Method for producing cell culture substrate

Abstract

To provide a method for producing a cell culture substrate that enables mask-free pattern formation and allows cell culture in an adherent state.SOLUTION: A method for producing a cell culture substrate comprises: a coating step of coating at least a part of a surface of a substrate with a composition containing a hydrophilic polymer to form a layer containing the hydrophilic polymer; a laminating step of laminating a protective film on the layer containing the hydrophilic polymer; a recess forming step of irradiating a part of a surface of the protective film with a laser to form a recess by thermal processing; a modification step of performing surface modification on surfaces of the protective film and the recess; and, after the modification step, a step of peeling the protective film from the layer containing the hydrophilic polymer.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for producing a cell culture substrate. [Background technology]

[0002] Pluripotent stem cells, such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), have the ability to differentiate into various tissues (pluripotency), and have attracted considerable attention as a cell source for regenerative medicine and drug discovery screening. To apply pluripotent stem cells to regenerative medicine and drug discovery screening, they must be differentiated into the desired cells. This process requires the formation of pluripotent stem cell aggregates. While pluripotent stem cells can differentiate into a variety of cells, it is known that the optimal size of the cell aggregates varies depending on the type of differentiated cell. Therefore, it is desirable to control the size and create uniformly sized cell aggregates.

[0003] Several cell culture substrates for forming cell aggregates and methods for producing the same have been proposed. Patent Document 1 discloses a method for producing a cell adhesion substrate, which sequentially includes the steps of: (a) applying a copolymer of 2-methacryloyloxyethyl phosphorylcholine (MPC) and butyl methacrylate to a cell culture substrate whose surface has been hydrophilically treated; (b) placing a mask with holes of a predetermined shape on the cell culture substrate coated with the copolymer of MPC and butyl methacrylate; and (c) plasma treating the cell culture substrate with the mask placed on it. Patent Document 2 discloses a culture method in which cells are seeded, cultured, the medium replaced, and recovered in a culture vessel having multiple depressions arranged on the bottom, and it is described that the depressions consist of a bottom and an opening and are treated to inhibit cell adhesion. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 7016150 [Patent Document 2] Patent No. 7219303 Summary of the Invention [Problem to be solved by the invention]

[0005] The method for producing a cell culture substrate disclosed in Patent Document 1 includes the steps of placing a predetermined mask and plasma-treating the cell culture substrate with the mask placed thereon, where the plasma-treated regions (areas of the holes in the mask) are patterned as cell-adhesion regions, and the non-plasma-treated regions (mask regions) are patterned as non-cell-adhesion regions. Thus, with patterning techniques that use a mask, changing the pattern requires changing the mask pattern specifications as well, making them less versatile.

[0006] In a cell suspension culture system using a cell culture substrate in which the inside of the recesses has been treated to make the cells less adhesive, such as the cell culture vessel disclosed in Patent Document 2, there is a high possibility that the cells will be sucked in when the culture medium is changed, making the operation complicated.

[0007] Therefore, an object of the present invention is to provide a method for producing a cell culture substrate that allows pattern formation without the need for a mask and that allows cells to be cultured in an adhesive state. [Means for solving the problem]

[0008] The present invention includes, for example, the following inventions. [1] A method for producing a cell culture substrate, a coating step of coating at least a portion of the surface of the substrate with a composition containing a hydrophilic polymer to form a layer containing the hydrophilic polymer; a lamination step of laminating a protective film on the layer containing the hydrophilic polymer; a recess forming step of irradiating a laser onto a portion of the surface of the protective film to form a recess by thermal processing; a modification step of performing a surface modification treatment on the surfaces of the protective film and the recesses after the recess formation step; The manufacturing method further comprises, after the modifying step, a peeling step of peeling the protective film from the layer containing the hydrophilic polymer. [2] The cell culture substrate is A substrate and a layer containing a hydrophilic polymer that covers at least a portion of the surface of the substrate, It has the following region (A) and the following region (B), (A) Region with cell adhesive and cell proliferation properties (B) a region adjacent to the region (A) and not having cell adhesiveness or cell proliferation properties The manufacturing method according to [1], wherein the region (A) comprises a recess formed in the cell culture substrate. [3] The manufacturing method according to [1] or [2], wherein the laser is a CO2 laser. [4] The manufacturing method according to any one of [1] to [3], wherein the surface modification treatment includes a plasma treatment. [Effects of the Invention]

[0009] According to the present invention, it is possible to provide a method for producing a cell culture substrate that allows pattern formation without the need for a mask and that allows cells to be cultured in an adherent state. [Brief explanation of the drawings]

[0010] [Figure 1] 1 is a flowchart of a method for producing a cell culture substrate according to one embodiment. [Figure 2] FIG. 1 is a schematic diagram (cross-sectional view) of a cell culture substrate according to one embodiment. [Figure 3] FIG. 2 is a schematic view (perspective view) of the cell culture substrate after the laminating step in the manufacturing method according to one embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0012] As used herein, the term "cell aggregate" refers to a three-dimensional aggregate of cells formed by the aggregation of multiple cells. The shape of the three-dimensional aggregate may be an ellipsoid such as a sphere, or may be a hemisphere or other shape. These shapes may have gaps formed by folding a sheet of cells, or may be hollow. An example of a cell aggregate is a spheroid.

[0013] In this specification, "temperature responsive" means that the degree of hydrophilicity / hydrophobicity changes with temperature. Furthermore, the boundary temperature at which the degree of hydrophilicity / hydrophobicity changes is referred to as the "responsive temperature."

[0014] As used herein, the term "biologically derived substance" refers to a substance present in the body of a living organism, or a chemically synthesized substance equivalent to such a substance. Substances present in the body of a living organism may be natural products, or may be artificially synthesized using recombinant DNA technology or the like. There are no particular limitations on biologically derived substances, and examples include nucleic acids, proteins, and polysaccharides, which are the basic materials that make up living organisms, and their constituent elements, such as nucleotides, nucleosides, amino acids, and various sugars, as well as lipids, vitamins, and hormones.

