JPWO2020158481A1 - Cell sheet manufacturing method and cell sheet - Google Patents

Abstract

The cell sheet of the method for producing a cell sheet is formed on the surface of a cell sheet forming member, and the surface has a shape extending in the first direction and the entire surface intersects the first direction. It is provided with a plurality of flat portions arranged in two directions and a plurality of uneven portions having a plurality of stepped structures that fill the space between the flat portions adjacent to each other, and the stepped structure is either a convex portion or a concave portion. .. In the manufacturing method, cells to which adhesion to either one of a flat portion and an uneven portion is dominant over adhesion to the other are adhered to the surface of the cell sheet forming member, and the cells are attached to the surface of the cell sheet forming member in the first direction. It involves forming a cell sheet with a three-dimensional tissue of oriented cells.

Description

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

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

Japanese Patent No. 5850419

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

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

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 such that cells have orientation.

In the method for producing a cell sheet according to one aspect of the present disclosure, the cell sheet is formed on the surface of a cell sheet forming member, and the surface has a shape extending in the first direction and is described above. The step is provided with a plurality of flat portions arranged in a second direction intersecting the first direction on the entire surface, and a plurality of uneven portions having a plurality of step structures that fill the space between the flat portions adjacent to each other. The structure is either a convex portion or a concave portion, and in the manufacturing method, a cell in which adhesion to either one of the flat portion and the concave-convex portion is dominant over adhesion to the other is used as a cell sheet forming member. It comprises forming a cell sheet with a three-dimensional tissue of cells oriented in the first direction on the surface of the cell sheet forming member, which is adhered to the surface.

According to the above configuration, a cell sheet having a three-dimensional tissue of cells oriented in the first direction can be easily formed.
In the method for producing a cell sheet, forming the cell sheet means culturing the cells to prepare cultured cells, and coating the cultured cells with an adhesive film composed of at least one coating film layer. The coated cultured cells are seeded on the surface of the cell sheet forming member, and the coated cultured cells are cultured and adhered to each other by the adhesive film to have the orientation. It may include 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 the cell elongation 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 method for producing a cell sheet, the cell sheet may be formed of cells having a collapsed layer structure as the distance from the cell sheet forming member increases in the thickness direction.

According to the above configuration, since a cell sheet capable of culturing cells in a state close to that of a living body can be produced, it can be expected that the drug responsiveness of the produced cell sheet will be close to that of the living body.
In the 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 producing a cell sheet, each uneven portion includes a plurality of the stepped structures that fill the space between the flat portions adjacent to each other, and the stepped structure may have a pitch of 100 nm or more and 10 μm or less.

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

The cell sheet according to one aspect of the present disclosure contains cells adhered to each other, the cells have orientation, and a three-dimensional tissue of the cells is constituted.
In the cell sheet, the cell may have an orientation inclined in a second direction intersecting the first direction with respect to a first direction in which the extension direction of the cell is a specific direction.

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

According to the above configuration, since the cells are cultured in a state close to that of a living body, it can be expected that the drug responsiveness or the like will be close to that of the living body.

(A) is a perspective view showing the structure of a cell sheet forming member for a cell sheet together with a petri dish, and (b) shows an enlarged part of the surface of the cell sheet forming member of FIG. 1 (a). It is a perspective view, (c) is an enlarged plan view which shows a part of the surface of the cell sheet forming member of FIG. 1 (b), and (d) is the cell sheet forming member of FIG. 1 (b). It is a partial cross-sectional view showing a part of. An image of the surface of the cell sheet forming member of FIG. 1 (a) taken by a scanning electron microscope. FIG. 1 is a process diagram for explaining an example of a method for manufacturing the cell sheet forming member of FIG. 1 (a). (A) to (c) are schematic views for explaining the manufacturing process of a cell sheet. (A) is a diagram showing cultured cells, (b) is a diagram showing a state in which the cultured cells of FIG. 5 (a) are covered with an adhesive membrane, and (c) is a diagram showing the culture of FIG. 5 (b). The figure which shows the state which the cell was three-dimensionally organized, (d) is the fluorescent staining image of the myoblast cultured using the cell sheet forming member. (A) to (c) are schematic views for explaining the manufacturing process of a cell sheet. (A) to (c) are schematic views for explaining the manufacturing process of a cell sheet.

