WO2020158481A1

WO2020158481A1 - Method for manufacturing cell sheet, and cell sheet

Info

Publication number: WO2020158481A1
Application number: PCT/JP2020/001733
Authority: WO
Inventors: 寿子得能; 啓篠塚; 紘太郎大; 凌峰沈
Original assignee: 王子ホールディングス株式会社
Priority date: 2019-01-28
Filing date: 2020-01-20
Publication date: 2020-08-06
Also published as: JP7192892B2; JPWO2020158481A1

Abstract

This cell sheet obtained by a method for manufacturing a cell sheet is formed on the surface of a cell sheet formation member, the surface having a shape extending in a first direction and being provided with a plurality of flat sections arranged on the entire surface in a second direction which intersects with the first direction, and a plurality of uneven sections provided with a plurality of step structures filling in between mutually adjacent flat sections, the step structures being any one of projections and recesses. This manufacturing method includes causing cells, in which adhesion to one of flat sections and uneven sections is more favored than adhesion to the other of flat sections and uneven sections, to adhere to the surface of the cell sheet formation member, and forming a cell sheet provided with a three-dimensional tissue of cells having orientation in one direction on the surface of the cell sheet formation member.

Description

Cell sheet manufacturing method and cell sheet

The present disclosure relates to a cell sheet manufacturing method and a cell sheet for forming a cell sheet.

A cell sheet in which a three-dimensional tissue of cells is constructed is known (see Patent Document 1). In order to construct a three-dimensional tissue of cells, there are a method of laminating cell sheets of a two-dimensional tissue and a method of laminating cells by a 3D printer. However, the method of stacking cell sheets is a complicated and time-consuming task of stacking cell sheets. Further, the method of using the 3D printer requires a dedicated device such as a 3D printer.

Patent No. 5850419

By the way, although some cells have an orientation, the coating method of the cells with an extracellular matrix of Patent Document 1 can form a three-dimensional tissue, but the orientation can be controlled. Have difficulty.

A method of constructing a three-dimensional tissue so that cells have orientation is to stack cell sheets of two-dimensional tissue constructed so that cells have orientation. However, such a method of stacking cell sheets is a complicated and time-consuming operation of stacking cell sheets.

An object of the present disclosure is to provide a method for producing a cell sheet and a cell sheet capable of easily producing a cell sheet having a three-dimensional tissue in which cells have an orientation.

In the method for producing a cell sheet according to an aspect of the present disclosure, the cell sheet is formed on a surface of a cell sheet forming member, the surface having a shape extending in a first direction, and A plurality of flat portions arranged in a second direction that intersects the first direction on the entire surface, and a plurality of uneven portions having a plurality of step structures that fill in between the flat portions adjacent to each other are provided. The structure is one of a convex portion and a concave portion, the manufacturing method, the adhesion to any one of the flat portion and the concavo-convex portion cells that are more predominant than the adhesion to the other of the cell sheet forming member. Adhering to the surface, forming a cell sheet having a three-dimensional tissue of cells oriented in the first direction on the surface of the cell sheet forming member.

According to the above configuration, it is possible to easily form the cell sheet including the three-dimensional tissue of cells having the orientation in the first direction.
In the above-mentioned method for producing a cell sheet, forming the cell sheet includes culturing the cells to produce cultured cells, and coating the cultured cells with an adhesive film composed of at least one coating film layer. And seeding the coated cultured cells on the surface of the cell sheet forming member, culturing the coated cultured cells and adhering them to each other by the adhesive film, and having the orientation Forming a three-dimensional tissue of cells.

In the method for producing a cell sheet, the cell sheet may include a three-dimensional tissue having an orientation in which a cell extending direction is inclined in the second direction with respect to the first direction. ..

In the method for producing a cell sheet, the cells of the cell sheet may be filled with other cells that are smaller than the cells and have an orientation in the first direction.
In the above-mentioned method for producing a cell sheet, the cell sheet may be formed of cells having a collapsed layer structure as the cell sheet is separated from the cell sheet forming member in the thickness direction.

According to the above configuration, a cell sheet capable of culturing cells in a state close to that of a living body can be manufactured, and thus it can be expected that the drug responsiveness of the manufactured cell sheet approaches that of a living body.
In the above-mentioned method for producing a cell sheet, the cells cultured in the cell sheet forming member may be at least one selected from the group consisting of myoblasts, fibroblasts, and cardiomyocytes.

In the method for manufacturing a cell sheet described above, each uneven portion may include a plurality of step structures that fill the space between the flat portions adjacent to each other, and the step structures may have a pitch of 100 nm or more and 10 μm or less.

According to the above configuration, the orientation of the cultured cells can be improved.
In the method for producing the cell sheet, the flat portion is a top surface of a convex portion, and the concave and convex portion is a concave portion sandwiched between the flat portions adjacent to each other, and a plurality of concave portions are provided on the bottom surface of the concave portion. And a convex portion.

