lattice, each lattice point on the hexagonal lattice, and further in the uneven portion 140 .
- The height of the uneven portion 140 may be the same as the height of the flat portion 130, may be higher, or may be lower.

[Second embodiment]
[Cell sheet forming member]
As shown in FIG. 4(a), the surface 211 of the cell sheet-forming member 200 in the second embodiment includes an orientation controller 201 for controlling the orientation of cells to be cultured, and a cell cluster for forming a cell cluster. and a forming portion 202 . A plurality of orientation control portions 201 are formed on the surface 211 , and a cell cluster forming portion 202 is formed adjacent to each orientation control portion 201 .

[Orientation control part]
As shown in FIG. 4B , the orientation control section 201 includes a plurality of first flat portions 230 and a plurality of first uneven portions 240 . The first uneven portion 240 extends in the first direction and is formed side by side in the second direction. Each first uneven portion 240 has a stepped structure (first stepped structure), and the stepped structure fills the space between the first flat portions 230 adjacent to each other. The stepped structure is a protrusion or a recess. The stepped structure in the present embodiment is a convex portion 241, and the first concave/convex portion 240 includes a concave portion sandwiched between adjacent first flat portions 230 and a plurality of convex portions 241 located on the bottom surface of the concave portion. and

Each first flat portion 230 is a flat surface extending in a first direction, which is one direction. The first flat portions 230 are arranged in a second direction orthogonal to the first direction over the entire surface 211 . The first uneven portion 240 also extends in the first direction and is aligned in the second direction.

As shown in FIG. 4C, each convex portion 241 forming the first concave-convex portion 240 is positioned, for example, at each vertex of a triangular lattice when viewed from the direction facing the surface 211 . Each first concave-convex portion 240 repeats such an arrangement of convex portions 241 in the first direction and the second direction. In the case of the first concave-convex portion 240 in which the convex portions 241 are located at the vertices of the triangular lattice, the original disk for forming the convex portions 241 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 241 has, for example, a circular shape when viewed from the direction facing the surface 211 . The mode of the distance between the centers of the convex portions 241 adjacent to each other is the pitch of the convex portions 241 . Also, the maximum width of the convex portion 241 in plan view is the diameter of the convex portion 241 .

A configuration in which the first pitch, which is the pitch of the projections 241, satisfies the following (C) and (D) is the extension direction of animal cells, particularly the above-described myoblasts, fibroblasts, and cardiomyocytes, in the first direction. It is suitable from the viewpoint of aligning. That is, the configuration in which the pitch of the convex portions 241 satisfies the following (C) and (D) is superior or inferior to the adhesion of animal cells, human or mouse myoblasts, fibroblasts, and cardiomyocytes. This is preferable in that the flat portion 230 and the first uneven portion 240 are clearly partitioned.

(C) First pitch of protrusions 241: 10 nm or more and 10 μm or less. Preferably, it is 100 nm or more and 10 μm or less. More preferably, it is 100 nm or more and 5 μm or less. Even more preferably, it is 500 nm or more and 2 μm or less.

(D) Diameter of convex portion 241: 50% or more and 100% or less of the pitch of convex portion 241;
The length of each first flat portion 230 in the second direction (short side direction) is the width of the first flat portion 230 . Also, the length in the second direction (short side direction) between the first flat portions 230 adjacent to each other is the width of the first uneven portion 240 .

The width of the first flat portion 230 and the width of the first uneven portion 240 are, for example, 1/10 to 10 times 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 230 and the width of the first uneven portion 240 satisfy the following (E) and (F) 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.

(E) Width of first flat portion 230: 10 μm or more and 50 μm or less.
(F) Width of first uneven portion 240: 10 μm or more and 50 μm or less.
The first concave-convex portion 240 may include convex portions 241 adjacent to each other, and concave portions 242 between the first flat portion 230 and the convex portion 241 adjacent thereto. Since the plurality of protrusions 241 are interspersed in the first uneven portion 240 , the recesses 242 that are spaces between the protrusions 241 are continuous in the first direction and the second direction in the first uneven portion 240 .