Hereinafter, a method for producing a cell sheet and an embodiment of the cell sheet will be described. First, the configuration of the cell sheet forming member will be described, and then a method for manufacturing the cell sheet forming member and a 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 in a petri dish culture dish 110. 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 directly processed and provided on a petri dish, the cell sheet forming member 100 is formed by, for example, injection molding the petri dish. The petri dish holds the cell suspension in a 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. 1 (b), 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 uneven portion 140 includes a plurality of stepped structures, and the plurality of stepped 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 a convex portion 141, and the concave-convex portion 140 includes a concave portion sandwiched between flat portions 130 adjacent to each other and a plurality of convex portions 141 located on the bottom surface of the concave portion.

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

Each convex portion 141 constituting the concave-convex portion 140 is located at each apex of the triangular lattice, for example, when viewed from the direction facing the surface 111. Each uneven portion 140 repeats such an arrangement of the convex portions 141 in the first direction and the second direction. In the case of the uneven portion 140 in which the convex portion 141 is located at each vertex of the triangular lattice, the master for forming the convex portion 141 is masked with a mask suitable for forming a minute repeating structure, for example, a single particle film. It is possible to form by the etching method.

When viewed from the direction facing the surface 111, each convex portion 141 has, for example, a circular shape. 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 in the plan view shape of the convex portion 141 is the diameter of the convex portion 141.

The configuration in which the pitch of the convex portion 141 satisfies the following (A) and (B) is such that the elongation direction of animal cells such as humans and mice, particularly the above-mentioned myoblasts, fibroblasts, and cardiomyocytes is the first direction. It is suitable from the viewpoint of alignment. That is, in the configuration in which the pitch of the convex portion 141 satisfies the following (A) and (B), the superiority or inferiority to the adhesion of animal cells, particularly the above-mentioned myoblasts, fibroblasts, and cardiomyocytes, is the same as that of the flat portion 130. It is suitable from the viewpoint of being clearly partitioned from the uneven portion 140.

(A) Pitch of convex portion 141: 100 nm or more and 10 μm or less (B) Diameter of convex portion 141: 50% or more and 100% or less of pitch of convex portion 141 Length of each flat portion 130 in the second direction (short side direction) 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 the size of the cells to be cultured (5 μm or more and 100 μm or less). The composition in which the width of the flat portion 130 and the width of the uneven portion 140 satisfy the following (C) and (D) is an animal cell such as a human mouse, particularly the above-mentioned myoblast, fibroblast, and cardiomyocyte. It is preferable from the viewpoint that it is easy to align the extension directions of the cells 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 recess 142 may be provided between the flat portion 130 and the convex portion 141 adjacent thereto. Since the plurality of convex portions 141 are scattered on the concave-convex portion 140, the concave portions 142, which are spaces between the convex portions 141, are continuous in the first direction and the second direction in the concave-convex portion 140.

In the thickness direction of the cell sheet forming member 100, the length between the bottom surface of the recess 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 height difference between the tip 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 convex portion 141 to the height of the convex portion 141 is the aspect ratio of the convex portion 141.

The configuration in which the boundary step satisfies the following (E) is suitable from the viewpoint of enhancing the flatness of the cell sheet. The configuration in which the height of the convex portion 141 satisfies the following (F) and the configuration in which the aspect ratio of the convex portion 141 satisfies the following (G) are from the viewpoint of enhancing the structural stability of the concave-convex portion 140, and also. It is suitable 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 And the above In the case of the configuration satisfying (A) and (B), the cell has priority over one structure regardless of whether the cell has predominantly adherent to the flat portion 130 or the predominantly adherent to the uneven portion 140. In combination with the inferior adhesion to the other structure, the cell elongation direction is aligned with the first direction, which is the extending direction of both structures. As a result, in the cell sheet spreading in the two-dimensional direction along the surface 111, it is possible to align the elongation directions of the cells in the one-dimensional direction, that is, to improve the orientation of the cells. Then, the cell sheets are piled up in the thickness direction in a state where the cultured cells are aligned in the one-dimensional direction, that is, in a state having orientation, to form a three-dimensional tissue. At this time, since 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 limited to the first direction. Instead, the cells are divided in the second direction and in the directions 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 of the same species as the cell but smaller than the cell and extend in the first direction. Filled with cells.