A cell sheet according to an aspect of the present disclosure includes cells adhered to each other, the cells have an orientation, and a three-dimensional tissue of the cells is configured.
In the above-mentioned cell sheet, the cells may have an orientation that is inclined in a second direction that intersects the first direction with respect to a first direction in which the elongation direction of the cells is a specific direction.

In the cell sheet, spaces between the cells may be filled with other cells that are smaller than the cells and have an orientation in the first direction.
In the cell sheet, the cell sheet may be formed of cells having a layered structure that collapses from the first surface of the cell sheet toward the second surface facing the first surface.

According to the above configuration, cells are cultivated in a state close to that of a living body, so that drug responsiveness can be expected to approach that of the living body.

1A is a perspective view showing a structure of a cell sheet forming member for a cell sheet together with a petri dish, and FIG. 1B is an enlarged view showing a part of the surface of the cell sheet forming member of FIG. 1A. It is a perspective view, (c) is a top view which expands and shows a part of surface of the cell sheet formation member of FIG.1(b), (d) is a cell sheet formation member of FIG.1(b). FIG. 3 is a partial cross-sectional view showing an enlarged part of FIG. The image which image|photographed the surface of the cell sheet formation member of FIG. 1 (a) with the scanning electron microscope. FIG. 3 is a process drawing for explaining an example of a method for manufacturing the cell sheet forming member of FIG. (A)-(c) is a schematic diagram for demonstrating the manufacturing process of a cell sheet. 5A is a diagram showing cultured cells, FIG. 5B is a diagram showing a state in which the cultured cells of FIG. 5A are covered with an adhesive film, and FIG. 5C is a culture of FIG. 5B. The figure which shows the state where the cells were three-dimensionally organized, (d) is a fluorescence-stained image of myoblasts cultured using a cell sheet-forming member. (A)-(c) is a schematic diagram for demonstrating the manufacturing process of a cell sheet. (A)-(c) is a schematic diagram for demonstrating the manufacturing process of a cell sheet.

Hereinafter, an embodiment of a cell sheet manufacturing method and a cell sheet will be described. First, the configuration 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. 1A, the cell sheet forming member 100 is, for example, a sheet material arranged 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 on a petri dish, the cell sheet forming member 100 is shaped by injection molding the petri dish, for example. The petri dish holds the cell suspension in the space surrounded by the culture dish 110 and the lid 120. Examples of cells contained in the cell suspension are myoblasts, fibroblasts, and cardiomyocytes.

As shown in FIG. 1B, the surface 111 of the cell sheet forming member 100 includes a plurality of flat portions 130 and a plurality of uneven portions 140. The concavo-convex portion 140 includes a plurality of step structures, and the plurality of step structures fill the space between the flat portions 130 adjacent to each other. The step structure is a convex portion or a concave portion. The step structure in the present embodiment is the convex portion 141, and the concave-convex portion 140 includes the concave portion sandwiched between the flat portions 130 adjacent to each other and the plurality of convex portions 141 located on the bottom surface of the concave portion.

As shown in FIG. 1C, each flat portion 130 is a flat surface extending in one direction, which is the first direction (the vertical direction in FIG. 1C). The flat portions 130 are arranged in the second direction (the left-right direction in FIG. 1C) orthogonal to the first direction on the entire surface 111. The flat portion 130 corresponds to the top surface of the convex portion (protrusion) extending in the first direction. The uneven portions 140 also extend in the first direction and are arranged in the second direction on the entire surface 111. This is clear from the image of the surface of the cell sheet forming member 100 taken by a scanning electron microscope as shown in FIG.

Each of the convex portions 141 forming the concave-convex portion 140 is located, for example, at each vertex of the triangular lattice when viewed from the direction facing the surface 111. Each concavo-convex part 140 repeats such arrangement|sequence of the convex part 141 in a 1st direction and a 2nd direction. In the case of the concavo-convex portion 140 in which the convex portion 141 is located at each apex of the triangular lattice, the master for forming the convex portion 141 is a mask suitable for forming a minute repeating structure, for example, a single particle film. Can be formed by the etching method described above.

Each protrusion 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 the plan view shape is the diameter of the convex portion 141.

The configuration in which the pitch of the convex portions 141 satisfies the following (A) and (B) is such that the elongation direction of animal cells such as human and mouse, particularly the above-mentioned myoblasts, fibroblasts, and cardiomyocytes is set to the first direction. It is suitable in terms of alignment. That is, in the configuration in which the pitch of the convex portions 141 satisfies the following (A) and (B), the superiority or inferiority with respect to the adhesion of animal cells, particularly the above-mentioned myoblasts, fibroblasts, cardiomyocytes, etc. It is preferable from the viewpoint of being clearly partitioned from the uneven portion 140.

(A) Pitch of convex part 141: 100 nm or more and 10 μm or less (B) Diameter of convex part 141: 50% or more and 100% or less of the pitch of convex part 141 The length of each flat part 130 in the second direction (short side direction) The width is the width of the flat portion 130. Further, the length in the second direction (short side direction) between the flat portions 130 adjacent to each other is the width of the uneven portion 140.

