Container for cell culture, and method for producing container for cell culture

Abstract

To provide a container for cell culture that is capable of exhibiting a stable and strong effect in promoting differentiation into osteoblasts, and a method for producing the container for cell culture.SOLUTION: A container for cell culture includes a bottom surface in contact with the culture liquid, and multiple columnar structures formed on the bottom surface, where each columnar structure has a diameter of 10 nm or more and less than 50 nm and a height of 20 nm or more. When the diameter of the columnar structure is D, and the arrangement pitch of the columnar structures is P1, P1 is in the range represented by the following formula (1): D<P1<2D.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a cell culture vessel and a method for manufacturing the cell culture vessel. [Background technology]

[0002] Conventionally, there has been a need to produce a large number of osteoblasts for the purpose of using them in regenerative medicine, etc.

[0003] For example, Non-Patent Document 1 describes a technique in which mesenchymal stem cells are differentiated into osteoblasts by culturing them on a highly rigid container bottom (matrix) of 25 kPa or more, particularly 34 kPa. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Engler et al., Matrix Elasticity Directs Stem Cell Lineage Specification. Cell,2006 P677-689. Summary of the Invention [Problem to be solved by the invention]

[0005] However, simply providing the bottom surface of the container with a strength equal to or greater than that described in Non-Patent Document 1 poses the problem of variation in the rate of differentiation into osteoblasts.

[0006] The present invention has been made in consideration of the above circumstances, and aims to provide a cell culture vessel capable of exerting a stable and high differentiation-promoting effect on osteoblasts, and a method for manufacturing the cell culture vessel. [Means for solving the problem]

[0007] In order to solve the above problems, one aspect of the present invention includes the following aspects.

[0008] [1] A cell culture vessel having a bottom surface in contact with a culture medium and a number of columnar structures formed on the bottom surface, each of which has a diameter of 10 nm or more and less than 50 nm and a height of 20 nm or more, wherein when the diameter is D and the arrangement interval of the columnar structures is P1, P1 is in the range represented by the following formula (1). D <P1<2D...(1)

[0009] [2] The cell culture vessel described in [1], wherein the height of the columnar structure is 1.5D or less.

[0010] [3] The cell culture vessel described in [1] or [2], wherein the numerous columnar structures are gold-coated.

[0011] [4] The cell culture vessel according to any one of [1] to [3], wherein the gold coating is coated with fibronectin or collagen.

[0012] [5] The cell culture vessel described in any one of [1] to [4], wherein the numerous columnar structures comprise an acrylic curable resin.

[0013] [6] A method for manufacturing a cell culture vessel, comprising the steps of: preparing an aluminum plate having a large number of depressions formed on its surface; filling the numerous depressions in the aluminum plate with a material and hardening it to form a substrate having a large number of columnar structures; applying a gold coating to the numerous columnar structures; and applying a coating of fibronectin or collagen to the numerous gold-coated columnar structures, wherein the opening of the depression has a width of 10 nm or more and less than 50 nm, and a depth of 20 nm or more, where W is the width and P2 is the spacing between the depressions, and P2 is in the range shown in the following formula (2). W <P2<2W...(2)

[0014] [7] The method for manufacturing a cell culture vessel according to [6], wherein the step of preparing the aluminum plate includes a step of forming a large number of recesses by repeatedly performing a process of forming a porous oxide film by anodization on the aluminum plate before the formation of the recesses, and a process of enlarging the pore size by etching the oxide film. Effect of the Invention

[0015] According to the present invention, it is possible to provide a cell culture vessel capable of exerting a stable and high effect of promoting differentiation into osteoblasts, and a method for producing the cell culture vessel. [Brief description of the drawings]

[0016] [Figure 1] FIG. 2 is a schematic perspective view showing a large number of columnar structures formed on the bottom surface of a cell culture vessel according to a preferred embodiment of the present invention. [Diagram 2] FIG. 2 is a schematic side view of a number of columnar structures shown in FIG. [Diagram 3] 1 is a flow chart showing a manufacturing process of a cell culture vessel according to a preferred embodiment of the present invention. [Figure 4] FIG. 2 is a schematic cross-sectional view showing a large number of depressions in an aluminum plate prepared in a formwork preparation process. [Diagram 5] FIG. 2 is an enlarged plan view of a columnar structure of the cell culture vessel of Example 1. [Figure 6] FIG. 2 is an enlarged side view of a columnar structure of the cell culture vessel of Example 1. [Figure 7] 1 shows images illustrating cell culture vessels of Example 1 and Comparative Examples 1 to 3. [Figure 8] 1 shows staining images using a chromogenic substrate, which show the expression of alkaline phosphatase in cells cultured in the cell culture vessels of Example 1 and Comparative Example 5. [Figure 9] 1 shows fluorescent immunostaining images showing the expression of osteocalcin in cells cultured in the cell culture vessels of Example 1 and Comparative Example 5. [Figure 10] 1 shows staining images using a chromogenic substrate, which show the expression of alkaline phosphatase in cells cultured in the cell culture vessels of Comparative Example 1 and Comparative Example 5. [Figure 11] 1 shows fluorescent immunostaining images showing the expression of osteocalcin in cells cultured in the cell culture vessels of Comparative Example 1 and Comparative Example 5. [Figure 12] 1 shows staining images using a chromogenic substrate, which show the expression of alkaline phosphatase in cells cultured in the cell culture vessels of Comparative Example 2 and Comparative Example 5. [Figure 13] 1 shows fluorescent immunostaining images showing the expression of osteocalcin in cells cultured in the cell culture vessels of Comparative Example 2 and Comparative Example 5. [Figure 14] 1 shows staining images using a chromogenic substrate, which show the expression of alkaline phosphatase in cells cultured in the cell culture vessels of Comparative Examples 3 to 5. [Figure 15] 1 is a graph showing the quantification of alkaline phosphatase expression in cells cultured in each of the cell culture vessels of Example 1 and Comparative Examples 1 to 3. [Figure 16] 1 is a graph showing the quantification of osteocalcin expression in cells cultured in each of the cell culture vessels of Example 1 and Comparative Examples 1 to 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Hereinafter, preferred embodiments of the present invention will be described in detail, with reference to the drawings where necessary.