[0015] As used herein, "cell adhesiveness" refers to the ease with which cells adhere to a substrate or cell culture substrate at culture temperatures, "having cell adhesiveness" refers to the ability of cells to adhere to a substrate or cell culture substrate at culture temperatures directly or via a biologically derived substance, and "not having cell adhesiveness" refers to the inability of cells to adhere to a substrate or cell culture substrate at culture temperatures.

[0016] As used herein, "cell proliferation" refers to the ease with which cells proliferate at the culture temperature, "having cell proliferation" refers to the ability of cells to proliferate at the culture temperature, and "not having cell proliferation" refers to the inability of cells to proliferate at the culture temperature. "High cell proliferation" refers to the proliferation of more cells when compared over the same culture period.

[0017] The method for producing a cell culture substrate according to this embodiment (hereinafter also referred to simply as "the production method according to this embodiment") includes the following steps: a coating step in which at least a portion of the surface of the substrate is coated with a composition containing a hydrophilic polymer to form a layer containing the hydrophilic polymer; a lamination step in which a protective film is laminated on the layer containing the hydrophilic polymer; a recess formation step in which a laser is irradiated onto a portion of the surface of the protective film to form recesses by thermal processing; a modification step in which a surface modification treatment is performed on the surfaces of the protective film and the recesses; and a peeling step in which, after the modification step, the protective film is peeled off from the layer containing the hydrophilic polymer. A flowchart of one embodiment of the production method is shown in Figure 1.

[0018] FIG. 2 is a schematic diagram (cross-sectional view) of a cell culture substrate obtained by a cell culture substrate manufacturing method according to one embodiment. The cell culture substrate 10 shown in FIG. 2 includes a substrate 1 and a layer 2 containing a hydrophilic polymer that coats the surface of the substrate 1. In FIG. 2, A and B respectively indicate a recess (which may be the (A) region described below) and the surface of the layer 2 containing a hydrophilic polymer (which may be the (B) region described below). Also, in FIG. 2, H indicates the height of the irregularities at the interface between the recess A and the surface of the layer 2 containing a hydrophilic polymer. The thickness of the layer 2 containing a hydrophilic polymer refers to the distance from the surface where the substrate 1 and the layer 2 containing a hydrophilic polymer meet to the surface of the layer 2 containing a hydrophilic polymer (the region indicated by B, which may be the (B) region described below). In addition, although the cell culture substrate 10 shown in FIG. 2 has the layer 2 containing a hydrophilic polymer on the surface of the recess, this is not limiting. For example, at least a portion or all of the surface of the (A) region may be the substrate 1.

[0019] In this specification, a clear distinction is made between the "substrate" and the "cell culture substrate." Referring to Figure 1, the substrate 1 is a member that comes into contact with the hydrophilic polymer layer 2, and the cell culture substrate 10 is the entire article for forming cell aggregates.

[0020] The coating step is a step of coating at least a portion of the surface of a substrate with a composition containing a hydrophilic polymer to form a layer containing the hydrophilic polymer. By forming a layer containing the hydrophilic polymer, it is possible to eliminate cell adhesiveness and cell proliferation. The method for forming the layer containing the hydrophilic polymer is not particularly limited, and examples include a method of forming the layer by applying a composition containing the hydrophilic polymer to at least a portion of the surface of the substrate. Examples of methods for applying the composition containing the hydrophilic polymer include various commonly known methods such as painting, brush coating, dip coating, spin coating, bar coating, flow coating, spray coating, roll coating, air knife coating, blade coating, gravure coating, microgravure coating, and slot die coating.

[0021] Fig. 1(A) is a cross-sectional view of a substrate having a layer containing a hydrophilic polymer formed on its surface after the coating process. In Fig. 1(A), a layer 2 containing a hydrophilic polymer is formed on the entire surface of the substrate 1.

[0022] The substrate is not particularly limited, but is preferably formed from at least one selected from the group consisting of polystyrene, polyethylene, polyethylene terephthalate, polycarbonate, cycloolefin polymer, cellulose acetate, nitrocellulose, and polyvinylidene fluoride, more preferably formed from at least one selected from the group consisting of polystyrene, polyethylene terephthalate, polycarbonate, and cycloolefin polymer, even more preferably formed from at least one selected from polystyrene, polyethylene terephthalate, and polycarbonate, and most preferably formed from polystyrene or polycarbonate.

[0023] The thickness of the substrate is preferably 0.01 mm to 0.5 mm, more preferably 0.05 mm to 0.4 mm, even more preferably 0.1 mm to 0.3 mm, and particularly preferably 0.15 mm to 0.2 mm. When the thickness of the substrate is within this range, the phase contrast image of the cells becomes clearer.

[0024] The shape of the substrate is not particularly limited, and may be a planar shape such as a plate or a film, or may be in the shape of a fiber, porous particle, porous membrane, hollow fiber, etc. The shape of the substrate may also be the shape of a container generally used for cell culture, etc. (cell culture dishes such as Petri dishes, flasks, plates, bags, etc.). From the perspective of ease of culture operations, the shape of the substrate is preferably a planar shape such as a plate or a film, or the shape of a flat porous membrane.

[0025] The thickness of the hydrophilic polymer-containing layer may be, for example, 10 nm to 500 nm. Having a thickness of 10 nm to 500 nm allows cells to adhere and proliferate only in the recesses, and also facilitates cell migration to gather in the recesses, thereby increasing the cell viability of cell aggregates. Here, the "thickness" of the hydrophilic polymer-containing layer refers to the out-of-plane length from the interface between the substrate and the hydrophilic polymer-containing layer to the interface of the hydrophilic polymer-containing layer on the opposite side of the substrate (excluding the recesses). For thicknesses exceeding 10 nm, the distance can be calculated by measuring cross-sectional images using a transmission electron microscope using ultrathin sections of the cell culture substrate prepared with a microtome, measuring the distances at 10 randomly selected points, and averaging the distances. Furthermore, for thicknesses of 10 nm or less, the distance can be measured using an ellipsometer. Because this is advantageous for inhibiting cell adhesion to areas other than the recesses, a thickness of 10 nm or more is more preferable, and a thickness of 50 nm or more is even more preferable. Furthermore, since this is suitable for increasing the cell survival rate of the cell aggregate by collecting cells in region (A) through cell migration, it is more preferable that the layer thickness is 200 nm or less.