The width of the flat portion 130 and the width of the uneven portion 140 are, for example, 1/10 times or more and 10 times or less of the size of cells to be cultured (5 μm or more and 100 μm or less). The configuration in which the width of the flat portion 130 and the width of the uneven portion 140 satisfy the following (C) and (D) is applied to animal cells such as humans and mice, particularly myoblasts, fibroblasts, and cardiomyocytes described above. It is preferable from the viewpoint of facilitating that the extension direction of is aligned with the first direction.

(C) Width of flat portion 130: 10 μm or more and 50 μm or less (D) Width of uneven portion 140: 10 μm or more and 50 μm or less As shown in FIG. A concave portion 142 may be provided between the flat portion 130 and the convex portion 141 adjacent to the flat portion 130. Since the plurality of convex portions 141 are scattered on the concave-convex portion 140, the concave portions 142 that are spaces between the convex portions 141 are continuous in the concave-convex portion 140 in the first direction and the second direction.

In the thickness direction of the cell sheet forming member 100, the length between the bottom surface of the concave portion 142 and the flat portion 130 is the height of the flat portion 130. Further, in the thickness direction of the cell sheet forming member 100, the difference in height between the tip end surface of each convex portion 141 and the 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 the 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 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.

The configuration in which the boundary step satisfies the following (E) is suitable from the viewpoint of improving the flatness of the cell sheet. A configuration in which the height of the convex portion 141 satisfies the following (F), and a configuration in which the aspect ratio of the convex portion 141 satisfies the following (G), can improve the structural stability of the concave and convex portion 140, and This is preferable from the viewpoint of facilitating the formation of the uneven portion 140.

(E) Boundary step: 0.5 μm or less, preferably 0.3 μm or less (F) Height of convex portion 141: 50 nm or more and 5 μm or less (G) Aspect ratio of convex portion 141: 0.1 or more and 10 or less As long as the structure satisfies (A) and (B), the cells have priority over one of the structures regardless of whether the cells are mainly attached to the flat portion 130 or the uneven portions 140. And the poor adhesion to the other structure, the cell extension direction is aligned with the first direction, which is the extending direction of both structures. As a result, in the cell sheet that spreads in the two-dimensional direction along the surface 111, it is possible to align the cell extension directions in the one-dimensional direction, that is, improve the orientation of the cells. Then, the cell sheet is stacked in the thickness direction in a state where the cultured cells are aligned in a one-dimensional direction, that is, in a state of having orientation, to form a three-dimensional tissue. At this time, since the other cells extending in the one-dimensional direction are cultured so as to overlap the cells extending in the one-dimensional direction, the size and shape of the cells extending in the one-dimensional direction are only in the first direction. However, it is divided in the second direction and the direction orthogonal to the first direction and the second direction. As a result, in the three-dimensional tissue, the cells extending in the first direction and adjacent to each other in the second direction are the same species as the cells, but are smaller than the cells, and extend in the first direction. Filled with cells.

In addition, in the cell sheet formed so as to cover the uneven portion 140 and the flat portion 130, the configuration satisfying the above (E), in particular, the configuration in which the tip end surface of each convex portion 141 and the flat portion 130 are flush with each other , It is possible to increase the flatness of it. Furthermore, the structure satisfying the above (F) can further enhance the flatness of the cell sheet.

Since the surface 111 of the cell sheet forming member 100 includes the flat portion 130 and the concave-convex portion 140, both the cells having a superior adhesion to the flat portion 130 and the cells having a superior adhesion to the concave-convex portion 140, It is also possible to apply a common cell sheet forming member 100. That is, the versatility of the cell sheet forming member 100 can be enhanced.

[Method of manufacturing cell sheet forming member]
Next, an example of a method of manufacturing the 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 plate 150 using the nanoimprint method will be described.

As shown in FIG. 3, the method for manufacturing the cell sheet forming member includes a step of forming the intaglio plate 150 and a step of forming the surface 111 of the cell sheet forming member 100 by transferring the intaglio plate 150.

The lower surface of the intaglio plate 150 has a shape extending in a first direction (a direction orthogonal to the paper surface) and a plurality of flat portions arranged in a second direction (left-right direction of the paper surface) intersecting with the first direction and mutually arranged. And a concavo-convex portion having a plurality of step structures that fill the space between the adjacent flat portions. The flat part of the intaglio plate 150 is a part for forming the flat part 130 of the cell sheet forming member 100 by transfer. The concavo-convex portion of the intaglio plate 150 is a portion for forming the concavo-convex portion 140 of the cell sheet forming member 100 by transfer.

The step structure of the intaglio plate 150 is a convex portion or a concave portion. The step structure of the intaglio plate 150 in the present embodiment is the concave portions 151 for forming the convex portions 141, and the pitch of the concave portions 151 is 100 nm or more and 10 μm or less. In the step of forming the intaglio plate 150, for example, a concavo-convex portion is formed on the silicon substrate for forming the intaglio plate 150 by using at least one of a photolithography method, a colloidal lithography method, an anodic oxidation method, and an interference exposure method. Is formed. Further, the intaglio plate 150 itself may be obtained by transferring the master plate once or plural times. A shape corresponding to the surface shape of the intaglio plate 150 is formed on the master by using at least one of a photolithography method, a colloidal lithography method, an anodic oxidation method, and an interference exposure method for a silicon substrate.