The length between the bottom surface of recess 242 and first flat portion 230 in the thickness direction of cell sheet-forming member 200 is the height of first flat portion 230 . In addition, in the thickness direction of the cell-sheet-forming member 200, the height difference between the tip surface of each convex portion 241 and the first flat portion 230 is a boundary step. The height difference between the bottom surface of the concave portion 242 and the tip surface of each convex portion 241 is the height of the convex portion 241 . In a configuration in which the tip surface of each convex portion 241 and the first flat portion 230 are flush with each other, the height of the first flat portion 230 and the height of the convex portion 241 are equal to each other. The ratio of the pitch of the protrusions 241 to the height of the protrusions 241 is the aspect ratio of the protrusions 241 .

A configuration in which the boundary step satisfies the following (G) is preferable from the viewpoint of enhancing the flatness of the cell sheet. The configuration in which the height of the convex portion 241 satisfies the following (H) and the configuration in which the aspect ratio of the convex portion 241 satisfies the following (I) are from the viewpoint of enhancing the structural stability of the first uneven portion 240. Moreover, it is preferable from the viewpoint of facilitating the formation of the first concave-convex portion 240 .

(G) Boundary step: 0.5 μm or less, preferably 0.3 μm or less.
(H) Height of convex portion 241: 5 nm or more and 5 μm or less. Preferably, it is 50 nm or more and 5 μm or less.

(I) Aspect ratio of convex portion 241: 0.1 or more and 10 or less.
In addition, it is preferable that one orientation control section 201 composed of the first flat section 230 and the first uneven section 240 satisfies the following conditions (J) and (K).

(J) Width of orientation control portion 201: 50 μm or more and 1 mm or less.
(K) Length of orientation control portion 201: 100 μm or more.
In a configuration in which each orientation control unit 201 satisfies (H) and (I), a region in which cells are oriented in the first direction can be formed in the cell sheet.

Then, if the configuration satisfies the above (J) and (K), the cell that adheres predominantly to the first flat portion 230 or the cell that adheres predominantly to the first uneven portion 240 has one structure. Cells adhere preferentially to the body and, combined with the inferior 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, in the cell sheet that spreads two-dimensionally along the surface 111, it is possible to align the extending directions of the cells in the one-dimensional direction, that is, to improve the orientation of the cells. The cell sheet can also form a three-dimensional tissue by piling up in the thickness direction in a state in which the cultured cells are aligned in a one-dimensional direction, that is, in a state in which the cells have orientation.

In addition, a configuration that satisfies the above (G), particularly a configuration in which the tip surface of each convex portion 241 and the first flat portion 230 are flush with each other, is arranged so that the first uneven portion 240 and the first flat portion 230 are covered. It is possible to increase the flatness of the formed cell sheet. Furthermore, the configuration that satisfies (H) and (I) above can further improve the flatness of the cell sheet.

Note that FIG. 4D is an image of the surface of the orientation control section 201 photographed with a scanning electron microscope.
[Cell cluster formation part]
The cell cluster formation section 202 includes a second flat section 221 and a second uneven section 222 adjacent to the orientation control section 201 . The second flat portion 221 extends in the first direction while being sandwiched between the pair of orientation control portions 201 . Specifically, the second flat portion 221 is adjacent to the first uneven portion 240 of each orientation control portion 201, and the first uneven portion 240 of one orientation control portion 201 and the other orientation control portion 201 and the first concave-convex portion 240 .

The width of the second flat portion 221 preferably satisfies the following conditions (L) and (M).
(L) Width of second flat portion 221: 50 μm or more and 200 μm or less.
(M) Length of second flat portion 221: 100 μm or more.

In the configuration where the second flat portion 221 satisfies (L) and (M), the cell mass 11 adheres to the second flat portion 221 and becomes difficult to separate. The width of the second flat portion 221 may not be the same at all positions.

The second uneven portion 222 is positioned between the first uneven portions 240 of two adjacent orientation control portions 201 . Also, the second uneven portion 222 is connected to both ends of the second flat portion 221 in the first direction. Each convex portion 223 that constitutes the second uneven portion 222 is positioned, for example, at each vertex of a triangular lattice when viewed from the direction facing the surface 211 . Each second uneven portion 222 repeats such an arrangement of the convex portions 223 in the first direction and the second direction. In the case of the second concave-convex portion 222 in which the convex portions 223 are located at the vertices of the triangular lattice, the original disk for forming the convex portions 223 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. Then, it becomes possible to form the convex portion 241 at the same time.