[0018] FIG. 1 is a schematic perspective view showing a number of columnar structures 8 formed on a bottom surface 5 of a cell culture vessel 1 in a preferred embodiment of the present invention, and FIG. 2 is a schematic side view of the number of columnar structures 8 shown in FIG. 1.

[0019] The form of the cell culture vessel 1 is not particularly limited as long as it can accommodate a culture medium and culture cells, and can be configured as, for example, a dish, a petri dish, a multi-well plate, a culture tube, a culture flask, etc. The cell culture vessel 1 of this embodiment is configured as a shallow dish.

[0020] The cell culture vessel 1 includes a flat plate portion 2, a side wall 3 extending upward from the edge of the flat plate portion 2, and a lid (not shown).

[0021] The upper surface of the flat plate portion 2 constituting the bottom surface 5 (inner bottom surface) of the cell culture vessel 1 and the inner surface 3a of the side wall 3 face the internal space 4 of the cell culture vessel 1. When a culture medium is contained in the internal space 4, the bottom surface 5 and the inner surface 3a of the side wall 3 come into contact with the culture medium.

[0022] The bottom surface 5 is preferably a horizontal and flat surface, but may be a gently curved surface. When the bottom surface 5 is formed as a curved surface, the inclination angle of the bottom surface 5 is set to an angle in which the change in height with respect to the horizontal length of the bottom surface 5 is less than 10%.

[0023] A large number of columnar structures (protrusions) 8 protruding substantially upward are formed on the bottom surface 5. The large number of columnar structures 8 are arranged at substantially equal intervals to form a dot pattern on the bottom surface 5.

[0024] The shape of each columnar structure 8 is not particularly limited, but examples include a cylindrical shape, a truncated cone shape, an elliptical cylinder shape, an elliptical truncated cone shape, a polygonal column shape, etc. In particular, the edge of the upper part of the columnar structure 8 may be a right angle or a rounded R shape in side view.

[0025] The diameter (thickness) D of each columnar structure 8 (see FIG. 2) is in the range of 10 nm or more and less than 50 nm. The diameter D of the columnar structure 8 refers to the diameter of the thickest part (diameter at the bottom end) of the columnar structure 8. The diameter D of the columnar structure 8 can be measured, for example, by comparing it with a scale bar in an image obtained by a scanning electron microscope.

[0026] The height H of each columnar structure 8 is set to 20 nm or more in order to prevent the spaces between the columnar structures 8 from being filled with protein components in the culture solution. There is no particular upper limit to the height H of each columnar structure 8. However, in terms of durability and ease of production, the ratio of the height H to the diameter D of the columnar structure 8 may be 3 or less, 2 or less, 1.5 or less, or 1 or less (in other words, the height H is the diameter D or less). For example, when the diameter D of the columnar structure 8 is 45 nm, the height H may be 135 nm or less, 90 nm or less, or 45 nm or less.

[0027] The arrangement interval (pitch) P1 of the columnar structures 8 is within the range represented by the following formula (1). D <P1<2D[nm]...(1) In addition, when there is a difference in diameter D between multiple columnar structures 8, the arrangement interval P1 between adjacent columnar structures 8 is calculated based on the diameter D of the columnar structure 8 with the larger diameter among the adjacent columnar structures 8.

[0028] The diameter of each columnar structure 8 at a height position 5 nm below the upper end thereof is preferably 5 nm or more, more preferably 8 nm or more, and most preferably 10 nm or more.

[0029] By setting the diameter D, height H, and arrangement interval P1 of the columnar structures 8 within the above ranges, differentiation into osteoblasts can be promoted when cells capable of differentiating into osteoblasts are cultured.

[0030] Although not wishing to be bound by any particular theory, it is believed that by setting the diameter D and spacing P1 of the columnar structures 8 within the above ranges, the columnar structures can be made to have a scale and spacing equivalent to the scale and spacing of the numerous adhesion complexes formed by cells, thereby promoting differentiation into osteoblasts.

[0031] Examples of cells that can differentiate into osteoblasts include, but are not limited to, mesenchymal stem cells, osteoblast progenitor cells, preosteoblasts, iPS cells, etc. The animals from which the cells are derived may be humans or other mammals.

[0032] The material of the cell culture vessel 1 is not particularly limited, but examples thereof include resin, metal, silica glass, ceramics, quartz, etc. Among these, in order to enable cells capable of differentiating into osteoblasts to adhere firmly to the bottom surface 5, it is particularly preferable that the material of the cell culture vessel 1 is resin or metal.

[0033] Examples of the resin include polystyrene resin, polypropylene resin, nylon resin, polycarbonate resin, ABS resin (Acrylonitrile Butadiene Styrene resin), polylactic acid resin, acrylic resin, phenol resin, melamine resin, fluororesin, etc. Among these, polystyrene resin and acrylic resin, which is a photocurable resin, are particularly preferable.

[0034] Examples of metals include gold, silver, copper, platinum, tungsten, titanium, nickel, molybdenum, aluminum, and alloys such as stainless steel, with gold being particularly suitable.

[0035] When a material other than metal (such as resin or glass) is used as the material for the cell culture vessel 1, the surface of the base material such as resin or glass may be coated with a metal such as gold, nickel, titanium, or platinum, or with fibronectin, collagen, etc. This can increase the adhesiveness of cells that can differentiate into osteoblasts, and can further promote differentiation into osteoblasts.