Next, the lower surface of the intaglio plate 150 is opposed to the surface 111 of the base material 160 for forming the cell sheet forming member 100. The forming material of the base material 160 is, for example, a thermoplastic resin or a photocurable resin. Then, the lower surface of the intaglio plate 150 is pressed against the surface 111 of the base material 160 while the base material 160 has fluidity. Next, the intaglio plate 150 is released from the surface 111 of the base material 160 with the fluidity of the base material 160 suppressed. As a result, the recess 151 of the intaglio plate 150 is transferred to the surface 111 of the base material 160, and the flat portion 130 and the uneven portion 140 are formed.

For the purpose of enhancing the adhesiveness of cells on the surface of the thermoplastic resin or the photocurable resin forming the base material 160, for example, laminin, collagen, gelatin, fibronectin, polylysine (PDL or PLL), hyaluronic acid, etc. An organic substance containing an adhesion factor such as extracellular matrix, polymer, or gel may be applied. Further, a biomaterial such as a polysaccharide or a protein may be used as a material for forming the base material 160.

In addition, a stimuli-responsive material may be applied to facilitate peeling and recovery of the cell sheet after the formation of the three-dimensional cell tissue. As the stimulus-responsive material, a temperature-responsive polymer whose water affinity changes with temperature changes is preferable. Specifically, poly-N-isopropylacrylamide (PIPAAm) is preferable. The stimulus-responsive material may be applied to the substrate using a conventional application method, or the structure treated on the substrate treated with the stimulus-responsive material may be processed using the method described below. Further, in order to facilitate the peeling and recovery of the cell sheet, ultrasonic treatment may be performed on the culture substrate on which the cell sheet is formed.

[Cell Sheet Manufacturing Method]
Next, a cell sheet manufactured using the cell sheet forming member 100 will be described. In culturing, a coating method using an extracellular matrix is used here.

As shown in FIG. 4A, the cell suspension located on the surface 111 of the cell sheet forming member 100 contains the cultured cells 1 that adhere to the flat portion 130, for example.
The cell suspension contains cultured cells 1 in which a first solution containing a cell adhesion first component and a second solution containing a cell adhesion second component that interacts with the first component are alternately coated. That is, the cultured cells 1 shown in FIG. 5A are covered with the adhesive film 2. 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. 5(b)). The combination of the first component and the second component is, for example, a combination of a polymer containing an arginine-glycine-aspartic acid (RGD) sequence to which an integrin binds and a polymer interacting with a polymer containing an RGD sequence. is there.

The polymer containing the RGD sequence containing the first component may be a protein having the RGD sequence or a protein chemically bound to the RGD sequence. Further, it may have a RGD sequence, and may be a naturally-occurring polymer other than protein or a synthetic polymer. Examples of the polymer containing an RGD sequence composed of a protein include conventionally known adhesive proteins such as fibronectin (molecular weight of about 500,000), vitronectin, laminin, cadherin and collagen.

As the polymer interacting with the polymer containing the RGD sequence containing the second component, both substances are capable of, for example, binding, adhering, adsorbing, and electron transfer due to the interaction with the polymer containing the RGD sequence. The substance is not particularly limited as long as it is a substance that can be brought into close proximity, and it is any one of a protein that interacts with a polymer containing an RGD sequence, a naturally-occurring polymer, and a synthetic polymer. Examples of the protein that interacts with the polymer containing the RGD sequence include water-soluble proteins, and specific examples thereof include collagen, gelatin (molecular weight of 100,000 as an example), proteoglycan, integrin, enzyme, antibody, etc. is there. Examples of naturally-occurring polymers that interact with polymers containing RGD sequences include water-soluble polypeptides, low-molecular peptides, polyamino acids such as α-polylysine and ε-polylysine (molecular weight 5,000), heparin and heparan sulfate, Examples include sugars such as dextran sulfate (molecular weight 500,000) and hyaluronic acid (molecular weight 1,000,000).

The combination of the first component and the second component is, for example, any of 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, a cultured cell 1 is prepared, and the entire surface thereof is coated with an adhesive film 2 to prepare a coated cell. Specifically, the prepared cultured cells 1 are put into a test tube.

Next, the first component is brought into contact with the cultured cell 1. Examples of the method for contacting the cultured cells with the first component include, for example, a method of directly adding the first component, a method of immersing the cell in a liquid containing the first component, and a dropping of the liquid containing the first component into the cultured cells 1. There is a method of spraying. Dipping is preferable because the operation is easy. The contact conditions can be appropriately determined depending on the contact method and the concentration of the contained liquid used. 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 further 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 further preferably 37° C.

When preparing the liquid containing the first component, the solvent may be an aqueous solvent such as water or a buffer, and the buffer may be, for example, a Tris buffer such as Tris-HCl buffer, a phosphate buffer, HEPES buffer, citrate-phosphate buffer, glycylglycine-sodium hydroxide buffer, Britton-Robinson buffer, GTA buffer and the like can be mentioned.