Also, the width of the second uneven portion 222 preferably satisfies the following condition (N).
(N) Width of the second concave-convex portion 222 (length between two adjacent orientation control portions 201, that is, length in the second direction of a portion sandwiched between two adjacent orientation control portions 201) : 50 μm or more and 1 mm or less.

The width of the second uneven portion 222 may not be the same at all positions.
Moreover, it is preferable that the pitch of the convex portions 223 forming the second uneven portion 222 satisfies the following condition (O).

(O) The pitch of the convex portions 223 in the second uneven portion 222: 10 nm or more and 10 μm or less. Preferably, it is 100 nm or more and 5 μm or less. More preferably, it is 300 nm or more and 5 μm or less. Even more preferably, it is 500 nm or more and 2 μm or less.

The pitch of the protrusions 223 may not be the same at all positions.
The pitch of the protrusions 223 in the second uneven portion 222 may be the same as the first pitch of the protrusions 241 in the first uneven portion 240, or may be larger or smaller. That is, the pitch of the projections 223 in the second projections and recesses 222 may be any dimension that facilitates aggregation of the cells. In addition, the width of the second concave-convex portion 222 is sufficient as long as it can hold the cells for forming the cell mass, and may be the same as the width of the first concave-convex portion 240, or it may be wider or narrower. good.

By satisfying the conditions (N) and (O), the second concave-convex portion 222 facilitates the aggregation of cells and can form cell clusters. At least a portion of the cell mass 11 is adhered to the second flat portion 221 by aggregating the cells separated from the second concave-convex portion 222 .

That is, on the surface 211, island-shaped regions configured by sandwiching one second flat portion 221 between two second concave-convex portions 222 are spaced apart and arranged in the second direction. An uneven portion 222 is formed. In such a cell sheet-forming member 200 , the orientation controller 201 can orient the cells in the first direction and culture them, and the cell cluster formation section 202 can form the cell clusters 11 .

[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 211 of the cell sheet forming member by transferring the intaglio 250 using the nanoimprint method will be described.

As shown in FIG. 5, the method for manufacturing the cell sheet-forming member includes the steps of forming an intaglio 250 and forming the surface 211 of the cell sheet-forming member 200 by transferring the intaglio 250 .

The lower surface of the intaglio 250 includes a first flat forming portion 251 for forming the first flat portion 230, a first uneven forming portion 252 for forming the first uneven portion 240, and a second flat forming portion for forming the second flat portion 221. and a second concave-convex forming portion 254 for forming the second concave-convex portion 222 . In the step of forming the intaglio 250, for example, a silicon substrate for forming the intaglio 250 is subjected to at least one of a photolithography method, a colloidal lithography method, an anodization method, and an interference exposure method. is formed. Also, the intaglio 250 itself may be obtained by one or more transfers from the master. A shape corresponding to the surface shape of the intaglio 250 is formed on the master by using, for example, at least one of photolithography, colloidal lithography, anodization, and interference exposure for a silicon substrate.

Next, the lower surface of the intaglio 250 is made to face the surface 211 of the substrate 260 for forming the cell sheet-forming member 200 . The material forming the base material 260 is, for example, a thermoplastic resin or a photocurable resin. Then, the lower surface of the intaglio 250 is pressed against the surface 211 of the substrate 260 while the substrate 260 has fluidity. Next, the intaglio 250 is released from the surface 211 of the substrate 260 while the fluidity of the substrate 260 is suppressed. As a result, the molded portions 251 to 254 of the intaglio 250 are transferred to the surface 211 of the substrate 260, and the first flat portion 230, the first uneven portion 240, the second flat portion 221 and the second uneven molded portion 254 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 260 for the purpose of increasing the adhesiveness of cells. Organic substances containing adhesion factors such as extracellular matrices, polymers, gels, etc. may be applied. Biomaterials such as polysaccharides and proteins may also be used as the material for forming the base material 260 .