Further, the configuration satisfying the above (E), particularly the configuration in which the tip surface of each convex portion 141 and the flat portion 130 are flush with each other, is a cell sheet formed so as to cover the uneven portion 140 and the flat portion 130. , Makes it possible to increase the flatness of it. Further, the configuration 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 uneven portion 140, both the cells in which the adhesion to the flat portion 130 is predominant and the cells in which the adhesion to the uneven portion 140 is predominant can be used. It is also possible to apply a common cell sheet forming member 100. That is, it is possible to increase the versatility of the cell sheet forming member 100.

[Manufacturing method of cell sheet forming member]
Next, an example of a method for manufacturing a cell sheet forming member will be described. In the following description, an example in which the surface 111 of the cell sheet forming member is formed by transcription of the intaglio 150 by using the nanoimprint method will be described.

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

The lower surface of the intaglio 150 has a shape extending in the first direction (direction orthogonal to the paper surface), and is mutually aligned with a plurality of flat portions arranged in the second direction (left-right direction of the paper surface) intersecting the first direction. It is provided with an uneven portion having a plurality of stepped structures that fills the space between adjacent flat portions. The flat portion of the intaglio 150 is a portion for forming the flat portion 130 of the cell sheet forming member 100 by transcription. The uneven portion of the intaglio 150 is a portion for forming the uneven portion 140 of the cell sheet forming member 100 by transfer.

The step structure of the intaglio 150 is a convex portion or a concave portion. The stepped structure of the intaglio 150 in the present embodiment is a concave portion 151 for forming the convex portion 141, and the pitch of the concave portion 151 is 100 nm or more and 10 μm or less. In the step of forming the intaglio 150, for example, at least one of a photolithography method, a colloidal lithography method, an anodizing method, and an interference exposure method is used on the silicon substrate for forming the intaglio 150. Is formed. Further, the intaglio 150 itself may be obtained by one or a plurality of transfers from the master. The master has a shape corresponding to the surface shape of the intaglio 150 using, for example, at least one of a photolithography method, a colloidal lithography method, an anodizing method, and an interference exposure method for a silicon substrate.

Next, the lower surface of the intaglio 150 is opposed to the surface 111 of the base material 160 for forming the cell sheet forming member 100. The material for forming the base material 160 is, for example, a thermoplastic resin or a photocurable resin. Then, with the base material 160 having fluidity, the lower surface of the intaglio 150 is pressed against the surface 111 of the base material 160. Next, the intaglio 150 is released from the surface 111 of the base material 160 while the fluidity of the base material 160 is suppressed. As a result, the recess 151 of the intaglio 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 cell adhesion on the surface of the thermoplastic resin or photocurable resin of the material for forming the base material 160, for example, laminin, collagen, gelatin, fibronectin, polylysine (PDL or PLL), hyaluronic acid, etc. Organic substances containing adhesion factors such as extracellular matrix, polymer, and gel may be applied. Further, as a material for forming the base material 160, a biomaterial such as a polysaccharide or a protein may be used.

Further, a stimulus-responsive material may be applied in order 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 change is preferable. Specifically, poly-N-isopropylacrylamide (PIPAAm) is preferable. The stimulus-responsive material may be applied to the substrate using a conventional coating method, or the structure may be processed on the substrate treated with the stimulus-responsive material by the method described below. Further, in order to facilitate the peeling and recovery of the cell sheet, the culture substrate on which the cell sheet is formed may be subjected to ultrasonic treatment.

[Method for manufacturing cell sheets]
Next, a cell sheet manufactured by 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, for example, cultured cells 1 that adhere to the flat portion 130.
The cell suspension contains cultured cells 1 that are alternately coated with 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. 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. be.