[0036] Whether or not to coat the surface of resin, glass, etc. can be determined based on the results of a preliminary test to confirm whether or not cells capable of differentiating into osteoblasts adhere and spread on uncoated resin or glass containers. If cells capable of differentiating into osteoblasts adhere and spread on the bottom surface of the container, coating is not particularly necessary. The degree of adhesion and spreading should preferably be compared with that of polystyrene cell culture containers, and if the adhesion and spreading properties are as good as or better than those of polystyrene cell culture containers, coating is not necessary.

[0037] When preliminary tests show that cells do not adhere or spread and coating is necessary, at least one member selected from the group consisting of gold, fibronectin, and collagen is particularly preferred as the coating agent.

[0038] An example of multiple coatings is, but is not limited to, coating the surface of the substrate with gold and then coating fibronectin on the gold. Another example is coating collagen on the gold.

[0039] The thickness of the coating on the substrate is not particularly limited, but in order to prevent the gaps between the columnar structures 8 from being filled, it is preferable that the total thickness of each coating is 30 nm or less. It is also preferable that the thickness of each coating is 5 nm or more. For example, when the substrate is coated with gold and fibronectin, it is preferable that the gold coat has a thickness of 5 nm or more, the fibronectin coat has a thickness of 5 nm or more, and the combined thickness of the gold coat and the fibronectin coat is 30 nm or less. When the substrate is coated with only fibronectin, it is preferable that the thickness of the fibronectin coat is in the range of 5 nm or more and 30 nm or less.

[0040] The surface hardness of the columnar structure 8 of the cell culture vessel 1 is not particularly limited, but as described in Non-Patent Document 1, it is preferably 34 kPa or more, and may be 50 kPa or more, or 80 kPa or more.

[0041] By culturing cells capable of differentiating into osteoblasts for several days to several weeks using the cell culture vessel 1 and culture medium constructed as described above, it is possible to stably and strongly promote the differentiation of cells capable of differentiating into osteoblasts into osteoblasts.

[0042] Examples of culture media used to culture cells capable of differentiating into osteoblasts include, but are not limited to, commercially available osteoblast differentiation-inducing media, DMEM media, αMEM media, etc. In addition, a differentiation inducer into osteoblasts may be added to the medium separately. Examples of such differentiation inducers include ascorbic acid and glucocorticoids.

[0043] <Method of manufacturing cell culture vessel 1> FIG. 3 is a flow chart showing the manufacturing process of the cell culture vessel 1 according to a preferred embodiment of the present invention.

[0044] The manufacturing method for cell culture vessel 1 includes a step of preparing an aluminum plate having numerous depressions formed therein (formula preparation step S1), a step of using the aluminum plate having numerous depressions formed therein as a formula to fill the numerous depressions with columnar structure material and harden it to form a substrate having numerous columnar structures 8 (substrate formation step S2), a step of applying a gold coating to the numerous columnar structures 8 (gold coating step S3), and a step of applying a fibronectin coating to the numerous gold-coated columnar structures 8 (fibronectin coating step S4).

[0045] It should be noted that it is not necessarily necessary to carry out the gold coating step S3 and the fibronectin coating step S4. If the results of the above-mentioned preliminary test indicate that coating the substrate is not necessary, then it is sufficient to carry out only the formwork preparation step S1 and the substrate formation step S2.

[0046] (Formwork preparation process S1) FIG. 4 is a schematic cross-sectional view showing a large number of depressions in an aluminum plate prepared in the formwork preparation step S1. In the formwork preparation process S1, an aluminum plate having a large number of depressions 9 formed therein that will serve as the formwork for a large number of columnar structures 8 within the above-mentioned range of diameter D, height H, and arrangement spacing P1 is obtained or such an aluminum plate is manufactured. The width W of the opening 9a of each dent 9 is 10 nm or more and less than 50 nm, the depth F of each dent 9 is 20 nm or more, and the formation interval P2 of the multiple dents 9 is within the range represented by the following formula (2). W <P2<2W...(2)

[0047] When manufacturing an aluminum plate having a large number of depressions 9 formed therein, it can be manufactured, for example, as follows.

[0048] First, an aluminum plate before the formation of the depressions is immersed in an acidic aqueous solution, and anodization is performed by applying a voltage to form an alumina film (oxide film). The oxide film formed has columnar cells extending in the depth direction from the surface of the aluminum plate, and has a structure in which a large number of such cells are accumulated in a direction parallel to the surface of the aluminum plate. Furthermore, it is known that the oxide film is a porous film in which each of the multiple cells has pores extending in the depth direction (the axial direction of the columnar cells). The acidic aqueous solution is preferably a sulfuric acid aqueous solution, but is not limited thereto, and other acidic aqueous solutions such as oxalic acid may be used.

[0049] Next, the portion of the aluminum plate that has become an alumina film by anodization is dissolved and removed by wet etching. At this time, the etchant penetrates into the pores of the oxide film and dissolves the oxide film from inside the pores, resulting in an oxide film with enlarged pore diameters. An aqueous solution (etchant) suitable for wet etching is a mixed solution of chromic acid and phosphoric acid, but is not limited thereto.

[0050] Furthermore, by subjecting the aluminum plate after wet etching to a second anodization and then wet etching, an oxide film with even larger pore diameters can be obtained.

[0051] Thereafter, the above-mentioned process of forming an alumina film by anodization and the process of dissolving and removing the alumina film by etching (pore diameter enlargement process) are repeated several times to make each pore deeper. A suitable aqueous solution for the second and subsequent anodization processes is a sulfuric acid aqueous solution, but is not limited to this. A suitable aqueous solution for the second and subsequent dissolving and removing processes is a phosphoric acid aqueous solution, but is not limited to this.