[0026] The hydrophilic polymer contains a phosphorylcholine group or a hydroxyl group. By including a phosphorylcholine group or a hydroxyl group in the hydrophilic polymer, it is possible to make the area coated with the hydrophilic polymer into an area where cells do not adhere. In addition, since such a hydrophilic polymer does not need to be completely decomposed and removed, the area can be made into an area with cell adhesive properties and cell proliferation properties by the modification process, and cells are less likely to be contaminated with decomposition products of the hydrophilic polymer. Other than containing a phosphorylcholine group or a hydroxyl group, the type of hydrophilic polymer is not particularly limited. Commercially available products include, for example, Lipidure® CM5206 (manufactured by NOF Corporation), Lipidure® CM2001 (manufactured by NOF Corporation), and BIOSURFINE®-AWP (manufactured by Toyo Gosei Co., Ltd.). In addition, commercially available substrates coated with a hydrophilic polymer include PrimeSurface(R) (manufactured by Sumitomo Bakelite Co., Ltd.), EZ-BindShut(R) (manufactured by AGC Technoglass Co., Ltd.), and EZ-BindShutII(R) (manufactured by AGC Technoglass Co., Ltd.).

[0027] The hydrophilic polymer preferably contains a compound represented by the following general formula (1), a compound represented by the following general formula (2), or a compound represented by the following general formula (3).

[0028] [ka] [In general formula (1), R 1 and R 2 each independently represents a hydrogen atom or a methyl group, and R 3 represents a hydrogen atom or an arbitrary organic group, and m and n each independently represent a positive integer.

[0029] [ka] [In general formula (2), R 4 , R 5 and R 6 each independently represents a hydrogen atom or a methyl group, and R 7represents a hydrogen atom or an arbitrary organic group, and x, y, and z each independently represent a positive integer.

[0030] [ka] [In general formula (3), R 8 R and 9 each independently represents a hydrogen atom or a methyl group, and R 10 represents a hydrogen atom or an arbitrary organic group, and a and b each independently represent a positive integer.

[0031] When the hydrophilic polymer contains a compound represented by the general formula (1), a compound represented by the general formula (2), or a compound represented by the general formula (3), cells can be easily attached and proliferated, and it is suitable for forming cell aggregates of uniform shape in the wells. 3 , R 7 , and R 10 are suitable for immobilizing hydrophilic polymers on substrates, and therefore are preferably hydrophobic groups or functional groups reactive to active energy rays (e.g., UV, electron beams, etc.). As the hydrophobic group, linear or cyclic alkyl groups such as methyl, ethyl, propyl, butyl, and cyclohexyl groups can be suitably used. Furthermore, as functional groups reactive to active energy rays (e.g., UV, electron beams, etc.), azide groups, acrylate groups, methacrylate groups, vinyl groups, and epoxy groups can be used, with azide groups being suitably used.

[0032] When the hydrophilic polymer is reactive to active energy rays, the coating step may include irradiating the layer containing the hydrophilic polymer with active energy rays to fix the layer containing the hydrophilic polymer to the surface of the substrate. Examples of the hydrophilic polymer reactive to active energy rays include the above-mentioned hydrophilic polymers having functional groups reactive to active energy rays (e.g., UV, electron beams, etc.). Examples of active energy rays include UV, electron beams, etc.

[0033] Irradiating a hydrophilic polymer that is reactive to active energy rays with active energy rays causes a chemical reaction between the hydrophilic polymers or between the hydrophilic polymer and the substrate, resulting in immobilization of a layer containing the hydrophilic polymer on the surface of the substrate. By immobilizing the layer containing the hydrophilic polymer on the surface of the substrate, a layer containing the hydrophilic polymer can be formed without deforming the layer containing the hydrophilic polymer when a composition containing the temperature-responsive polymer is applied in the temperature-responsive layer-forming step described below. Furthermore, by immobilizing the layer containing the hydrophilic polymer on the surface of the substrate, the shape of the recesses formed in the recess-forming step described below can be maintained.

[0034] The lamination process is a process of laminating a protective film on a layer containing a hydrophilic polymer. By laminating the protective film, it is possible to perform a surface modification treatment only on the surface of the recesses formed in a later process. In Figure 1(B), a layer 2 containing a hydrophilic polymer is formed on the entire surface of the substrate 1, and a protective film 3 is further laminated on the layer 2 containing a hydrophilic polymer.

[0035] The protective film is not particularly limited as long as it can be melted and removed by thermal processing using a laser, but it is preferable that it be easily peeled off from the layer containing a hydrophilic polymer after the modification process described below. For example, it may be one that adheres to the layer containing a hydrophilic polymer by electrostatic action, or one that does not leave any adhesive residue on the layer containing a hydrophilic polymer. The material of the protective film may be any material that melts with the heat of laser irradiation, such as polyethylene terephthalate (PET), polypropylene (PP), polyimide (PI), acrylic, etc. The thickness of the protective film may be, for example, 50 μm or more and 100 μm or less.

[0036] The recess formation step is a step in which a laser is irradiated onto a portion of the surface of the protective film to form a recess by thermal processing. The bottom of the recess may be in a layer containing a hydrophilic polymer, or part or all of the bottom may be in the substrate. Figure 1(C) is a schematic diagram of the recess formation step. In Figure 1(C), a laser 4 is irradiated onto a portion of the surface of the protective film 3 to form a recess A by thermal processing. Because the recess formation step is performed by thermal processing, the processed surface becomes flat, which is thought to improve cell observation when the produced cell culture agent is used.

[0037] The laser irradiation conditions (e.g., laser wavelength, output, pulse width, irradiation time, etc.) may be any conditions that allow thermal processing, and can be appropriately set by a person skilled in the art. In this specification, thermal processing refers to processing in which the substrate is absorbed by the laser, causing a temperature rise and melting the substrate to remove it, and is different from ablation processing, which is substantially not affected by thermal diffusion.