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

As a polymer that interacts with a polymer containing an RGD sequence containing a second component, both substances can, for example, bind, adhere, adsorb, and transfer electrons by interacting with a polymer containing an RGD sequence. It is not particularly limited as long as it is a substance that can be brought close to each other to some extent, but 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 100,000 as an example), proteoglycan, integrin, enzyme, antibody, and the like. be. Naturally-derived macromolecules that interact with macromolecules containing RGD sequences include, for example, water-soluble polypeptides, low molecular weight peptides, polyamino acids such as α-polylysine and ε-polylysine (molecular weight 5,000), heparin and heparan sulfate, and the like. Sugars such as dextran sulfate (molecular weight 500,000) and hyaluronic acid (molecular weight 1 million ~).

The combination of the first component and the second component is, for example, fibronectin and gelatin, fibronectin and heparin, fibronectin and dextran sulfate, and laminin and collagen.

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

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

When preparing the solution containing the first component, examples of the solvent include aqueous solvents such as water and a buffer, and examples of the buffer include a Tris buffer such as Tris-HCl buffer, a phosphate buffer, and the like. Examples include HEEPS buffer, citrate-phosphate buffer, glycylglycine-sodium hydroxide buffer, Britton-Robinson buffer, GTA buffer and the like.

In this way, the cultured cells 1 are covered with the first membrane 2a composed of the first component, and then the free first component is removed. As a method for removing the first component, the free first component is removed by centrifuging using the above solvent, separating the cultured cells 1 from the solution containing the first component, and removing the supernatant. There is a method of removing it. Thereby, the cultured cells 1 coated with the first membrane 2a composed of the first component can be obtained.

When forming a laminated film composed of the first film 2a of the first component and the second film 2b consisting of the second component as the adhesive film 2, the cultured cells 1 coated with the first component are brought into contact with the second component. Thereby, it is covered with the second film 2b composed of the second component. The contact method with the second component can be carried out in the same manner as that of the first component, and in the case of contacting with the second component-containing liquid, the same as the method for preparing the first component-containing liquid. A liquid containing the second component can be prepared. As a result, the cultured cells 1 coated with the adhesive film 2 composed of the first film 2a composed of the first component and the second film 2b composed of the second component can be obtained. The laminated film composed of the first film 2a composed of the first component and the second film 2b composed 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 onto the surface 111 of the cell sheet forming member 100, and the cultured cells 1 are seeded. After that, the cultured cells 1 are cultured.

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. Time, more preferably 3-12 hours. Thereby, the cultured cells 1 can be adhered to each other or the grown cultured cells 1 can be adhered to each other via the adhesive membrane 2 to produce a cell sheet 10 having a three-dimensional tissue (FIG. 5 (FIG. 5). c) See.).

In the cell sheet 10, other cultured cells extending in the one-dimensional direction are cultured so as to overlap the cultured cells extending in the one-dimensional direction. Therefore, the size and shape of the cultured cells extending in the one-dimensional direction are large. , Not only in the first direction, but also in the second direction and in 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 filled with other cells smaller than the cells and extending in the first direction. Therefore, in the three-dimensional tissue, a layer structure is observed at the first layer constituting the first surface adjacent to the surface 111 and at a position close to the first layer, but the surface 111 (the first layer) due to variations in the size and shape of the cultured cells 1. The layer structure collapses as the surface moves away from the first surface and approaches the second surface facing the first surface, and the layers are stacked so that another cultured cell 1 enters the gap between the cultured cells 1. The three-dimensional tissue in which the cells are oriented can be cultured in a state close to that of a living body. Further, as shown in FIG. 5D, the orientation of the cells of the cultured cells 1 is such that the cell elongation direction is directed to, for example, the first direction, which is a specific direction, or the first direction to the second direction. It has the property of aligning in a slightly tilted direction. That is, the cultured cells 1 have an orientation in which the elongation direction of the cultured cells 1 is a one-dimensional direction, 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 of the size of a general 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 the cell length in the first direction. Axial directions are arranged and they are connected in a straight line. That is, the elongation 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, so that the cells are arranged in a random direction in the long axis direction.

Further, as shown in FIG. 6A, the cell suspension located on the surface 111 of the cell sheet forming member 100 includes, for example, the cultured cells 1 that adhere 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 linearly arranged in the long axis direction of the cells in the first direction. To be connected to. That is, the elongation direction of the cultured cells 1 is controlled so as to be aligned with the long side direction of the uneven portion 140. As shown in FIG. 6 (c), in the cell sheet-forming substrate that does not satisfy the above (A), the orientation of the cultured cells 1 is not controlled, so that the long axis direction of the cells exists in a random direction.

