Method for producing cell culture substrate

Abstract

To provide a simple method for producing a cell culture substrate that allows cell culture in an adherent state.SOLUTION: There is provided a method for producing a cell culture substrate, the cell culture substrate having the following regions (A) and (B): (A) a region having cell adhesive and cell proliferative properties, and (B) a region adjacent to the region (A) and having no cell adhesive or cell proliferative properties, wherein the region (A) consists of recesses formed in the cell culture substrate, the method comprising a patterning step of forming the region (A) by irradiating a part of a surface of the substrate with a laser, but not including a surface modification step for the recesses.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, whereby the plasma-treated regions (areas of the holes in the mask) are patterned as cell-adherent regions, and the non-plasma-treated regions (masked regions) are patterned as non-cell-adherent regions. Thus, in a method for producing a cell culture substrate using a patterning technique that uses a mask, changing the pattern requires also changing the pattern specifications of the mask, which is cumbersome.

[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 simple method for producing a cell culture substrate that allows cells to be cultured in an adherent 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, comprising: The cell culture substrate 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 region (A) is a recess formed in the cell culture substrate, a patterning step of irradiating a part of the surface of the substrate with a laser to form the region (A), A method for producing a cell culture substrate, which does not include a surface modification treatment step for the recesses. [2] The manufacturing method according to [1], wherein the patterning step is a step of forming the region (A) by ablation processing. [3] The manufacturing method according to [1] or [2], wherein the laser is a femtosecond laser. [4] The manufacturing method according to any one of [1] to [3], wherein the peak power of the laser is 1.250 MW or more. [5] The manufacturing method according to any one of [1] to [4], wherein the peak wavelength of the laser is within the range of 300 nm or more and 700 nm or less. [Effects of the Invention]

[0009] According to the present invention, a simple method for producing a cell culture substrate that allows cells to be cultured in an adherent state can be provided. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram (cross-sectional view) of a cell culture substrate according to one embodiment. [Figure 2] 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. [Figure 3] 1 shows an image of the cell culture substrate of Example 1 observed with a phase contrast microscope. [Figure 4] 1 shows an image of the cell culture substrate of Comparative Example 1 observed with a phase contrast microscope. [Figure 5] 1 shows a fluorescence microscope image and a phase-contrast microscope image of cells cultured on the cell culture substrate of Example 1. [Figure 6] 1 shows a fluorescence microscope image and a phase-contrast microscope image of cells cultured on the cell culture substrate of Comparative Example 1. 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 cell culture substrate obtained by the method for producing a cell culture substrate according to this embodiment (hereinafter also simply referred to as the "production method according to this embodiment") has the following region (A) and 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 Here, the region (A) is composed of a recess formed in the cell culture substrate.

[0018] 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 from them.

[0019] The method for producing a cell culture substrate according to this embodiment includes a patterning step of irradiating a part of the surface of the substrate with a laser to form the region (A), but does not include a surface modification step for the recesses.

[0020] Figure 1 is a schematic diagram (cross-sectional view) of a cell culture substrate obtained by a method for producing a cell culture substrate according to one embodiment. The cell culture substrate 10 shown in Figure 1 has regions (A) and (B), indicated by A and B in Figure 1, formed on a substrate 1. H indicates the maximum depth of region (A).

[0021] In this specification, a clear distinction is made between a "substrate" and a "cell culture substrate." Explaining with reference to Figure 1, a substrate 1 is a member (material) used to manufacture a cell culture substrate, and a cell culture substrate 10 is the entire article used to form cell aggregates.

[0022] The patterning step is a step of irradiating a portion of the surface of the cell culture substrate with a laser to form region (A). The patterning step can be performed by appropriately setting the laser irradiation conditions (e.g., laser wavelength, output, pulse width, irradiation time, etc.). The patterning step is preferably a step of forming region (A) (recesses) by ablation processing, since this easily gives the surface of the recesses a finely textured shape that allows cells to adhere appropriately.

[0023] The laser to be irradiated may be a laser having a peak wavelength in the ultraviolet to infrared wavelength range, or may be a laser having a peak wavelength in the green to infrared wavelength range. Specifically, the peak wavelength of the irradiated laser may be, for example, in the range of 100 nm to 1000 nm. Since this makes it easier to give the surface of the well a finely uneven shape (surface roughness) that allows cells to appropriately adhere, the peak wavelength of the irradiated laser may be in the range of 150 nm to 950 nm, 200 nm to 900 nm, 250 nm to 850 nm, 300 nm to 800 nm, 300 nm to 750 nm, or 300 nm to 700 nm.