[0038] The laser to be irradiated may be a laser with a wavelength in the ultraviolet to infrared wavelength range, or may be a laser with a wavelength in the green to infrared wavelength range, and is preferably a laser with a wavelength in the infrared wavelength range, since this enables efficient thermal processing. Specifically, the wavelength of the laser to be irradiated may be, for example, 100 nm or more and 11,000 nm or less. Since this enables efficient thermal processing, the wavelength of the laser to be irradiated may be 1,000 nm or more and 11,000 nm or less, 2,500 nm or more and 11,000 nm or less, 5,000 nm or more and 11,000 nm or less, 7,000 nm or more and 11,000 nm or less, 8,000 nm or more and 11,000 nm or less, or 9,200 nm or more and 11,000 nm or less.

[0039] The output of the laser to be irradiated may be, for example, 1.0 W or more and 120 W or less.

[0040] The power density of the irradiated laser is, for example, 4000 W / cm 2 More than 470000W / cm 2 In this specification, the "laser power density" is measured at the laser irradiation point (the surface of the cell culture substrate irradiated with the laser).

[0041] The irradiated laser may be a pulsed laser or a continuous wave laser. When the irradiated laser is a pulsed laser, the pulse width may be, for example, 1 nanosecond or more, or 1 microsecond or more.

[0042] The laser to be irradiated may be, for example, a CO2 laser, a YAG laser, a fiber laser, a green laser, or the like.

[0043] The maximum depth of the recesses to be formed refers to the out-of-plane distance between the bottom of the recesses and the surface of the layer containing the hydrophilic polymer. The maximum depth of the recesses may be, for example, 1 nm to 2000 nm, or 1 nm to 500 nm. A maximum depth of the recesses of 500 nm or less can further reduce the number of dead cells trapped by the unevenness, thereby further reducing the number of dead cells mixed into the cell aggregates. This can further increase the cell viability of the cell aggregates. Furthermore, a maximum depth of the recesses of 500 nm or less further reduces the adhesion of air bubbles to the uneven portions. Suppressing the adhesion of air bubbles eliminates the need for degassing or repeated dispensing and aspirating of the medium using a pipetter to remove air bubbles, thereby further improving operability. A maximum depth of the recesses of 1 nm or more allows live cells that spontaneously migrate (migrate) on the cell culture substrate to more easily gather in the recesses, thereby further increasing the cell viability of the cell aggregates. The maximum depth of the recess is more preferably 400 nm or less, and even more preferably 350 nm or less, as this is suitable for further increasing the cell survival rate of the cell aggregates that are formed.

[0044] The recesses are recognized as island-like regions when the cell culture substrate is observed from the side of the layer containing the hydrophilic polymer in a direction perpendicular to the substrate. In this case, the periphery of the island-like region corresponds to the boundary between the recesses and areas other than the recesses. In this specification, the planar region defined by the boundary between the recesses and areas other than the recesses (i.e., the periphery of the island-like region) is sometimes referred to as an opening. Note that an island-like region indicates that the region exists independently from areas other than the recesses. When region (A) is an island-like region as described above, living cells are concentrated in region (A) compared to when the region is not an island-like region as described above (for example, in the case of a striped structure, etc.), and cell aggregates can be produced more efficiently.

[0045] The shape of the opening (the planar area defined by the boundary between the recess and the area other than the recess) is not particularly limited and can be set appropriately depending on the shape of the desired cell aggregate, but examples include an ellipse (including a circle), a polygon, or a closed shape formed by straight lines and curves. Furthermore, since this is suitable for producing cell aggregates with a shape close to a sphere, the shape of the opening is preferably an ellipse (including a circle) or a polygon, more preferably an ellipse (including a circle) or a rectangle, even more preferably an ellipse (including a circle) or a square, and most preferably an ellipse (including a circle).

[0046] Since this method is suitable for producing cell aggregates with a shape close to a sphere, the aspect ratio of the shape of the opening (the planar area defined by the boundary between the recess and the area other than the recess) is preferably 1 to 2, more preferably 1 to 1.5, even more preferably 1 to 1.1, and most preferably 1 to 1.05. Here, "aspect ratio" refers to the ratio of the maximum diameter (major diameter) to the minimum diameter (minor diameter) of the shape, i.e., major diameter / minor diameter.

[0047] Since this is suitable for producing cell aggregates of uniform size and shape, it is preferable that the standard deviation / average aspect ratio of the aspect ratio of the shape of the opening (the planar area defined by the boundary between the recess and the area other than the recess) is 80% or less, more preferably 50% or less, even more preferably 20% or less, and most preferably 5% or less.

[0048] The area of ​​the opening (the flat area defined by the boundary between the recess and the area other than the recess) is, for example, 0.001 mm 2 More than 5mm 2 The area may be less than 0.005 mm 2 More than 1mm 2 The area of ​​0.01 mm or less is preferable. 2 More than 0.5mm 2 Less than 0.015mm is more preferable. 2 More than 0.25mm 2 More preferably, the area is 0.02 mm or less. 2 More than 0.2mm 2 The following are most preferred:

[0049] Since this is suitable for producing cell aggregates of uniform size and shape, it is preferable that the standard deviation / average area of ​​the opening (the planar area defined by the boundary between the recess and the area other than the recess) is 80% or less, more preferably 50% or less, even more preferably 20% or less, and most preferably 5% or less.

[0050] The shape of the bottom of the well may be the same as or different from the shape of the opening. The shape of the bottom of the well is not particularly limited and can be set appropriately depending on the shape of the desired cell aggregate, and examples thereof include an ellipse (including a circle), a polygon, or a closed shape formed by straight lines and curves. Furthermore, since this is suitable for producing cell aggregates with a shape close to a sphere, the shape of the opening is preferably an ellipse (including a circle) or a polygon, more preferably an ellipse (including a circle) or a rectangle, even more preferably an ellipse (including a circle) or a square, and most preferably an ellipse (including a circle).