[0052] The pores in the oxide film thus formed correspond to the "numerous depressions" in the present invention. Also, an aluminum plate having an oxide film with pores formed on its surface corresponds to the "aluminum plate having a large number of depressions formed on its surface" in the present invention.

[0053] The depth F of each recess 9 (in other words, the height of the columnar structure 8) can be controlled by the time of anodization.

[0054] The diameter W of each recess 9 (in other words, the diameter of the columnar structure 8) can be controlled by the concentration of the aqueous solution used for dissolving and removing and the time for dissolving and removing.

[0055] The arrangement interval between the depressions 9 (in other words, the arrangement interval between the columnar structures 8) is proportional to the voltage during the initial anodization, and the arrangement interval can be increased by 2.5 nm for every 1 V increase.

[0056] In this way, by changing the conditions of anodization or wet etching (dissolution and removal), the depth F, diameter W, and formation spacing P2 of the recess 9 in the formwork can be adjusted. Therefore, a person skilled in the art can set the conditions appropriately and manufacture a container having columnar structures on the bottom surface of any diameter, height, and spacing. As described above, the formwork preparation step S1 includes a step of forming a large number of depressions 9 by repeating the formation of an alumina film by anodizing an aluminum plate and the dissolution and removal of the alumina film by etching (pore diameter enlarging process).

[0057] (Base material formation process S2) In the substrate forming step S2 according to the present embodiment, a liquid photocurable resin before curing is added to the surface of a flat plate member or the bottom surface of a container having a flat plate portion and side walls, and the aluminum plate obtained or manufactured in the mold preparation step S1 is used as a mold, and the added photocurable resin is filled into many depressions 9 in the aluminum plate, and ultraviolet light or the like is irradiated to form a substrate having many columnar structures 8. As a material for the flat plate member, a resin such as PET (polyethylene terephthalate) is preferable, but is not limited to this. As a material for a container without columnar structures, for example, a resin such as PET or acrylic resin can be mentioned, but is not limited to these.

[0058] In the nanoimprint method, which uses an aluminum plate having numerous depressions 9 and photocurable resin, the aluminum plate is pressed against the photocurable resin as a mold to produce columnar structures 8. This allows the mold to be reused repeatedly, achieving a high yield.

[0059] Conventional containers having microstructures have been manufactured by a combination of photolithography or electron beam lithography and dry or wet etching. This manufacturing method has the problem that the photolithography or electron beam lithography process requires time and costs. In addition, in the case of the exposure method through a photomask, there is a limit to the area that can be uniformly irradiated with little unevenness in illuminance. In addition, in the case of the direct writing method that does not use a photomask, beam scanning is required at the resolution of the fine structure to be formed, but there is a technical limit to achieving both accuracy and working distance in driving the beam scanning unit and stage moving device. For these reasons, it was difficult to increase the area of ​​the container using the conventional manufacturing method that combines photolithography or electron beam lithography with dry etching or wet etching.

[0060] In contrast, according to the manufacturing method of this embodiment, a porous alumina film is formed by anodization, followed by wet etching, and used as a mold to manufacture a cell culture vessel 1 having fine columnar structures 8 by photo-nanoimprinting. Therefore, the time and cost required for manufacturing the vessel can be effectively reduced, and the vessel having the columnar structures 8 can be made large in area.

[0061] (Gold coating process S3) In the gold coating step S3, a gold coating is applied to the numerous columnar structures 8 formed in the base material forming step S2. The method of gold coating is not particularly limited, but examples thereof include sputtering and coating using an ion coater.

[0062] (Fibronectin coating step S4) In the fibronectin coating step S4, a fibronectin coating is further applied to the numerous gold-coated columnar structures 8. The method of coating with fibronectin is not particularly limited, but examples thereof include adding fibronectin to the columnar structures 8, and immersing the gold-coated substrate in a fibronectin-containing solution and then leaving it to dry. In the case where a photocurable resin is added to the surface of a flat plate member and a form is pressed against the plate member to form the columnar structure 8 in the base material forming step S2, the flat plate member is fixed to the flat bottom surface of a container without a columnar structure before or after the gold coating step S3 or after the fibronectin coating step S4. The container without a columnar structure needs to have a flat plate portion and a side wall.

[0063] Although the preferred embodiment has been described in detail above, the present invention is not limited to the above embodiment, and various modifications are possible within the scope of the invention described in the claims.

[0064] For example, in the embodiment of the manufacturing method shown in Figure 3, a step of coating a large number of columnar structures 8 with fibronectin is provided after the gold coating step S3, but collagen may be coated onto a large number of columnar structures 8 instead of fibronectin.

[0065] 3, in the base material forming step S2, the depressions in the aluminum plate are filled with a photocurable resin as the material for the columnar structures, but the material for the columnar structures in the base material forming step S2 is not limited to a photocurable resin. For example, a large number of columnar structures may be formed by filling the depressions in the aluminum plate with a thermosetting resin and then applying heat.

[0066] It is also possible to fill the depressions in the aluminum plate with methyl methacrylate monomer containing benzoic peroxide. In this case, after filling, the monomer is polymerized and hardened, and then the aluminum plate as a formwork is immersed in an aqueous sodium hydroxide solution to dissolve the aluminum plate, allowing the columnar structure to be removed from the aluminum plate.

[0067] The above-mentioned peroxybenzoic acid is preferably contained in an amount of, for example, 5% by mass relative to the methyl methacrylate monomer, but the concentration is not particularly limited. The polymerization conditions for the methyl methacrylate monomer include, for example, polymerization at 50° C. for 12 hours. The concentration of the above-mentioned aqueous sodium hydroxide solution is, for example, 10% by mass.

[0068] Alternatively, a coating of spin-on-glass material (SOG film) may be formed on a glass substrate, and the resulting SOG film may be pressed against the depressions in an aluminum plate to fill the depressions with the SOG material. In this case, the SOG material is dried after filling, then hardened by heat treatment and released from the aluminum plate. The SOG material is then heat-treated again to completely remove the organic components and polymerize the silicon material, turning it into glass (silica glass).