[Method for producing cell sheet]
Next, a cell sheet manufactured using the cell sheet forming member 200 will be described.
As shown in FIG. 6(a), the cell suspension located on the surface 211 of the cell sheet forming member 200 contains cells S1 adhered to the first flat portion 230, for example. At this time, in the orientation control section 201, each first flat portion 230 extends in the long-side direction (first direction) of the first concave-convex portion 240, and the width of each first flat portion 230 is equal to that of a general cell. It is about one to several times the size. Therefore, as shown in FIG. 6(b), the positions of the cells S1 are preferentially distributed within the range of the first flat portion 230, 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 230 . FIG. 6 shows an example of myoblasts cultured using the cell sheet-forming member 200. In the example shown in FIG. 6, the myoblasts are controlled to align in one direction. FIG. 9 shows an example of myoblasts cultured in the orientation control section 201 of the cell sheet forming member 200. In the example shown in FIG. It is

As shown in FIG. 6(c), in the cell cluster formation substrate that does not satisfy (C) 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. 10 shows an example of myoblasts cultured using a commercially available cell culture petri dish serving as a reference example. In the example shown in FIG. 10, the myoblasts are randomly arranged in the extending direction.

As shown in FIG. 7(a), the cell suspension located on the surface 211 of the cell sheet forming member 200 contains cells S2 adhered to the first uneven portion 240, for example. At this time, in the orientation control section 201, each first uneven portion 240 extends in the long side direction (first direction) of the first uneven portion 240, and the width of each first uneven portion 240 is the same as that of a general cell. It is about one to several times the size. Therefore, as shown in FIG. 7B, the positions of the cells S2 are preferentially distributed within the range of the first concave-convex portion 240, and the long axis direction of the cells S2 is arranged in the first direction. connected in a straight line. That is, the extending direction of the cells S2 is controlled so as to be aligned with the long-side direction of the first concave-convex portion 240 .

As shown in FIG. 7(c), in the cell sheet-forming member that does not satisfy (A) above, since the orientation of the cells S2 is not controlled, the long axis directions of the cells exist in random directions.
The cell suspension located on the surface 111 of the cell sheet forming member 100 contains, for example, cells forming a cell sheet. As shown in FIG. 8( a ), the cells are cells S1 that preferentially adhere to the first flat portion 230 , and although inferior to the first flat portion 230 , adhere to the first uneven portion 240 . is also a cell S2 that has been allowed to The cells seeded on the surface 111 are cultured in a monolayer spreading state on the surface 111 at the initial stage of culture. In the orientation control section 201, as shown in FIG. 8B, the first flat portions 230 and the first uneven portions 240 extend in the first direction and are alternately arranged in the second direction. Therefore, on the surface 211 of the cell-sheet-forming member, for example, the orientation of the cells S1 preferentially adhered to the first flat portion 230 is determined by the structure of the first flat portion 230 and the first unevenness that partitions it. controlled by the structure of section 240;

In the first uneven portion 240 sandwiched between the first flat portions 230 adjacent to each other, the cell S2 adhering to the first uneven portion 240 is inferior to the first flat portion 230, but the flat portion Orientation control by 130 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. 8(c).

In the cell sheet forming member 200 shown in FIGS. 6(a) to 8(c), the cells are cultured in the orientation control section 201 so as to have the orientation in the first direction. On the other hand, in the cell cluster forming portion 202, after a certain period of time, the cells positioned on the second concave-convex portion 222 naturally aggregate, and at least on the second flat portion 221, spherical or rod-shaped cell clusters are formed. 11 is formed. At least a part of the cell mass 11 should be adhered to the second flat portion 221 so as not to separate. The cell mass 11 may be wholly adhered to the second flat portion 221 or partially adhered to the second flat portion 221 . Furthermore, the cell cluster 11 is a portion protruding from the orientation control section 201 to the cell cluster formation section 202 . In either case, the cell mass 11 does not detach from the surface 211 and is difficult to release.

<Example 2>
Example 2 of the cell-sheet-forming member, the method for producing the cell-sheet-forming member, and the method for producing the cell-sheet according to the above embodiments will be described below.

<Preparation of cell sheet forming member>
As shown in FIGS. 4A and 4B, a second uneven portion 222 is formed on the outer peripheral portion, and the orientation control portion 201 (the first flat portion 230 and the first uneven portion 240) is formed inside the second uneven portion 222. Then, a nickel intaglio plate for forming the cell sheet forming member 200 having the second flat portion 221 sandwiched between the two orientation control portions 201 by transfer was produced. Next, using a nickel intaglio stamper as a stamper, the polystyrene sheet is processed into the second uneven portion 222, the orientation control portion 201 (the first flat portion 230 and the first uneven portion 240), and the second flat portion 221 by nanoimprinting. and thereby, the cell sheet forming member 200 of Example 2 was produced.