[0051] The aspect ratio of the shape of the bottom of the recess may be the same as or different from the aspect ratio of the shape of the opening. Since this is suitable for producing cell aggregates with a shape close to a sphere, the aspect ratio of the shape of the bottom of region (A) is preferably 1 to 2, more preferably 1 to 1.5, even more preferably 1 to 1.1, and most preferably 1 to 1.05.

[0052] The area of ​​the bottom surface of the recess may be the same as or different from the area of ​​the opening. For example, the area of ​​the bottom surface of the recess is 0.001 mm 2 More than 6mm 2 The area may be 0.001 mm or less. 2 More than 5mm 2 Less than 0.005mm is preferable 2 More than 1mm 2 Less than 0.01mm is more preferable. 2 More than 0.5mm 2 More preferably, the area is 0.015 mm or less. 2 More than 0.25mm 2 Even more preferably, the area is 0.02 mm or less.2 More than 0.2mm 2 The following are most preferred:

[0053] Since this is suitable for producing cell aggregates of uniform size and shape, it is preferable that the standard deviation / average area of ​​the bottom surface of the well is 80% or less, more preferably 50% or less, even more preferably 20% or less, and most preferably 5% or less.

[0054] Furthermore, since this is suitable for increasing the oxygen concentration around the cells and increasing the survival rate of cell aggregates, the minimum distance between recesses is preferably 400 μm or more and 10,000 μm or less, more preferably 500 μm or more and 8,000 μm or less, even more preferably 1,000 μm or more and 5,000 μm or less, and most preferably 2,000 μm or more and 4,000 μm or less.

[0055] The area ratio of the recesses to the areas other than the recesses is not particularly limited, but since this is suitable for increasing the number of cell aggregates that can be produced per unit area of ​​the cell culture substrate, the area of ​​the recesses is preferably 10% or more, more preferably 30% or more, even more preferably 50% or more, and most preferably 70% or more of the total area of ​​the recesses and the areas other than the recesses. Furthermore, since this is suitable for providing a sufficient distance between multiple recesses and preventing the cell aggregates in multiple recesses from fusing together to form a non-uniform shape, the area other than the recesses is preferably 20% or more, more preferably 40% or more, even more preferably 60% or more, and most preferably 80% or more of the total area of ​​the recesses and the areas other than the recesses.

[0056] The modification step is a step of performing a surface modification treatment on the surfaces of the protective film and the recesses. FIG. 1(D) is a schematic diagram of the modification step. In FIG. 1(D), the surfaces of the protective film 3 and the recesses A are subjected to a surface modification treatment 5, and the surfaces of the protective film 3 and the recesses A are modified into surface-modified regions 6. The surface modification treatment may be, for example, at least one treatment selected from the group consisting of plasma treatment, corona discharge treatment, and UV treatment. Plasma treatment is preferably included because it can be performed in a short time and can improve the mass productivity of the cell culture substrate. Surface modification treatments such as plasma treatment, corona treatment, and UV treatment can be performed under conditions commonly used to treat the surface of a cell culture substrate to impart cell adhesiveness and cell proliferation properties. Region (A) is a region modified by such treatment, etc., and therefore, region (A) has cell adhesiveness and cell proliferation properties.

[0057] When the modification step includes plasma treatment, the plasma irradiation time is preferably 5 seconds to 10 minutes, more preferably 10 seconds to 5 minutes, and most preferably 30 seconds to 3 minutes. The gas introduced during the plasma treatment is preferably air, nitrogen, or oxygen, and more preferably oxygen.

[0058] The peeling process is a process in which the protective film is peeled off from the layer containing the hydrophilic polymer after the modification process. Figure 1(E) is a cross-sectional view of the cell culture substrate after the peeling process. Since the modification process was performed with the protective film still laminated, only the surface of the recesses can be made into a surface-modified region. The region indicated by B is an area that was not surface-modified because it was covered with the protective film during the modification process.

[0059] In addition to the coating step, lamination step, recess formation step, modification step, and peeling step, the manufacturing method according to this embodiment may further include one or more steps selected from the group consisting of a bonding step, a temperature-responsive layer formation step, a biological substance-containing layer formation step, and a sterilization step, as necessary.

[0060] The lamination process is carried out after the modification process, with the cross-sectional area in the in-plane direction being 0.05 cm2 More than 100cm 2 This is a process of bonding a plate having the following through-holes to a substrate on the side of the substrate coated with a layer containing a hydrophilic polymer. 2 More than 100cm 2 By laminating a plate having the following through-holes to a substrate, it is possible to produce a plate having a space for containing a culture medium with high mass productivity. FIG. 3 is a schematic diagram (perspective view) of the cell culture substrate after the lamination step. The cell culture substrate 11 shown in FIG. 3 is, for example, a cell culture substrate 10 shown in FIG. 1(E) or FIG. 2, etc., with a partition plate 20 (with a cross-sectional area in the in-plane direction of 0.05 cm2). 2 More than 100cm 2 The cross-sectional area in the in-plane direction is 0.05 cm 2 More than 100cm 2 The plate having the following through-holes may be, for example, a partition member having a plurality of cylindrical partition walls that can partition the surface on the side where cells are cultured. 2 More than 100cm 2 The side walls of the following through-holes function as cylindrical partition walls.

[0061] The temperature-responsive layer-forming step is a step in which the surface of the hydrophilic polymer-containing layer after the modification step is coated with a composition containing a temperature-responsive polymer to form a layer containing a temperature-responsive polymer. Forming the layer containing the temperature-responsive polymer to a thickness of 100 nm or less allows the molecular chains of the temperature-responsive polymer to be sparsely coated on the surface of the formed recesses, which is suitable for imparting temperature responsiveness while maintaining the functionality of the recesses (cell adhesiveness and cell proliferation). Furthermore, making the layer containing the temperature-responsive polymer to a thickness of 1 nm or more is suitable for imparting sufficient temperature responsiveness and producing a cell culture substrate capable of rapid formation of cell aggregates.