[0069] The pressure to which the aluminum plate is pressed is not particularly limited, but for example, 1 kg / cm 2 Examples of the drying time for the SOG material include, but are not limited to, 12 hours. Examples of the heat treatment of the SOG material before demolding include heat treatment at 200°C for 1 hour. Examples of the heat treatment of the SOG material after demolding include heat treatment at 450°C for 2 hours. Demolding can be facilitated by coating the aluminum plate with fluoroalkylsilane or the like before filling with the SOG material.

[0070] Furthermore, in the embodiment shown in Figure 3, a columnar structure 8 was formed by adding uncured liquid photocurable resin to the surface of a flat plate member or the bottom surface of a container having a flat plate portion and sidewalls, but uncured photocurable resin may also be added to the surface of a thin plate having a curved surface or the bottom surface of a container having a curved plate portion and sidewalls. In this case, in the form preparation step S1, an aluminum plate having a curved surface with the same curvature as that of the thin or flat plate portion is anodized to form an alumina film, and the alumina film is dissolved and removed by etching (pore size enlargement process), so that a large number of depressions can be formed on the curved surface of the aluminum plate. Therefore, it becomes possible to form columnar structures on the thin or flat plate portion having a curved surface. EXAMPLES

[0071] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0072] Table 1 shows the configurations of the cell culture vessels of Example 1 and Comparative Examples 1-5.

[0073] [Table 1]

[0074] The cell culture vessels of Example 1 and Comparative Examples 1 and 2 were prepared by subjecting the surface of an acrylic curable resin as a base material having a columnar structure to gold sputtering and then coating with fibronectin. The dimensions of the columnar structures shown in Table 1 are all dimensions after gold sputtering and fibronectin coating were applied to the acrylic curable resin as a base material, and were measured using the scale bar in the image obtained by a scanning electron microscope set at 70,000 and 100,000 magnifications as shown in Figures 5 and 6.

[0075] The columnar structures of Example 1 have a diameter D of 45 nm, a height H of 45 nm, and an arrangement interval P1 of 50 nm.

[0076] The columnar structures of Comparative Example 1 have a diameter D of 100 nm, a height H of 150 nm, and an arrangement interval P1 of 200 nm.

[0077] The columnar structures of Comparative Example 2 have a diameter D of 200 nm, a height H of 390 nm, and an arrangement interval P1 of 400 nm.

[0078] The cell culture vessels of Comparative Examples 3 and 4 were made by subjecting the surface of a flat acrylic curable resin on which no columnar structures were formed to gold sputtering or nickel sputtering, and then coating the surface with fibronectin.

[0079] The cell culture vessel of Comparative Example 5 was a commercially available polystyrene culture dish (4-well plate) for tissue culture, which served as a positive control that was expected to have a certain degree of effect of promoting differentiation into osteoblasts.

[0080] The cell culture vessels of Example 1 and Comparative Examples 1 to 5 all have a flat bottom surface extending substantially horizontally, and a side wall extending upward from the edge of the bottom surface.

[0081] <Manufacturing of cell culture vessels> (Cell Culture Vessel of Example 1) An aluminum plate with a purity of 99.99% was anodized for one hour using an 8M (mol / L) aqueous sulfuric acid solution as the electrolyte at a bath temperature of 17°C and a chemical voltage of 18V to form an alumina film.

[0082] Next, the alumina film was dissolved and removed in a mixed solution of 1.8 mass % chromic acid and 6 mass % phosphoric acid at a bath temperature of 70° C. for 2 hours.

[0083] Thereafter, the alumina was anodized for 30 seconds in a 0.3 M sulfuric acid bath at 18 V, and then immersed in 5% by mass phosphoric acid at 30°C for 5 minutes to dissolve and remove the alumina (pore size enlargement treatment). The formation of an alumina film by anodization in the sulfuric acid bath and the dissolution and removal of the alumina film were repeated a total of five times to obtain porous alumina with tapered pores (depressions).

[0084] The obtained porous alumina mold was immersed in a 0.1% OPTOOL solution to perform a treatment for facilitating demolding.

[0085] Next, acrylic photocurable resin PAK-02 (manufactured by Toyo Gosei Co., Ltd.) was added to the surface of a flat PET sheet (an example of the above-mentioned flat plate member), a porous alumina mold was pressed against it, and photoimprinting was performed by irradiating with ultraviolet light to produce a thin plate-shaped substrate having tapered columnar structures.

[0086] Then, the surface of the substrate including the columnar structure was gold sputtered using an Ar ion beam sputtering method under the conditions of 7 kV and 5 mA, and the thickness of the gold sputtered coating was 10 nm.

[0087] Next, fibronectin was further coated on the gold sputtered layer. In detail, the gold-sputtered substrate was first immersed in 70% ethanol as a sterilization treatment, washed with ultrapure water (MilliQ) for 5 minutes three times, and then dried at 37°C for 1 hour. Next, 0.5 mg / mL fibronectin (WTQ1780, 163-05591, Fujifilm Wako Pure Chemical Industries, Ltd.) was diluted 1 / 25 with phosphate buffered saline (PBS) (-) to obtain 0.02 mg / mL fibronectin, which was then added and allowed to stand for 1 hour. The thickness of the fibronectin coated on the substrate in this manner was 5 nm or more but less than 20 nm, and the total thickness, including the gold coating, was less than 30 nm. After that, after washing twice with PBS(-), the thin plate was immersed in Poweredby10 (Glycotechnica) to allow the columnar structures to become familiar with the medium, and then left to soak for 30 minutes at 37°C in a 5% CO2 environment. The obtained thin plate with columnar structures was attached to the bottom of a commercially available culture dish to create a simple cell culture vessel.