On the other hand, in the cell sheet-forming substrate satisfying the above (A), as shown in FIG. 7 (a), the cultured cells 1 of the cell suspension held on the cell sheet-forming member 100 are attached to the flat portion 130. It is a cell that preferentially adheres, and is inferior to the flat portion 130, but is also a cell that is allowed to adhere to the uneven portion 140. Alternatively, the cultured cells 1 of the cell suspension held in the cell sheet forming member 100 are cells that preferentially adhere to the uneven portion 140, which is inferior to the uneven portion 140 but 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 portion 130 and the uneven portion 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 that partitions the flat portion 130. Be controlled.

The uneven portion 140 sandwiched between the flat portions 130 adjacent to each other is inferior to the flat portion 130, but the cultured cells 1 adhered to the uneven portion 140 control the orientation by the flat portion 130. It will be reflected. As a result, as shown in FIG. 7 (c), the cultured cells 1 whose orientation is controlled so that the long axis direction of the cells is aligned in a one-dimensional direction such as the first direction spreads over the entire surface 111. To form the cell sheet 10.

Alternatively, the orientation of the cultured cells 1 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 the uneven portion 140. The flat portion 130 sandwiched between the uneven portions 140 adjacent to each other is inferior to the uneven portion 140, but the cultured cells 1 adhered to the flat portion 130 can control the orientation by the uneven portion 140. It will be reflected. As a result, the cultured cells 1 whose orientation is controlled so that the long-axis directions of the cells are aligned in a one-dimensional direction such as the first direction spread over the entire surface 111, thereby forming the cell sheet 10.

Examples 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 embodiment will be described below.
<Example 1>
<Preparation of cell sheet forming member>
First, a nickel intaglio was prepared for forming the uneven portion 140 of the cell sheet forming member 100 by transfer. Next, using a nickel intaglio plate as a stamper, the polystyrene sheet was processed with an uneven portion 140 by a nanoimprint method, whereby the cell sheet forming member 100 of Example 1 was produced. The flat portion 130 in the cell sheet forming member 100 of Example 1 has a shape extending in the first direction, and is aligned in the second direction intersecting the first direction as a whole on the surface of the cell sheet forming member 100. The width (length in the second direction) of each flat portion 130 was 10 μm. Each uneven portion 140 has a plurality of step structures that fill the space between the flat portions 130 that are adjacent to each other, and the length between the flat portions 130 that are adjacent to each other in the second direction is 10 μm, and the convex portion in the uneven portion 140. The pitch of the portion 141 was 300 nm. The height of each convex portion in the concave-convex portion 140 was measured by using 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 is cut into a circle having a diameter of 8.8 mm, the cut cell sheet forming member 100 is irradiated with UV, and after this sterilization treatment, it is used for a cell culture test. bottom.

<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 modified Eagle's medium) supplemented with 10% FBS (fetal bovine serum) at 37 ° C. and 5% CO ₂ atmosphere. Trypsin was used for cell recovery, and the procedure was followed. The number of collected cells was counted using a hemocytometer.

The operation of coating the collected cells with two types of cell adhesion components was carried out using a cell lamination kit CellFeille (registered trademark) (Sumitomo Bakelite Co., Ltd.) according to a protocol. 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) is placed 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 a washing solution, and after centrifugation, 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 a washing solution, and after centrifugation, the washing solution was removed.

By the above operation, a cell having a layered coating of component A and component B on the cell surface was obtained. Subsequently, the above coating operation was performed three times. Finally, it was suspended in Solution A, and after removing Solution A by centrifugation, it was suspended in a washing solution.

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

Next, 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 (48 holes) for cell culture. After dispensing 0.2 ml of the cleaning solution attached to the kit to the multi-well plate on which the cell sheet forming member 100 was installed, the cleaning solution was removed, 0.2 ml of solution C was dispensed, and CO _{2 was set at 37 ° C.} The cells were allowed 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.