[0062] The wells may be temperature-responsive, as they are suitable for detaching cultured cell aggregates. When the wells are temperature-responsive, the cells can be cultured on the cell culture substrate at a temperature close to body temperature, so the response temperature is preferably 50°C or lower, more preferably 35°C or lower. Furthermore, the response temperature is particularly preferably 25°C or lower, as this is suitable for preventing cells from detaching when performing operations such as changing the culture medium during culture. Furthermore, the response temperature is preferably 4°C or higher, more preferably 10°C or higher, and even more preferably 15°C or higher, as this allows cell aggregates to be formed by cooling at a temperature that does not damage the cells.

[0063] When the recesses are temperature-responsive, for example, a layer containing a temperature-responsive polymer with a layer thickness of 1 nm to 100 nm may be further provided on the surface (including the surface of the recesses and the surface other than the recesses) of the layer containing a hydrophilic polymer. By providing a layer containing a temperature-responsive polymer with a layer thickness of 1 nm to 100 nm, temperature responsiveness can be imparted to the recesses without impairing the properties of the recesses and the surface other than the recesses formed on the surface of the layer containing a hydrophilic polymer. To be suitable for imparting temperature responsiveness to the recesses without impairing the properties of the recesses and the surface other than the recesses, the layer thickness of the layer containing a temperature-responsive polymer is more preferably 3 nm to 50 nm, even more preferably 5 nm to 40 nm, and most preferably 10 nm to 35 nm. The layer thickness of the temperature-responsive polymer suitable for impairing cell proliferation and enabling cells to be detached and recovered by temperature responsiveness after culture varies depending on the cells to be cultured and can be appropriately adjusted within the range of layer thickness exemplified above.

[0064] The temperature-responsive polymer is preferably a block copolymer having a water-insoluble block segment and a temperature-responsive block segment. When the temperature-responsive polymer is such a block copolymer, mass productivity of the cell culture substrate is improved and contamination of the produced cell aggregates with the temperature-responsive polymer can be suppressed. The ratio of the constituent units of the temperature-responsive block segment contained in the temperature-responsive polymer is preferably 70 wt% or more, more preferably 80 wt% or more, particularly preferably 90 wt% or more, and most preferably 92 wt% or more, because this is suitable for rapid detachment of cell aggregates from the cell culture substrate.

[0065] Furthermore, since the temperature-responsive polymer is a block copolymer having a water-insoluble block segment and a temperature-responsive block segment, temperature responsiveness can be imparted to the surface of the cell culture substrate by the simple method of dropping a solution containing the temperature-responsive polymer onto the surface of the cell culture substrate and drying it. Furthermore, since the layer formed in this manner has the preferred thickness of the temperature-responsive polymer, even if the entire surface of the layer containing the hydrophilic polymer is coated with the temperature-responsive polymer, the properties of the recesses and areas other than the recesses are less impaired.

[0066] Examples of the monomer unit constituting the temperature-responsive block segment include (meth)acrylamide compounds such as acrylamide and methacrylamide; N-alkyl-substituted (meth)acrylamide derivatives such as N,N-diethylacrylamide, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, Nt-butylacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, and N-tetrahydrofurfurylmethacrylamide; N,N-dialkyl-substituted (meth)acrylamide derivatives such as N,N-dimethyl(meth)acrylamide, N,N-ethylmethylacrylamide, and N,N-diethylacrylamide; 1-(1-oxo-2-propenyl)-pyrrolidine, 1-(1-oxo-2-propenyl)-pyrrolidine, and the like. Examples of the cyclic acrylamide derivatives include (meth)acrylamide derivatives having a cyclic group such as 1-(1-oxo-2-methyl-2-propenyl)-piperidine, 4-(1-oxo-2-methyl-2-propenyl)-morpholine, 1-(1-oxo-2-methyl-2-propenyl)-pyrrolidine, 1-(1-oxo-2-methyl-2-propenyl)-piperidine, and 4-(1-oxo-2-methyl-2-propenyl)-morpholine; vinyl ethers such as methyl vinyl ether; and proline derivatives such as N-proline methyl ester acrylamide. Since they are suitable for setting the response temperature to 0 to 50°C, N,N-diethylacrylamide, Nn-propylacrylamide, N-isopropylacrylamide, Nn-propylmethacrylamide, N-ethoxyethylacrylamide, N-tetrahydrofurfurylacrylamide, and N-tetrahydrofurfurylmethacrylamide are preferred, Nn-propylacrylamide and N-isopropylacrylamide are more preferred, and N-isopropylacrylamide is particularly preferred. Furthermore, when a room temperature medium is used during medium replacement in a culture operation, Nn-propylacrylamide and N-proline methyl ester acrylamide are preferred because they are suitable for setting the response temperature of the block copolymer to a temperature lower than room temperature.

[0067] Examples of monomer units constituting the water-insoluble block segment include n-butyl acrylate, n-butyl methacrylate, isobutyl acrylate, isobutyl methacrylate, t-butyl acrylate, t-butyl methacrylate, n-hexyl acrylate, n-hexyl methacrylate, n-octyl acrylate, n-octyl methacrylate, n-decyl acrylate, n-decyl methacrylate, n-dodecyl acrylate, n-dodecyl methacrylate, n-tetradecyl acrylate, and n-tetradecyl methacrylate. Furthermore, those having a reactive group are preferred because they are suitable for firmly immobilizing the block copolymer on a substrate, and examples thereof include 4-azidophenyl acrylate, 4-azidophenyl methacrylate, 2-((4-azidobenzoyl)oxy)ethyl acrylate, and 2-((4-azidobenzoyl)oxy)ethyl methacrylate. Furthermore, structures having an aromatic ring are preferred because they are suitable for enhancing cell proliferation, and examples thereof include 2-hydroxyphenyl acrylate, 2-hydroxyphenyl methacrylate, 3-hydroxyphenyl acrylate, 3-hydroxyphenyl methacrylate, 4-hydroxyphenyl acrylate, 4-hydroxyphenyl methacrylate, N-(2-hydroxyphenyl)acrylamide, N-(2-hydroxyphenyl)methacrylamide, N-(3-hydroxyphenyl)acrylamide, N-(3-hydroxyphenyl)methacrylamide, N-(4-hydroxyphenyl)acrylamide, N-(4-hydroxyphenyl)methacrylamide, and styrene.