[0088] An enlarged planar image of the columnar structure of the cell culture vessel of Example 1 produced as described above is shown in Fig. 5, and an enlarged side image is shown in Fig. 6. Note that Figs. 5 and 6 are images taken using a scanning electron microscope.

[0089] (Cell Culture Vessel of Comparative Example 1) An aluminum plate with a purity of 99.99% was anodized for 17 hours using a 0.3 M aqueous oxalic acid solution as the electrolyte at a bath temperature of 17°C and a chemical voltage of 40 V to form an alumina film.

[0090] Next, the alumina film was dissolved and removed in a mixed solution of 1.8 mass % chromic acid and 6 mass % phosphoric acid at a bath temperature of 70° C. for 2 hours.

[0091] Thereafter, under the same conditions as the first anodization, an alumina film was formed by anodizing for 30 seconds, and then the alumina film was dissolved and removed (pore size enlargement treatment) by immersing the sample in 5 mass% phosphoric acid at 30°C for 11 minutes. The formation of the alumina film by anodizing for 30 seconds in an oxalic acid aqueous solution bath and the dissolution and removal of the alumina film were repeated a total of five times to obtain porous alumina with tapered pores (depressions).

[0092] The obtained porous alumina mold was immersed in a 0.1% OPTOOL solution to perform a treatment for facilitating demolding.

[0093] Next, the above-mentioned photocurable resin PAK-02 was added to the surface of the PET sheet, a porous alumina mold was pressed against it, and photo-imprinting was performed by irradiating with ultraviolet light to produce a thin plate-shaped substrate with tapered columnar structures.

[0094] Thereafter, the substrate was subjected to gold sputtering in the same manner as in Example 1. The thickness of the gold sputtered coating was 10 nm. Next, the plate was coated with fibronectin in the same manner as in Example 1, immersed in Poweredby10, and allowed to stand for 30 minutes in an environment of 37°C and 5% CO2. The obtained thin plate with columnar structures was attached to the bottom of a commercially available culture dish to create a simple cell culture vessel.

[0095] (Cell Culture Vessel of Comparative Example 2) An aluminum plate with a purity of 99.99% was anodized for one hour using a 0.1M aqueous phosphoric acid solution as the electrolyte at a bath temperature of 17°C and a chemical voltage of 160V to form an alumina film.

[0096] Next, the alumina film was dissolved and removed in a mixed solution of 1.8 mass % chromic acid and 6 mass % phosphoric acid at a bath temperature of 70° C. for 2 hours.

[0097] Thereafter, under the same conditions as the first anodization, an alumina film was formed by anodizing for 300 seconds, and then the alumina film was dissolved and removed (pore size expansion treatment) by immersing the sample in 10% by mass phosphoric acid at 30°C for 10 minutes. The formation of the alumina film by anodizing for 300 seconds in the phosphoric acid aqueous solution and the dissolution and removal of the alumina film (pore size expansion treatment) were repeated a total of five times to obtain porous alumina with tapered pores (depressions).

[0098] The obtained porous alumina mold was immersed in a 0.1% OPTOOL solution to perform a treatment for facilitating demolding.

[0099] Next, the above-mentioned photocurable resin PAK-02 was added to the surface of the PET sheet, a porous alumina mold was pressed against it, and photo-imprinting was performed by irradiating with ultraviolet light to produce a thin plate-shaped substrate with tapered columnar structures.

[0100] Thereafter, the substrate was subjected to gold sputtering in the same manner as in Example 1. The thickness of the gold sputtered coating was 10 nm. Next, the plate was coated with fibronectin in the same manner as in Example 1, immersed in Poweredby10, and allowed to stand for 30 minutes in an environment of 37°C and 5% CO2. The obtained thin plate with columnar structures was attached to the bottom of a commercially available culture dish to create a simple cell culture vessel.

[0101] (Cell culture vessels of Comparative Examples 3 and 4) A thin plate made of acrylic curable resin was subjected to gold sputtering or nickel sputtering by Ar ion beam sputtering. The sputtering thickness was 10 nm. Thereafter, the substrate was coated with fibronectin in the same manner as in Example 1, and immersed in Poweredby10 for 30 minutes. The obtained thin plate was attached to the bottom of a commercially available culture dish to create a simple cell culture vessel.

[0102] Of the cell culture vessels of Example 1 and Comparative Examples 1 to 4 manufactured as described above, plan views of the cell culture vessels of Example 1 and Comparative Examples 1 to 3 are shown in Fig. 7. In Fig. 7, in order to easily distinguish the thin plate from a commercially available culture dish to which the thin plate is attached, the alkaline phosphatase of the cells on the thin plate is stained, as described in detail later.

[0103] <Culture using various cell culture vessels> Human bone marrow-derived mesenchymal stem cell line UE6E7-16 (passage number 33, RIKEN BioResource Research Center) was seeded and cultured on the bottom (thin plate) of the cell culture vessels of Example 1 and Comparative Examples 1 to 5. Culturing was performed twice for each cell culture vessel (n=2).

[0104] The medium (culture solution) used was MEMα medium (#12561-056, Life Technologies) supplemented with 10% fetal bovine serum (FBS, Lot No. 27419001, Corning), 100 IU / mL penicillin, and 0.2 mg / mL streptomycin (Penicillin-Streptomycin Solution, 200920, 168-24191, Wako). The mesenchymal stem cells were cultured in this medium at 37°C under 5% CO2 for 14 days. MEMα medium is known as a neutral medium that has no advantageous or disadvantageous effect on differentiation into osteoblasts.

[0105] <Staining> The cells on days 0, 7, and 14 of culture using the cell culture vessels of each of Example 1 and Comparative Examples 1 to 5 were stained for osteoblast differentiation markers alkaline phosphatase and osteocalcin, and for cell nuclei. The degree of differentiation into osteoblasts was confirmed based on the degree of staining (expression level) of alkaline phosphatase and osteocalcin.