After coating the cultured cells 1 with the first membrane 2a composed of the first component in this manner, the released first component is removed. As a method for removing the first component, centrifugation is performed using the above solvent, the cultured cells 1 and the liquid containing the first component are separated, and the supernatant is removed. A method of removing is mentioned. As a result, the cultured cells 1 covered with the first film 2a composed of the first component can be obtained.

When a laminated film composed of the first film 2a of the first component and the second film 2b of the second component is formed 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, the second film 2b made of the second component is coated. The method of contacting with the second component can be carried out in the same manner as the first component. When contacting with the liquid containing the second component, it is carried out in the same manner as the method for preparing the liquid containing the first component. A liquid containing the second component can be prepared. As a result, the cultured cells 1 coated with the adhesive film 2 including the first film 2a including the first component and the second film 2b including the second component can be 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 peritoneal layer is, for example, 1 to 20 layers, preferably 1 to 10 layers, and more preferably 1 to 5 layers. Then, the cell suspension containing the cultured cells 1 is dropped on the surface 111 of the cell sheet forming member 100, and the cultured cells 1 are seeded. Then, the cultured cell 1 is cultured.

The culture conditions for completing the three-dimensional cell sheet of the 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. Time, more preferably 3 to 12 hours. As a result, the cell sheets 10 having a three-dimensional tissue can be manufactured by adhering the cultured cells 1 to each other or the proliferated cultured cells 1 to each other via the adhesive film 2 (see FIG. 5 ( See c).).

In the cell sheet 10, other cultured cells extending in the one-dimensional direction are cultivated so as to overlap the cultured cells extending in the one-dimensional direction. Therefore, 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 space between the cultured cells that extend in the first direction and are adjacent to each other in the second direction is smaller than the cells and filled with other cells that extend in the first direction. Therefore, in the three-dimensional tissue, a layer structure is recognized in the first layer constituting the first surface adjacent to the surface 111 or a position close to the first layer. The layer structure collapses as it moves away from the first surface) and approaches the second surface facing the first surface, and the other cultured cells 1 are stacked so that another cultured cell 1 enters the gap between the cultured cells 1. The three-dimensional tissue in which the cells have the orientation can culture the cells in a state close to that of a living body. Further, as shown in FIG. 5(d), the orientation of the cells of the cultured cells 1 is such that the cell extension direction is, for example, a first direction which is a specific direction, or a direction from the first direction to the second direction. It has the property of aligning in a slightly inclined direction. That is, the cultured cells 1 have a one-dimensional extension direction of the cultured cells 1, and have, for example, a first direction which is a specific direction, or an orientation having an inclination in the second direction with respect to the first direction. ..

As shown in FIG. 4A, each flat portion 130 extends in the long side direction (first direction) of the uneven portion 140, and the width of each flat portion 130 is 1 to several times the size of a typical cell. It is about double. Therefore, as shown in FIG. 4B, the positions of the cultured cells 1 adhering to the flat portion 130 are preferentially distributed within the range of the flat portion 130, and the cultured cells 1 have cell lengths in the first direction. The axial direction is arranged and they are linearly connected. That is, the extension direction of the cultured cells 1 is controlled so as to be aligned with the long side direction of the flat portion 130. As shown in FIG. 4(c), in the cell sheet-forming substrate that does not satisfy the above (A), the orientation of the cultured cells 1 is not controlled, and thus the cells are arranged in a long axis direction at random.

Further, as shown in FIG. 6A, the cell suspension located on the surface 111 of the cell sheet forming member 100 contains, for example, the cultured cells 1 adhered to the uneven portion 140. At this time, each uneven portion 140 extends in the long side direction (first direction) of the uneven portion 140, and the width of each uneven portion 140 is about 1 to several times the size of a general cell. Therefore, as shown in FIG. 6B, the cultured cells 1 are preferentially distributed within the range of the uneven portion 140, and the cultured cells 1 are arranged linearly with the long axis direction of the cells arranged in the first direction. Connected to. That is, the extending direction of the cultured cells 1 is controlled so as to be aligned with the long side direction of the uneven portion 140. In addition, as shown in FIG. 6C, in the cell sheet forming substrate that does not satisfy the above (A), the orientation of the cultured cells 1 is not controlled, and thus the long axis direction of the cells exists in a random direction.

On the other hand, in the cell sheet forming base material that satisfies the above (A), as shown in FIG. The cells preferentially adhere to each other, and although they are inferior to the flat portion 130, they are cells that are allowed to adhere to the uneven portion 140. Alternatively, the cultured cells 1 of the cell suspension retained in the cell sheet forming member 100 are cells that preferentially adhere to the uneven portion 140, and are inferior to the uneven portion 140, but with respect to the flat portion 130. It is also a cell that is allowed to adhere.

In such a case, as shown in FIG. 7B, the flat portions 130 and the 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 cultured cells 1 preferentially adhered to the flat portion 130 depends on the structure of the flat portion 130 and the structure of the uneven portion 140 partitioning the flat portion 130. Controlled.