[0068] The water-insoluble block segment may also contain a repeating unit that controls the response temperature of the block copolymer. Examples of the repeating unit that controls the response temperature of the block copolymer include hydrophilic or hydrophobic components, and are not particularly limited to those having an amino group, such as 2-dimethylaminoethyl acrylate, 2-dimethylaminoethyl methacrylate, 2-diethylaminoethyl acrylate, 2-diethylaminoethyl methacrylate, and N-[3-(dimethylamino)propyl]acrylamide; those having a betaine, such as N-(3-sulfopropyl)-N-methacryloyloxyethyl-N,N-dimethylammonium betaine and N-methacryloyloxyethyl-N,N-dimethylammonium-α-N-methylcarboxybetaine; hydroxyethyl acrylate, hydroxyethyl methacrylate, N-(2-hydroxyethyl)acrylamide, polyethylene glycol monoacrylate, polyethylene glycol monomethacrylate, polypropylene glycol monoacrylate, polypropylene glycol monomethacrylate, methoxypolyethylene glycol monoacrylate, methoxypolyethylene glycol monoacrylate, methoxypolyethylene glycol monoacrylate, methoxypolyethylene glycol monoacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate, N-(2-hydroxyethyl)acrylamide, polyethylene glycol monoacrylate, polyethylene glycol monomethacrylate, polypropylene glycol monoacrylate, polypropylene glycol monometh ... those having a polyethylene glycol group or a methoxyethyl group, such as polyethylene glycol monomethacrylate, diethylene glycol monomethyl ether acrylate, diethylene glycol monomethyl ether methacrylate, diethylene glycol monoethyl ether acrylate, diethylene glycol monoethyl ether methacrylate, 2-methoxyethyl acrylate, 2-methoxyethyl methacrylate, 2-ethoxyethyl acrylate, 2-ethoxyethyl methacrylate, 3-butoxyethyl acrylate, 3-butoxyethyl methacrylate, 3-butoxyethyl acrylamide, furfuryl acrylate, furfuryl methacrylate, tetrahydrofurfuryl acrylate, and tetrahydrofurfuryl methacrylate; those having an acrylate group, such as methoxymethyl acrylate, methoxymethyl methacrylate, 2-ethoxymethyl acrylate, 2-ethoxymethyl methacrylate, 3-butoxymethyl acrylate, 3-butoxymethyl methacrylate, and 3-butoxymethyl acrylamide;Examples of phosphorylcholine groups include 2-methacryloyloxyethyl phosphorylcholine, 2-acryloyloxyethyl phosphorylcholine, 3-(meth)acryloyloxypropyl phosphorylcholine, 4-(meth)acryloyloxybutyl phosphorylcholine, 6-(meth)acryloyloxyhexyl phosphorylcholine, 10-(meth)acryloyloxydecyl phosphorylcholine, ω-(meth)acryloyl(poly)oxyethylene phosphorylcholine, 2-acrylamidoethyl phosphorylcholine, 3-acrylamidopropyl phosphorylcholine, 4-acrylamidobutyl phosphorylcholine, 6-acrylamidohexyl phosphorylcholine, 10-acrylamidodecyl phosphorylcholine, and ω-(meth)acrylamido(poly)oxyethylene phosphorylcholine;

[0069] As a method for coating with a composition containing a temperature-responsive polymer, the same method as the above-mentioned method for applying a composition containing a hydrophilic polymer can be suitably used.

[0070] As a method for coating with a composition containing a temperature-responsive polymer, it is also preferable to coat the entire surface of the cell culture substrate with a temperature-responsive substance. When coating with a composition containing a temperature-responsive polymer, coating using a commonly used coating method without patterning can improve the mass productivity of the cell culture substrate. Furthermore, by coating the entire surface of the cell culture substrate with a composition containing a temperature-responsive polymer, temperature responsiveness is imparted to the recesses, and the temperature-responsive polymer is also coated in areas other than the recesses. By coating the temperature-responsive polymer in areas other than the recesses, cell adhesion in areas other than the recesses can be reduced.

[0071] The biological substance-containing layer forming step is a step of coating the surface of the cell culture substrate after the modification step with a composition containing a biological substance to form a layer containing the biological substance. The layer containing the biological substance may be present on the entire surface of the cell culture substrate after the modification step, or may be present only on the surface of the recesses. The biological substance is not particularly limited, but examples include Matrigel, laminin, fibronectin, vitronectin, collagen, etc.

[0072] These biological substances may be natural products, or may be artificially synthesized using genetic engineering techniques, or may be fragments obtained by cleavage with restriction enzymes, or synthetic proteins or peptides obtained by chemically synthesizing substances equivalent to these biological substances.

[0073] As Matrigel, commercially available products such as Matrigel (manufactured by Corning Incorporated) and Geltrex (manufactured by Thermo Fisher Scientific) can be suitably used due to their ease of availability.

[0074] The type of laminin is not particularly limited, but examples that can be used include laminin 511, laminin 521, and laminin 511-E8 fragment, which have been reported to exhibit high activity against α6β1 integrin expressed on the surface of human iPS cells. Laminin may be a natural product, artificially synthesized using genetic engineering techniques, or a synthetic protein or peptide obtained by chemically synthesizing a substance equivalent to laminin. Commercially available products such as iMatrix-511 (manufactured by Nippi Corporation) are suitable for use due to their ease of availability.

[0075] Vitronectin may be a natural product, artificially synthesized using genetic recombination technology, or a synthetic protein or peptide obtained by chemically synthesizing a substance equivalent to vitronectin. Commercially available products such as human plasma-derived vitronectin (manufactured by Wako Pure Chemical Industries, Ltd.), Synthemax (manufactured by Corning Incorporated), and Vitronectin (VTN-N) (manufactured by Thermo Fisher Scientific) are suitable for use due to their availability.

[0076] Fibronectin may be a natural product, artificially synthesized by genetic recombination technology, or a synthetic protein or peptide obtained by chemically synthesizing a substance equivalent to fibronectin. In view of ease of availability, commercially available products such as fibronectin solution, human plasma-derived fibronectin (manufactured by Wako Pure Chemical Industries, Ltd.), and Retronectin (manufactured by Takara Bio Inc.) can be preferably used.