[0106] (Alkaline phosphatase staining) The cells were fixed by immersion in 4% paraformaldehyde (PFA, 163-20145, Wako) for 15 minutes.

[0107] Thereafter, the plate was washed three times for 5 minutes with PBS(-), 07269-84 (Nacalai Tesque), and three times for 5 minutes with ultrapure water (MilliQ).

[0108] Next, a color-developing substrate for alkaline phosphatase (BCIP-NBT solution (Ready To Use), 19880-84, manufactured by Nacalai Tesques) was added and left to stand for 2 hours at 25° C. After that, the plate was washed twice with ultrapure water (MilliQ) for 5 minutes each to complete the staining of alkaline phosphatase.

[0109] Next, 5 mg / mL DAPI staining solution (DAPI [4',6-diamidino-2-phenylindole, dihydrochloridel], D1306, Invitrogen) was diluted 5000-fold and added, and the mixture was allowed to stand for 10 minutes to stain the cell nuclei.

[0110] (Osteocalcin immunofluorescence staining) First, the cells were immersed in 4% paraformaldehyde for 15 minutes to fix the cells, and then washed three times with PBS(-) for 5 minutes each.

[0111] Next, the cells were immersed in 0.1% Triton-X (T8787, Sigma-Aldrich) for 5 minutes to make holes in the cell membrane, and then washed three times with PBS(-) for 5 minutes each.

[0112] The sections were then immersed in 0.2% gelatin (Nacalai Tesque) for 1 hour for blocking, and then a 0.5 mg / mL osteocalcin (OCN) primary antibody solution (Human / Rat Osteocalcin Antibody, MAB1419, R&D Systems) was diluted 300-fold and allowed to stand at room temperature for 1 hour.

[0113] This was followed by three 5-min washes with 0.2% gelatin.

[0114] Then, a 2 mg / mL secondary antibody solution (Alexa Fluor 488 goat anti-mouse IgG (H+L), A-11029, Invitrogen) was diluted 1000-fold and allowed to stand at room temperature for 1 hour.

[0115] Finally, the sections were washed three times with 0.2% gelatin for 5 minutes each, and then a 5 mg / mL DAPI staining solution was diluted 5000-fold to stain the cell nuclei, followed by leaving the sections to stand for 10 minutes to stain the cell nuclei.

[0116] <Culture results> 8 to 14 described below show stained images observed with an upright microscope using a 4x lens (NA: 0.13) or a 10x lens (NA: 0.3).

[0117] FIG. 8 is a stained image using a chromogenic substrate showing the expression of alkaline phosphatase in the cells cultured in the cell culture vessels of Example 1 and Comparative Example 5, and FIG. 9 is a fluorescent immunostained image showing the expression of osteocalcin in the cells cultured in the cell culture vessels of Example 1 and Comparative Example 5.

[0118] In FIG. 8, the dark areas are stained alkaline phosphatase, and in FIG. 9, the light areas are stained osteocalcin. As shown in FIG. 8, more cells expressing alkaline phosphatase were observed in the cell culture vessel of Example 1 than in the cell culture vessel of Comparative Example 5, which was the positive control.

[0119] Furthermore, as shown in FIG. 9, in the cell culture vessel of Example 1, more cells expressing osteocalcin were observed than in the cell culture vessel of Comparative Example 5.

[0120] Furthermore, in the cell culture vessel of Example 1, the number of cells expressing both alkaline phosphatase and osteocalcin was clearly greater than in Comparative Example 5 already on day 0, the day the culture was started.

[0121] As described above, from the stained images shown in FIG. 8 and FIG. 9, it was revealed that in the cell culture vessel of Example 1, differentiation into osteoblasts was promoted.

[0122] On the other hand, FIG. 10 is a stained image using a chromogenic substrate showing the expression of alkaline phosphatase in cells cultured in the cell culture vessels of Comparative Example 1 and Comparative Example 5, and FIG. 11 is a fluorescent immunostained image showing the expression of osteocalcin in cells cultured in the cell culture vessels of Comparative Example 1 and Comparative Example 5.

[0123] As shown in FIGS. 10 and 11, the cell culture vessel of Comparative Example 1 had fewer cells expressing alkaline phosphatase and osteocalcin than the cell culture vessel of Comparative Example 5, which was the positive control.

[0124] This revealed that in the cell culture vessel of Comparative Example 1, differentiation into osteoblasts was inhibited or the effect of promoting differentiation was weak.

[0125] FIG. 12 is a stained image using a chromogenic substrate showing the expression of alkaline phosphatase in cells cultured in the cell culture vessels of Comparative Example 2 and Comparative Example 5, and FIG. 13 is a fluorescent immunostained image showing the expression of osteocalcin in cells cultured in the cell culture vessels of Comparative Example 2 and Comparative Example 5.

[0126] 12 and 13, the number of cells expressing alkaline phosphatase and osteocalcin was smaller in the cell culture vessel of Comparative Example 2 than in the cell culture vessel of Comparative Example 5, which is the positive control. In particular, the difference in the number of cells expressing these proteins on day 14 was remarkable.

[0127] This revealed that in the cell culture vessel of Comparative Example 2, differentiation into osteoblasts was suppressed or the effect of promoting differentiation was weak.

[0128] FIG. 14 shows staining images using a chromogenic substrate, which show the expression of alkaline phosphatase in the cells cultured in the cell culture vessels of Comparative Examples 3 to 5.

[0129] In the cell culture vessel of Comparative Example 3, in which the surface of a flat acrylic curable resin was gold sputtered and coated with fibronectin, there were slightly fewer cells expressing alkaline phosphatase than in the cell culture vessel of Comparative Example 5, which was the positive control.