The uneven portion 140 sandwiched between the flat portions 130 adjacent to each other is inferior to the flat portion 130, but in the cultured cells 1 adhered to the uneven portion 140, the orientation of the flat portion 130 can be controlled. Reflected. As a result, as shown in FIG. 7C, the cultured cells 1 whose orientation is controlled so that the long axis direction of the cells are aligned in a one-dimensional direction such as the first direction spreads over the entire surface 111, and Thus, the cell sheet 10 is formed.

Alternatively, the orientation of the cultured cells 1 that are preferentially adhered to the uneven portion 140 is controlled by the structure of the uneven portion 140 and the structure of the flat portion 130 that partitions it. In the flat part 130 sandwiched between the concavo-convex parts 140 adjacent to each other, the orientation of the cultured cells 1 adhered to the flat part 130 can be controlled by the concavo-convex part 140, although it is inferior to the concavo-convex part 140. Reflected. As a result, the cultured cells 1 whose orientation is controlled so that the long axis direction of the cells are aligned in the one-dimensional direction such as the first direction spreads over the entire surface 111, whereby the cell sheet 10 is formed.

Examples of the cell sheet forming member, the method for manufacturing the cell sheet forming member, and the method for manufacturing the cell sheet described in the above embodiment will be described below.
<Example 1>
<Production of cell sheet forming member>
First, a nickel intaglio plate for forming the uneven portion 140 of the cell sheet forming member 100 by transfer was produced. Next, using the nickel intaglio plate as a stamper, the concavo-convex portion 140 was processed on the polystyrene sheet by the nanoimprint method, whereby the cell sheet forming member 100 of Example 1 was produced. The flat portions 130 of the cell sheet forming member 100 of Example 1 have a shape extending in the first direction, and the entire surface of the cell sheet forming member 100 is arranged in the second direction intersecting the first direction. The width (length in the second direction) of each flat portion 130 was 10 μm. Each concavo-convex portion 140 includes a plurality of step structures that fill the space between the mutually adjacent flat portions 130, and the length in the second direction between the mutually adjacent flat portions 130 is 10 μm. The pitch of the parts 141 was 300 nm. The height of each convex portion in the concave-convex portion 140 was measured using an AFM, and the average height from the bottom surface of the concave portion to the tip of the convex portion was 446 nm. The average height from the bottom surface of the recess to the flat portion was 455 nm. Then, the cell sheet forming member 100 of Example 1 was cut into a circle having a diameter of 8.8 mm, the cut cell sheet forming member 100 was irradiated with UV, and after this sterilization treatment, used for a cell culture test. did.

<Cell culture test>
First, mouse-derived myoblasts (C2C12 cells, manufactured by DS Pharma Biomedical) were cultured in a cell culture flask (25 cm ² ). The culture conditions were DMEM (Dulbecco's modified Eagle medium) supplemented with 10% FBS (fetal bovine serum) at 37° C. in a 5% CO ₂ atmosphere. Trypsin was used to recover the cells, and the cells were collected according to a standard method. The number of cells in the collected cells was measured using a hemocytometer.

The operation of coating the collected cells with two types of cell adhesion components was performed according to the protocol using a cell stacking kit CellFeille (registered trademark) (Sumitomo Bakelite Co., Ltd.). Specifically, the cell suspension is centrifuged (200 g), the supernatant is removed, the cell pellet is suspended in solution A (corresponding to the first solution), and component A (corresponding to the first component) on the cell surface. Was coated. Next, the cell suspension of solution A was centrifuged to remove solution A. The remaining cell pellet was suspended in the washing solution, centrifuged, and then the washing solution was removed.

Next, the cell pellet was suspended in solution B (corresponding to the second solution), and the cell surface was coated with component B (corresponding to the second component). Next, the cell suspension of solution B was centrifuged to remove solution B. The remaining cell pellet was suspended in the washing solution, centrifuged, and then the washing solution was removed.

By the above operation, cells in which the component A and the component B were layer-coated on the cell surface were obtained. Subsequently, the above coating operation was performed three times. Finally, the suspension was suspended in the solution A, the solution A was removed by centrifugation, and the suspension was suspended in the washing solution.

By the above operation, cells in which the component A and the component B were alternately coated on the cell surface were obtained. After counting the number of cells using a hemocytometer, the supernatant was removed, and the cell suspension was adjusted to a cell concentration of 1×10 ⁶ cells/ml using a medium.

Then, the cell sheet forming member 100 of Example 1 cut into a circle having a diameter of 8.8 mm was placed on the bottom surface of the multi-well plate for cell culture (48 holes). A 0.2 ml portion of the washing solution attached to the kit was dispensed into the multi-well plate on which the cell sheet forming member 100 was installed, the washing fluid was removed, and 0.2 ml portions of the solution C were dispensed, and CO ₂ was set at 37°C. The plate was left to stand in an incubator (5% CO ₂ ) for 1 hr. The solution C is a scaffolding material containing collagen as a main component for enhancing cell adhesion.