[0077] The type of collagen is not particularly limited, and examples thereof include type I collagen and type IV collagen. Collagen may be a natural product, artificially synthesized using genetic engineering techniques, or a synthetic peptide obtained by chemically synthesizing a substance equivalent to collagen. Commercially available products such as collagen I, human (manufactured by Corning Incorporated) and collagen IV, human (manufactured by Corning Incorporated) are suitable for use due to their ease of availability.

[0078] From the viewpoint of suppressing denaturation of the biological substance and enhancing cell proliferation, the biological substance is preferably immobilized on the cell culture substrate by a non-covalent bond. Here, "non-covalent bond" refers to a bonding force other than a covalent bond derived from intermolecular forces, such as electrostatic interaction, water-insoluble interaction, hydrogen bond, π-π interaction, dipole-dipole interaction, London dispersion force, or other van der Waals interaction. The biological substance may be immobilized on the block copolymer by a single bonding force or a combination of multiple bonding forces.

[0079] The method for immobilizing biological substances is not particularly limited, but suitable methods include, for example, applying a solution of the biological substance to a cell culture substrate for a predetermined period of time to immobilize the substance, and adding the biological substance to the culture medium when culturing cells to allow the biological substance to be adsorbed onto the cell culture substrate and immobilized.

[0080] The sterilization step is a step of sterilizing the cell culture substrate. There are no particular limitations on the sterilization method, but high-pressure steam sterilization, UV sterilization, gamma-ray sterilization, ethylene oxide gas sterilization, etc. can be used. From the viewpoint of suppressing denaturation of the block copolymer, high-pressure steam sterilization, UV sterilization, and ethylene oxide gas sterilization are preferred. From the viewpoint of suppressing deformation of the substrate, UV sterilization or ethylene oxide gas sterilization are more preferred. From the viewpoint of excellent mass productivity, ethylene oxide gas sterilization is preferred.

[0081] The cells to be cultured using the cell culture substrate obtained by the production method of this embodiment are not particularly limited, as long as they can adhere to the surface before the temperature drop stimulus. Examples include various established cell lines such as Chinese hamster ovary-derived CHO cells, mouse connective tissue L929 cells, human embryonic kidney-derived HEK293 cells, and human cervical cancer-derived HeLa cells, as well as epithelial cells and endothelial cells that constitute various tissues and organs in the body, contractile skeletal muscle cells, smooth muscle cells, and cardiac muscle cells, neuronal cells, glial cells, and fibroblasts that constitute the nervous system, hepatic parenchymal cells, non-hepatic parenchymal cells, and adipocytes that are involved in the metabolism of the body, as well as differentiation-competent stem cells present in various tissues such as mesenchymal stem cells, bone marrow cells, and Muse cells, as well as pluripotent stem cells (pluripotent stem cells) such as ES cells and iPS cells, and cells induced to differentiate therefrom.

[0082] The cell culture substrate obtained by the manufacturing method according to the present embodiment comprises a substrate and a layer containing a hydrophilic polymer that covers at least a portion of the surface of the substrate, and may have the following region (A) and region (B): (A) Region with cell adhesive and cell proliferation properties (B) A region adjacent to region (A) that does not have cell adhesive or cell proliferation properties. Here, the (A) region consists of a recess formed in the cell culture substrate.

[0083] Region (A) is the recessed portion described above, and becomes a region having cell adhesive properties and cell proliferation properties by carrying out a modification process under conditions commonly used when treating the surface of a cell culture substrate to have cell adhesive properties and cell proliferation properties.

[0084] Region (B) is adjacent to region (A), has not been surface-modified with a protective film, and is a layer whose surface contains a hydrophilic polymer, and therefore does not have cell adhesiveness or cell proliferation properties. If region (B) is adjacent to region (A) and does not have cell proliferation properties, when cells are cultured, it is possible to form cell aggregates only in region (A), and to create a state in which no cells are present around part or all of region (A). Furthermore, since this is suitable for uniforming the size and shape of the cell aggregates produced, it is preferable that region (B) not only has no cell proliferation properties but also no cell adhesiveness.

[0085] The shape of region (B) is not limited other than being adjacent to region (A), but since this is suitable for producing cell aggregates of uniform size and shape, it is preferable that region (B) be adjacent to region (A) for at least 20% of its length, more preferably at least 50%, even more preferably at least 80%, and it is most preferable that region (A) is completely surrounded by region (B). [Explanation of symbols]

[0086] A...recess (area (A)), B...surface of the layer containing a hydrophilic polymer (area (B)), H...height of the unevenness at the boundary between the recess and the surface of the layer containing a hydrophilic polymer, 1...substrate, 2...layer containing a hydrophilic polymer, 3...protective film, 4...laser, 5...surface modification treatment, 6...surface-modified area, 10, 11...cell culture substrate, 20...partition plate.

Claims

1. A method for producing a cell culture substrate, a coating step of coating at least a portion of the surface of a substrate with a composition containing a hydrophilic polymer to form a layer containing the hydrophilic polymer; a lamination step of laminating a protective film on the layer containing the hydrophilic polymer; a recess forming step of irradiating a laser onto a portion of the surface of the protective film to form a recess by thermal processing; a modification step of performing a surface modification treatment on the surfaces of the protective film and the recesses after the recess formation step; The manufacturing method further comprises, after the modifying step, a peeling step of peeling the protective film from the layer containing the hydrophilic polymer.

2. The cell culture substrate is A substrate and a layer containing a hydrophilic polymer that covers at least a portion of the surface of the substrate, It has the following region (A) and the following region (B), (A) Region having cell adhesive properties and cell proliferation properties (B) a region adjacent to the region (A) and not having cell adhesiveness or cell proliferation properties The method according to claim 1 , wherein the region (A) comprises a recess formed in the cell culture substrate.

3. The laser is CO ２ The manufacturing method according to claim 1 or 2, wherein the method is a laser.

4. The manufacturing method according to claim 1 or 2, wherein the surface modification treatment includes a plasma treatment.

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