[0130] Furthermore, in the cell culture vessel of Comparative Example 4, in which the surface of a flat acrylic curable resin was nickel sputtered and coated with fibronectin, there were significantly fewer cells expressing alkaline phosphatase than in the cell culture vessel of Comparative Example 5, which was the positive control.

[0131] From the above, it was revealed that the promotion effect was slightly lower in Comparative Example 3, in which gold sputtering was performed, than in Comparative Example 5. It was also revealed that in the cell culture vessel of Comparative Example 4, in which nickel sputtering was performed, differentiation into osteoblasts was inhibited or the differentiation promotion effect was weak.

[0132] <Quantification of culture results> First, for each of the cell culture vessels in Example 1 and Comparative Examples 1 to 3, the number N of cells in which alkaline phosphatase expression was confirmed in the stained image was determined as represented by the following formula (3). ALP+ is the total number of cells being cultured, N totalDivide by P to get the alkaline phosphatase positive percentage. ALP+ The number of cells evaluated exceeded 20 for all cell culture vessels. P ALP+ =N ALP+ / N total ...(3) Then, the alkaline phosphatase positive rate P ALP+ The expression of alkaline phosphatase was quantified by dividing the above by the alkaline phosphatase positive rate in the cell culture vessel of Comparative Example 5.

[0133] For each of the cell culture vessels in Example 1 and Comparative Examples 1 to 3, the average fluorescence brightness within the region in which the cells existed in the stained image of osteocalcin was calculated as shown in the following formula (4). The number of frames of the stained image exceeded 4 for all of the cell culture vessels.

[0134]

number

[0135] Thereafter, the average fluorescence brightness of each of Example 1 and Comparative Examples 1 to 3 was divided by the average fluorescence brightness of the stained image of Comparative Example 5 to quantify the expression of osteocalcin.

[0136] FIG. 15 is a graph quantifying the expression of alkaline phosphatase in cells cultured in each of the cell culture vessels of Example 1 and Comparative Examples 1 to 3, and FIG. 16 is a graph quantifying the expression of osteocalcin in cells cultured in each of the cell culture vessels of Example 1 and Comparative Examples 1 to 3.

[0137] 15 and 16, the expression of alkaline phosphatase or osteocalcin in the cells obtained as a result of the above-mentioned "two times" of culture using each of the cell culture vessels of Example 1 and Comparative Examples 1 to 3 is shown by separate bars. In Figs. 15 and 16, when the quantified vertical axis value is 1, it means that the expression is equivalent to that of Comparative Example 1, which is the positive control, and differentiation into osteoblasts is promoted to the same extent. When the quantified vertical axis value exceeds 1, it is understood that differentiation into osteoblasts is promoted more than in Comparative Example 1.

[0138] As shown in Figures 15 and 16, in the cell culture vessel of Example 1, expression values ​​for both alkaline phosphatase and osteocalcin were confirmed to be significantly greater than 1 on average in two cultures, excluding day 0 of culture. Moreover, it can be seen that in the cell culture vessel of Example 1, the difference in results between the two cultures was small, regardless of the number of days of culture and the difference in markers.

[0139] In contrast, in Comparative Examples 1 to 3, the expression of both alkaline phosphatase and osteocalcin did not exceed 1 on average in two cultures.

[0140] In addition, in Comparative Example 3, although some samples were found to have a high effect of promoting differentiation into osteoblasts, it can be seen that the results were not stable after two cultures.

[0141] From the above, it was revealed that not only from the stained images but also quantitatively, a cell culture vessel having a large number of columnar structures formed on the inner bottom surface of the vessel, the columnar structures having a diameter D of 10 nm or more and less than 50 nm, a height H of 20 nm or more, and an arrangement interval of diameter D or more and less than 2D, has the highest effect of promoting differentiation into osteoblasts and exhibits this effect most stably. [Explanation of symbols]

[0142] 1: Cell culture vessel 2: Flat plate 3: Side wall 4: Internal space 5: Bottom surface 8: Columnar structure D: Diameter of columnar structure H: Height of columnar structure P1: Arrangement interval of columnar structures P2: Formation interval of depressions in aluminum plate

Claims

1. A bottom surface in contact with the culture medium; A number of columnar structures formed on the bottom surface, Each columnar structure has a diameter of 10 nm or more and less than 50 nm, and a height of 20 nm or more; The diameter is D, and the arrangement interval of the columnar structures is P. １ When the above P １ is in the range represented by the following formula (1). D<P １ <2D...(1)

2. The cell culture vessel according to claim 1 , wherein the height of the columnar structure is 1.5D or less.

3. The cell culture vessel according to claim 1 , wherein the numerous columnar structures are coated with gold.

4. 4. The cell culture vessel according to claim 3, further comprising a coating of fibronectin or collagen on said gold coating.

5. The cell culture vessel according to claim 1 , wherein the numerous columnar structures comprise an acrylic curable resin.

6. A step of preparing an aluminum plate having a large number of depressions formed on a surface thereof; A step of filling the numerous cavities in the aluminum plate with a material and hardening the material to form a substrate having numerous columnar structures; applying a gold coating to the plurality of columnar structures; and coating the gold-coated columnar structures with fibronectin or collagen. The width of the opening of the recess is 10 nm or more and less than 50 nm, and the depth is 20 nm or more, the width is W, and the formation interval of the recess is P ２ When the above P ２ is within the range represented by the following formula (2). W<P ２ <2W...(2)

7. 7. The method for manufacturing a cell culture vessel according to claim 6, wherein the step of preparing the aluminum plate includes a step of forming the numerous recesses by repeatedly subjecting the aluminum plate before the formation of the recesses to a process of forming a porous oxide film by anodization and a process of enlarging the pore size of the oxide film by etching.

Images