Solution C was removed after standing for 1 hr and washed with 0.2 ml of a washing solution, and then 1 ml of myoblasts coated with a cell adhesion component was seeded. After culturing for 24 hours or more in a CO ₂ incubator, the medium was exchanged, and the medium was exchanged every day.

3 days after the start of culture, the cells that had been subjected to layered culture were collected and used for observation. The cells cultured in a laminated manner were fixed to the cell sheet forming member 100 with 4% paraformaldehyde (phosphate buffered saline) at room temperature for 10 minutes, and after removing the solution used for fixation, phosphoric acid was added. Washed with buffered saline.

Subsequently, a phosphate buffered saline containing 0.5% Triton X-100 was used for treatment at room temperature for 5 minutes for permeabilization. After washing with phosphate buffered saline, it was immersed in an acti-stain 488 phalloidin (manufactured by Cytoskeleton) diluted to 100 nM for 30 minutes for staining. After the stained cell sheet forming member 100 was washed with phosphate buffered saline, it was attached to a slide glass onto which a mounting medium (Antifade mounting medium, Fluka) was dropped to prepare a slide glass for observation. Then, the orientation of the cells was observed using a confocal laser microscope (Olympus). As a result, in the cell sheet forming member 100, the state in which the cells were elongated in the one-dimensional direction was recognized. FIG. 5D is a fluorescence-stained image of myoblasts. From the surface of the myoblast, the orientation in which the elongation direction of the cultured cell 1 is inclined in the second direction with respect to the first direction which is the specific direction is recognized.

The cells cultured in a laminated manner were fixed to the cell sheet forming member 100 with 4% paraformaldehyde (phosphate buffered saline) at room temperature for 10 minutes, and after removing the solution used for fixation, phosphoric acid was added. Washed with buffered saline. Then, for dehydration of the tissue, a 50% ethanol solution, a 70% ethanol solution, an 80% ethanol solution, a 90% ethanol solution, a 95% ethanol solution, and a 100% ethanol solution were soaked in this order for half a day, respectively, and the operation was performed. ..

Next, the tissue was embedded using Technovit7100 (Kulzer). Specifically, the pre-replacement liquid is used to remove ethanol, the replacement liquid and hardener II are mixed at a ratio of 11:1, and the cells that have been layer-cultured are embedded in the cell sheet forming member 100 in an adhered state. did. The embedded tissue was trimmed to an appropriate size, and then a section was prepared using a microtome, and the cross section of the tissue was observed.

As described above, according to the above embodiment, the effects listed below can be obtained.
(1) The cell sheet 10 having a three-dimensional tissue having orientation can be manufactured.

(2) The cultured cells 1 are three-dimensionally organized through the adhesive film 2.
(3) The cells can be cultured so that the cell extension direction is oriented along the first direction or the cell extension direction is oriented along a direction slightly inclined from the first direction. ..

(4) The three-dimensional tissue has a layer structure in the first layer adjacent to the surface 111 or a position close to the first layer, but the layer structure collapses as the distance from the surface 111 increases, and the three-dimensional tissue is cultivated due to variations in the size of the cultured cells 1. The cells 1 may be stacked so that another cultured cell 1 enters the gap between the cells 1.

(5) Since the cell sheet forming member 100 includes the flat portion 130 and the concave-convex portion 140 on the surface 111, both the cells having a superior adhesion to the flat portion 130 and the cells having a superior adhesion to the concave-convex portion 140. Can be applied to. Thereby, the versatility of the cell sheet forming member 100 can be improved.

(6) It is possible to manufacture a cell sheet having a three-dimensional tissue having orientation such as myoblasts, fibroblasts, and cardiomyocytes.
(7) Since the three-dimensional tissue having orientation has cells cultivated in a state close to a living body, it is expected that drug responsiveness and the like are close to a living body. Therefore, an effect can be expected also in the transplant tissue in the regenerative dormitory.

(8) The risk of contamination of the cell sheets can be reduced in that the work of stacking the cell sheets of the two-dimensional tissue is unnecessary.
The above embodiment may be modified and implemented as follows.

-The cell sheet 10 can be produced from oriented three-dimensional tissues such as myoblasts, fibroblasts, and cardiomyocytes, and further, the oriented cells include endothelial cells such as vascular endothelial cells, Producing a cell sheet 10 having a three-dimensional tissue by mixing and seeding epithelial cells such as intestinal epithelial cells and stem cells such as iPS cells and mesenchymal stem cells or seeding between tissue layers of cells having orientation Can also

The surface 111 of the cell sheet forming member 100 is, for the purpose of enhancing the adhesiveness of cells, for example, an extracellular matrix such as laminin, collagen, gelatin, fibronectin, polylysine (PDL or PLL), hyaluronic acid, a polymer, a gel. An organic substance containing an adhesion factor such as the above may be applied, or the surface may be made of metal. Further, the surface 111 of the cell sheet forming member 100 may have hydrophilicity or hydrophobicity for the purpose of enhancing the adhesiveness of cells and the flatness of the cell sheet.