The second uneven portion 222 in the cell-sheet-forming member 200 of Example 2 had a plurality of stepped structures, and the pitch of the protrusions 223 in the second uneven portion 222 was 300 nm. The height of each convex portion 223 in the second concave-convex portion 222 was measured using an AFM, and the average height from the bottom surface of the concave portion to the tip of the convex portion 223 was 446 nm.

The orientation control section 201 includes a first flat portion 230 and a first uneven portion 240. Each first flat portion 230 has a shape extending in the first direction, and the first flat portion 230 has a shape of a cell sheet. The width (the length in the second direction) of each first flat portion 230 arranged in the second direction intersecting the first direction in the region on the surface of the forming member 200 excluding the outer peripheral portion was 10 μm. Each first uneven portion 240 has a plurality of stepped structures that fill spaces between adjacent first flat portions 230, and the length between adjacent first flat portions 230 in the second direction (first uneven portion 240 width) was 10 μm, and the pitch of the convex portions 241 in the first concave-convex portion 240 was 300 nm. The height of each convex portion 241 in the first uneven portion 240 was measured using an AFM, and the average height from the bottom surface of the concave portion to the tip of the convex portion 241 was 446 nm. Also, the average height from the bottom surface of the concave portion to the first flat portion 230 was 455 nm. The long side of the second flat portion 221 was adjacent to the long side of the first uneven portion 240, and the width of each second flat portion 221 was 100 μm. Then, the cell sheet-forming member 200 of Example 2 was cut into a circle with a diameter of 8.8 mm, UV irradiation was performed as a sterilization treatment, and then used in a cell culture test.

<Cell culture test>
First, mouse-derived cardiomyocytes (manufactured by Cosmo Bio) were cultured in a cell culture flask (25 cm ² ). The culture conditions were 37° C., 5% CO ₂ atmosphere, using a dedicated medium. Cells were collected using trypsin according to a standard method. The collected cells were counted using a hemacytometer.

Next, the cell sheet-forming member 200 of Example 2 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). Cardiomyocytes adjusted to a concentration of 4×10 ⁵ cells/ml were seeded in 0.2 ml portions. After culturing in a CO ₂ incubator for 24 hours, the medium was changed, and the shape of the cells was observed after changing the medium every other day.

After 4 days from the day 0 of the start of culture, cell clusters were formed in which the cells aggregated into rods. The cell mass adhered to the second flat portion 221 and was not released from the substrate by the medium exchange.

According to the said 2nd Embodiment, the effect enumerated below is acquired.
(2-1) The orientation control unit 201 can align the extending direction of the cells with the first direction. As a result, it is possible to improve the orientation of the cells in the cell sheet that spreads in two dimensions. In addition, in the cell mass forming portion 202 , the cells positioned on the second uneven portion 222 aggregate on the second flat portion 221 , and at least some of the cells adhere to the second flat portion 221 . As a result, the cell mass 11 is formed on the second flat portion 221, and the cell mass 11 adheres to the second flat portion 221, making it difficult to separate. Therefore, in a state where the cell mass 11 is adhered to the second flat portion 221, identification of the cell mass 11 and measurement of the size of the cell mass 11 and movement such as pulsation are facilitated. In addition, analysis such as image processing is facilitated.

(2-2) The second flat portion 221 is sandwiched between the two first uneven portions 240 . Therefore, cell clusters 11 can be formed near oriented cells.
(2-3) The second concave-convex portion 222 is sandwiched between the two first concave-convex portions 240 . Therefore, the cell mass 11 can be formed with cells near the oriented cells.

(2-4) The second flat portion 221 has a length of 50 μm or more and 200 μm or less in the second direction, and a length of 100 μm or more in the first direction. As a result, the cells positioned on the second concave-convex portion 222 aggregate on the second flat portion 221 , and at least part of the cell mass 11 can adhere to the second flat portion 221 .

(2-5) In the second concave-convex portion 222, the length in the second direction of the portion sandwiched between two adjacent orientation control portions 201 is 50 μm or more and 1 mm or less. Also, the pitch of the protrusions in the second uneven portion 222 is 10 nm or more and 10 μm or less. Therefore, the cells of the second concave-convex portion 222 easily aggregate.