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

Three days after the start of the culture, the cells that had been laminated and cultured were collected and used for observation. The stacked and cultured cells were fixed on 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 fixing, phosphoric acid was used. Washed with buffered saline.

Subsequently, a phosphate buffered saline solution containing 0.5% Triton X-100 was used for treatment at room temperature for 5 minutes for permeation treatment. After washing with phosphate buffered saline, it was immersed in a solution of acti-stein488 phalloidin (manufactured by Cytoskeleton) diluted to 100 nM for 30 minutes for staining. The stained cell sheet forming member 100 was washed with a phosphate buffered saline solution, and then attached to a slide glass to which an encapsulant (Antifade mounting medium, manufactured by Fluka) was dropped to prepare a slide glass for observation. Then, the orientation of the cells was observed using a confocal laser scanning microscope (manufactured by Olympus). As a result, in the cell sheet forming member 100, a state in which the extending directions of the cells were aligned in the one-dimensional direction was observed. FIG. 5D is a fluorescence-stained image of myoblasts. From the surface of the myoblasts, an orientation having an inclination in the second direction with respect to the first direction in which the elongation direction of the cultured cells 1 is a specific direction is recognized.

The stacked and cultured cells were fixed on 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 fixing, phosphoric acid was used. Washed with buffered saline. Then, for tissue dehydration, 50% ethanol solution, 70% ethanol solution, 80% ethanol solution, 90% ethanol solution, 95% ethanol solution, and 100% ethanol solution were immersed in this order for half a day, and replaced. ..

Next, tissue embedding was performed using Technovit7100 (Kulzer). Specifically, deethanolation is performed using a preliminary replacement solution, the replacement solution and hardenerII are mixed at a ratio of 11: 1, and the cells laminated and cultured are embedded in a state of being adhered to the cell sheet forming member 100. bottom. The embedded tissue was trimmed to an appropriate size, 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) A cell sheet 10 having an oriented three-dimensional tissue can be produced.

(2) The cultured cells 1 are three-dimensionally organized via the adhesive membrane 2.
(3) The cells can be cultured so that the elongation direction of the cells is oriented along the first direction and the elongation direction of the cells is oriented along a direction having a slight inclination from the first direction. ..

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

(5) Since the cell sheet forming member 100 includes the flat portion 130 and the uneven portion 140 on the surface 111, both the cell in which the adhesion to the flat portion 130 is predominant and the cell in which the adhesion to the uneven portion 140 is predominant. Can be applied to. Thereby, the versatility of the cell sheet forming member 100 can be enhanced.

(6) A cell sheet having oriented three-dimensional tissues such as myoblasts, fibroblasts, and cardiomyocytes can be produced.
(7) Since the three-dimensional tissue having orientation cultivates cells in a state close to that of a living body, it is expected that the drug responsiveness or the like is close to that of a living body. Therefore, it can be expected to be effective in transplantation organizations in regenerative dormitories.

(8) The risk of cell sheet contamination can be reduced in that the work of stacking the cell sheets of the two-dimensional tissue is not required.
The above embodiment may be modified as follows.

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

The surface 111 of the cell sheet forming member 100 is an extracellular matrix such as laminin, collagen, gelatin, fibronectin, polylysine (PDL or PLL), hyaluronic acid, polymer, gel, for the purpose of enhancing cell adhesion. An organic substance containing an adhesion factor such as the above may be applied, or the surface may be composed of a 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 cell sheets.

-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. The ascorbic acid is preferably L-form.

[Cell sheet forming member]
-The shape of the convex portion 141 can be any one of a cone such as a cone and a pyramid, a column such as a cylinder and a pyramid, a frustum such as a truncated cone and a frustum, and a hemispherical shape. be.