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

[Cell sheet forming member]
The shape of the convex portion 141 can be any one of a cone 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. is there.

The position of the convex portion 141 may be irregular at each lattice point on the square lattice, at each lattice point on the hexagonal lattice, and at the irregular portion 140.
The shape of the concavo-convex portion 140 is not limited to the linear shape extending in the first direction, but may be changed to a polygonal line 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 uneven portion 140 and the flat portion 130 to be flush, that is, to change the base end portion of the convex portion 141 and the flat portion 130 to be flush. In addition, as described above, the configuration in which the tip end surface of the uneven portion 140 and the flat portion 130 are flush with each other is suitable from the viewpoint of improving the flatness of the cell sheet.

The stepped structure that constitutes the concave-convex portion 140 can be changed to a concave portion or both a concave portion and a convex portion. For example, the concavo-convex portion 140 can be changed to a structure in which one side surface that is continuous with the flat portion 130 is provided and a plurality of concave portions are formed on the side surface.

The width of one uneven portion 140 and the width of another uneven portion 140 may be different from each other or may be equal to each other. If the width of one uneven portion 140 and the width of another uneven portion 140 are equal to each other, the characteristics of the cell sheet can be more uniform in the second direction. ..

The width of one flat portion 130 and the width of the other flat portion 130 may be different from each other or may be equal to each other. If the width of one flat portion 130 and the width of the other flat portion 130 are equal to each other, the characteristics of the cell sheet can be more uniform in the second direction. ..

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

The second direction in which the flat portions 130 and the concavo-convex portions 140 are alternately arranged is not limited to the direction orthogonal to the first direction, and if the direction intersects the first direction, for example, the angle formed with the first direction is It is also possible that the direction is 45°.

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

The cell sheet forming member can be applied to a multi-well plate, a petri dish, a flask, a chamber slide, etc., as long as it can hold a cell suspension.

1... Cultured cell, 2... Adhesive film, 2a... 1st film, 2b... 2nd film, 10... Cell sheet, 100... Cell sheet forming member, 110... Culture dish, 111... Surface, 120... Lid, 130... Flat Parts, 140... uneven parts, 141... convex parts, 142... concave parts, 150... intaglio, 151... concave parts, 160... substrate.

Claims

A method of manufacturing a cell sheet, comprising:
The cell sheet is formed on the surface of the cell sheet forming member,
The surface has a shape that extends in the first direction, and fills a space between the plurality of flat portions that are arranged in the second direction that intersects the first direction on the entire surface and the flat portions that are adjacent to each other. A plurality of uneven portions having a plurality of step structures,
The step structure is one of a convex portion and a concave portion,
The manufacturing method, the adhesion to any one of the flat portion and the uneven portion is predominantly adherent cells to the other is adhered to the surface of the cell sheet forming member, to the surface of the cell sheet forming member. Forming a cell sheet having a three-dimensional organization of cells having an orientation in the first direction,
Cell sheet manufacturing method.
Forming the cell sheet comprises
Culturing the cells to produce cultured cells,
Coating the cultured cells with an adhesive film comprising at least one coating film layer;
Seeding the coated cultured cells on the surface of the cell sheet forming member,
Culturing the coated cultured cells and adhering them to each other by the adhesive film to form a three-dimensional tissue of the cells having the orientation.
The method for producing the cell sheet according to claim 1.
The method for producing a cell sheet according to claim 2, wherein the cell sheet includes a three-dimensional tissue having an orientation in which a cell extension direction is inclined in the second direction with respect to the first direction.
The method for producing a cell sheet according to claim 3, wherein spaces between the cells of the cell sheet are smaller than the cells and are filled with other cells having an orientation in the first direction.
The method for producing a cell sheet according to claim 3, wherein the cell sheet is formed of cells having a collapsed layer structure as the cell sheet is separated from the cell sheet forming member in the thickness direction.
The cells cultured in the cell sheet forming member are at least one selected from the group consisting of myoblasts, fibroblasts, and cardiomyocytes. Cell sheet manufacturing method.
Each uneven part is provided with a plurality of step structure which fills up between the flat parts which adjoin mutually, and the step structure has a pitch of 100 nm or more and 10 micrometers or less. Cell sheet manufacturing method.
The flat portion is the top surface of the convex portion,
The said uneven|corrugated|grooved part is equipped with the recessed part pinched|interposed by the said flat part which mutually adjoins, and the some convex part provided in the bottom face of the said recessed part. Cell sheet manufacturing method.
Contains cells adhered to each other,
The cells have orientation, and the three-dimensional tissue of the cells is constituted,
Cell sheet.
The cell sheet according to claim 9, wherein the cells have an orientation in which a direction in which the cells extend is a specific direction that is inclined in a second direction that intersects the first direction.
The cell sheet according to claim 9 or 10, wherein spaces between the cells are smaller than the cells and are filled with other cells having an orientation in the first direction.
12. The cell sheet is formed of cells having a layered structure that collapses toward the second surface facing the first surface from the first surface of the cell sheet. The described cell sheet.