(2-6) The first uneven portion 240 has a length of 10 μm or more and 50 μm or less in the second direction, and a length of 100 μm or more in the first direction. As a result, the orientation of the cells can be improved in the cell sheet that spreads in two dimensions.

According to the said 2nd Embodiment, you may change as follows and may implement.
If it is possible to improve the orientation of the cells in the cell sheet that spreads in the two-dimensional direction, the length of the first uneven portion 240 in the second direction is 10 μm or more and 50 μm or less, and the length in the first direction is 100 μm. It doesn't have to be that much.

If the aggregation of the cells positioned on the second uneven portion 222 is possible, the length of the second uneven portion 222 in the second direction is 50 μm or more and 1 mm or less, and the length of the convex portion 223 in the second uneven portion 222 is The pitch does not have to be 10 nm or more and 10 μm or less.

If at least part of the cell mass 11 can be adhered to the second flat portion 221, the second flat portion 221 has a length of 50 μm or more and 200 μm or less in the second direction and a length of 50 μm or more and 200 μm or less in the first direction It does not have to be 100 μm or more.

As long as the cell mass 11 can be formed by cells near the oriented cells, the second concave-convex portion 222 does not have to be sandwiched between the first concave-convex portions 240 .
- The second flat portion 221 does not have to be sandwiched between the first concave-convex portions 240 as long as a cell cluster can be formed near the oriented cells.

[Orientation Control Unit of Cell Sheet Forming Member in Second Embodiment]
The shape of the convex portion 241 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 241 can 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 240 .
- The shape of the first concave-convex portion 240 is not limited to a linear shape extending in the first direction, and may be changed to a polygonal line 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 240 and the first flat portion 230 to be flush, that is, to change the base end portion of the convex portion 241 and the first flat portion 230 to be flush. . In addition, as described above, the configuration in which the tip surface of the first concave-convex portion 240 and the first flat portion 230 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 240 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 240 may have one side surface continuous with the first flat portion 230 and may be modified to have a plurality of concave portions formed on the side surface.

- The width of one first uneven portion 240 and the width of the other first uneven portion 240 may be different or equal to each other. Note that if the width of one first uneven portion 240 and the width of the other first uneven portion 240 are equal to each other, it is possible to improve the uniformity of the properties of the cell sheet in the second direction. Become.

- The width of one first flat portion 230 and the width of the other first flat portion 230 may be different or equal to each other. Note that if the width of one first flat portion 230 and the width of the other first flat portion 230 are equal to each other, it is possible to improve the uniformity of the properties of the cell sheet in the second direction. Become.

- The width of the first flat portion 230 and the width of the first uneven portion 240 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 230, the width of the first flat portion 230 is within a range in which the orientation can be controlled and is larger than the width of the first uneven portion 240. preferred. Further, when cell adhesion is predominant in the first uneven portion 240, the width of the first uneven portion 240 is within a range in which the orientation can be controlled and is larger than the width of the first flat portion 230. preferred.

The second direction in which the first flat portions 230 and the first concave-convex portions 240 are alternately arranged is not limited to the direction orthogonal to the first direction. Directions forming an angle of 45° are also possible.

[Cell cluster forming part of cell sheet forming member in second embodiment]
In the second uneven portion 222, 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 222 , the positions of the convex portions may be irregular at each lattice point on the square lattice, each lattice point on the hexagonal lattice, and further at the first uneven portion 240 .
- The height of the second uneven portion 222 may be the same as, higher, or lower than the height of the first flat portion 230 and/or the first uneven portion 240 .

If the cell cluster formation section 202 is provided with a second flat section 221 adjacent to the orientation control section 201, and the second flat section 221 is connected to the second uneven section 222, for example, the second unevenness The portion 222 does not have to be adjacent to the orientation control portion 201 . This is because it is sufficient that the cell mass 11 is formed near the oriented cells.

Furthermore, the above-described first and second embodiments may be modified and implemented as follows.
- Surfaces 111 and 211 of cell sheet forming members 100 and 200 are coated with an extracellular matrix such as laminin, collagen, gelatin, fibronectin, polylysine (PDL or PLL), and hyaluronic acid for the purpose of enhancing cell adhesiveness. Organics containing adhesion factors such as , polymers, gels, etc. may be applied. Alternatively, the surface may be made of metal. In addition, the surfaces 111 and 211 of the cell sheet-forming members 100 and 200 may be hydrophilic or hydrophobic for the purpose of enhancing cell adhesiveness and cell sheet flatness.