The position of the convex portion 141 can be irregular at each grid point on the square grid, each grid point on the hexagonal lattice, and further at the uneven portion 140.
The shape of the uneven portion 140 is not limited to a straight line extending in the first direction, but can be changed to a polygonal line extending in the first direction or a curved line 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 with each other, that is, to change the base end portion of the convex portion 141 and the flat portion 130 to be flush with each other. As described above, the configuration in which the tip 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 step structure constituting the uneven portion 140 can be changed to a concave portion, and can be changed to both a concave portion and a convex portion. For example, the uneven portion 140 may be changed to a structure in which one side surface 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 the other 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 the other uneven portion 140 are equal to each other, it is possible to enhance the uniformity of the characteristics of the cell sheet 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, it is possible to enhance the uniformity of the characteristics of the cell sheet 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 is within a range in which the orientation can be controlled and is larger than the width of the uneven portion 140. Further, when cell adhesion is predominant 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 portion 130 and the uneven portion 140 are alternately arranged is not limited to the direction orthogonal to the first direction, but if the direction intersects the first direction, for example, the angle formed with the first direction is It is also possible to set the direction to be 45 °.

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

-The cell sheet forming member can be applied to any one capable of holding a cell suspension, such as a multi-well plate, a petri dish, a flask, and a chamber slide.

1 ... cultured cells, 2 ... adhesive membranes, 2a ... first membranes, 2b ... second membranes, 10 ... cell sheets, 100 ... cell sheet forming members, 110 ... culture dishes, 111 ... surfaces, 120 ... lids, 130 ... flat Part, 140 ... Concavo-convex part, 141 ... Convex part, 142 ... Concave, 150 ... Concave plate, 151 ... Concave, 160 ... Base material.

Claims (12)

It is a method for manufacturing cell sheets.
The cell sheet is formed on the surface of the cell sheet forming member, and is formed on the surface of the cell sheet forming member.
The surface has a shape extending in the first direction, and fills the space between a plurality of flat portions arranged in the second direction intersecting the first direction and the flat portions adjacent to each other. With multiple uneven parts with multiple step structures,
The step structure is either a convex portion or a concave portion, and the step structure is one of a convex portion and a concave portion.
In the manufacturing method, a cell in which adhesion to either one of the flat portion and the uneven portion is superior to adhesion to the other is adhered to the surface of the cell sheet forming member, and the cell is attached to the surface of the cell sheet forming member. Includes forming a cell sheet with a three-dimensional tissue of cells oriented in the first direction.
How to make a cell sheet. Forming the cell sheet is
To produce cultured cells by culturing the cells,
By covering the cultured cells with an adhesive membrane composed of at least one coating film layer,
Seeding the coated cultured cells on the surface of the cell sheet forming member, and
It comprises culturing the coated cultured cells and adhering them to each other by the adhesive membrane to form a three-dimensional tissue of the cells having the orientation.
The method for producing a cell sheet according to claim 1. The method for producing a cell sheet according to claim 2, wherein the cell sheet comprises a three-dimensional tissue having an orientation in which the cell elongation 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 the cells of the cell sheet are filled with other cells that are smaller than the cells and have orientation in the first direction. The method for producing a cell sheet according to claim 3 or 4, wherein the cell sheet is formed of cells having a collapsed layer structure as the distance from the cell sheet forming member increases in the thickness direction. The cell according to any one of claims 1 to 5, wherein the cell cultured in the cell sheet forming member is at least one selected from the group consisting of myoblasts, fibroblasts, and cardiomyocytes. How to make a cell sheet. The method according to any one of claims 1 to 6, wherein each uneven portion includes a plurality of the stepped structures that fill the space between the flat portions adjacent to each other, and the stepped structure has a pitch of 100 nm or more and 10 μm or less. How to make a cell sheet. The flat portion is the top surface of the convex portion.
The method according to any one of claims 1 to 7, wherein the uneven portion includes a concave portion sandwiched between the flat portions adjacent to each other and a plurality of convex portions provided on the bottom surface of the concave portion. How to make a cell sheet. Contains cells that are adhered to each other
The cell has orientation and constitutes a three-dimensional tissue of the cell.
Cell sheet. The cell sheet according to claim 9, wherein the cells have an orientation inclined in a second direction intersecting the first direction with respect to a first direction in which the extension direction of the cells is a specific direction. The cell sheet according to claim 9 or 10, wherein the space between the cells is smaller than that of the cells and is filled with other cells oriented in the first direction. The cell sheet according to any one of claims 9 to 11, wherein the cell sheet is formed of cells having a layered structure that collapses as it approaches the second surface facing the first surface from the first surface of the cell sheet. The cell sheet of the description.

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