- 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.

- A stimuli-responsive material may be applied to the surfaces 111 and 211 of the cell sheet forming members 100 and 200 in order to facilitate the peeling and collection of the cell sheets formed on the surfaces 111 and 211 of the cell sheet forming members 100 and 200 . 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 coating methods, or the substrate treated with the stimulus-responsive material may be structured using the methods described below. In addition, in order to facilitate detachment and collection of cell aggregates, the culture substrate on which the cell aggregates are formed may be subjected to ultrasonic treatment.

- The cell sheet-forming members 100 and 200 are not limited to transfer bodies using an intaglio plate, but may be transfer bodies 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.

[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 11... Cell mass, 100... Cell sheet formation member, 110... Culture dish, 111... Surface, 120... Cover, 130... Flat part, 140... Uneven part, 141... Convex part, 150... Intaglio, 151... Flat molding part, 152... Concavo-convex forming part, 160... Base material, 200... Cell sheet forming member, 201... Orientation control part, 202... Cell mass forming part, 211... Surface, 221... Second flat part, 222... Second uneven part, 223... Convex part 230... First flat part 240... First uneven part 241... Convex part 242... Concave part 250... Intaglio 251... First flat molded part 252... First uneven molded part 253... Second flat molded portion 254 Second concave-convex molded portion 260 Base material.

Claims (9)

having a surface for forming a cell sheet,
The surface comprises an orientation control section for controlling the orientation of cells, and a cell cluster forming section for forming cell clusters by aggregating the cells,
The orientation control part has a shape extending in a first direction, and fills a gap between the first flat parts arranged in a second direction intersecting the first direction and the first flat parts adjacent to each other. and a first uneven portion,
The cell cluster forming portion includes a second flat portion adjacent to the orientation control portion, and a second uneven portion adjacent to the orientation control portion and connected to the second flat portion,
At least a part of the cell mass is formed on the second flat portion ,
The pitch of the convex portions in the first uneven portion is 10 nm or more and 10 μm or less.
Cell sheet forming member. The cell sheet-forming member according to claim 1 , wherein the second flat portion is sandwiched between the first uneven portions. The cell sheet-forming member according to claim 2, wherein the second uneven portion is sandwiched between the first uneven portions. The cell sheet formation according to any one of claims 1 to 3 , wherein the second flat portion has a length of 50 µm or more and 200 µm or less in the second direction and a length of 100 µm or more in the first direction. Element. In the second concave-convex portion, a portion sandwiched between the adjacent orientation control portions has a length of 50 μm or more and 1 mm or less in the second direction,
The cell sheet-forming member according to any one of claims 1 to 4 , wherein the pitch of the protrusions in the second uneven portion is 10 nm or more and 10 µm or less. The cell sheet formation according to any one of claims 1 to 5 , wherein the first uneven portion has a length of 10 µm or more and 50 µm or less in the second direction and a length of 100 µm or more in the first direction. Element. A substrate comprising the cell sheet-forming member according to any one of claims 1 to 6 and having cell clusters formed on the surface thereof. The substrate according to claim 7 , wherein cells having orientation in the first direction are formed in the orientation control section. forming an intaglio;
forming the surface of the cell sheet forming member for forming the cell sheet by transferring the intaglio;
The surface comprises an orientation control section for controlling the orientation of cells, and a cell cluster forming section for forming cell clusters by aggregating the cells,
The orientation control part has a shape extending in a first direction, and fills a gap between the first flat parts arranged in a second direction intersecting the first direction and the first flat parts adjacent to each other. and a first uneven portion,
The cell cluster forming portion includes a second flat portion adjacent to the orientation control portion, and a second uneven portion adjacent to the orientation control portion and connected to the second flat portion,
At least a part of the cell mass is formed on the second flat portion,
The pitch of the convex portions in the first uneven portion is 10 nm or more and 10 μm or less,
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 flat forming part that forms the second flat part;
and a second irregularity forming part that forms the second irregularity part,
Forming the intaglio includes forming the first concave-convex molded portion and the second concave-convex molded portion using at least one of a photolithography method, a colloidal lithography method, an anodization method, and an interference exposure method. including,
A method for producing a cell sheet-forming member.

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