[0024] The output power of the laser to be irradiated may be, for example, 0.1W or more and 10W or less, 0.1W or more and 5W or less, 0.1W or more and 2W or less, 0.1W or more and 1W or less, or 0.1W or more and 0.5W or less.

[0025] The energy of the irradiated laser may be 1.0 μJ / pulse or more and 5.0 μJ / pulse or less. When the peak power is 1.250 MW or more, the device stability during processing is excellent, which contributes to the productivity of the cell culture substrate.

[0026] The power density of the irradiated laser is, for example, 2.546 J / cm 2 More than 12.73J / 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).

[0027] The laser to be irradiated is preferably a pulsed laser, and particularly preferably a femtosecond laser. Here, in this specification, "femtosecond laser" refers to a laser with a pulse width of 1 femtosecond to 1000 femtoseconds. The pulse width is, for example, preferably 1 femtosecond to 1000 femtoseconds, more preferably 100 femtoseconds to 900 femtoseconds, and even more preferably 100 femtoseconds to 500 femtoseconds.

[0028] When forming the (A) region by laser irradiation as described below, the stronger the treatment conditions, such as increasing the laser output and prolonging the treatment time, the more the surface of the recess ((A) region) of the cell culture substrate can be made to have a fine uneven shape (surface roughness) that allows cells to adhere appropriately easily.

[0029] The maximum depth of region (A) refers to the out-of-plane distance between the bottom surface of region (A) and the surface of region (B). The maximum depth of region (A) may be, for example, 1 nm or more and 500 nm or less. A maximum depth of region (A) 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 aggregate. This can further increase the cell viability of the cell aggregate. Furthermore, a maximum depth of region (A) of 500 nm or less further reduces the adhesion of air bubbles to the unevenness. Suppressing the adhesion of air bubbles eliminates the need for degassing or repeated dispensing and aspirating of the medium using a pipette to remove air bubbles, thereby further improving operability. A maximum depth of region (A) of 1 nm or more can more easily attract live cells that spontaneously migrate (migrate) on the cell culture substrate to region (A), thereby further increasing the cell viability of the cell aggregate. The maximum depth of region (A) is more preferably 400 nm or less, and even more preferably 350 nm or less, since this is suitable for further increasing the cell viability of the cell aggregates that are formed.

[0030] The substrate used for producing the cell culture 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.

[0031] 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.

[0032] 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.

[0033] Region (A) consists of a recess formed in the cell culture substrate. Region (A) is recognized as an island-like region when the cell culture substrate is observed from the layer side where region (A) is present in a direction perpendicular to the substrate. In this case, the periphery of the island-like region corresponds to the boundary between region (A) and region (B). In this specification, the planar region defined by the boundary between region (A) and region (B) (i.e., the periphery of the island-like region) is sometimes referred to as an opening.

[0034] The shape of the opening (the planar region defined by the boundary between region (A) and region (B)) 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).

[0035] Since this is suitable for producing cell aggregates with a shape close to a sphere, the aspect ratio of the shape of the opening (the planar region defined by the boundary between region (A) and region (B)) 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.

[0036] 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 area (A) and area (B)) is 80% or less, more preferably 50% or less, even more preferably 20% or less, and most preferably 5% or less.

[0037] The area of ​​the opening (the planar area defined by the boundary between the (A) and (B) areas) 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:

[0038] 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 area (A) and area (B)) is 80% or less, more preferably 50% or less, even more preferably 20% or less, and most preferably 5% or less.

[0039] The shape of the bottom of region (A) may be the same as or different from the shape of the opening. The shape of the bottom of region (A) 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).

[0040] The aspect ratio of the shape of the bottom surface of region (A) may be the same as or different from the aspect ratio of the shape of the opening. Because this is suitable for producing cell aggregates with a shape close to a sphere, the aspect ratio of the shape of the bottom surface 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.

[0041] 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 bottom surface of region (A) is 80% or less, more preferably 50% or less, even more preferably 20% or less, and most preferably 5% or less.

[0042] The area of ​​the bottom surface of the (A) region may be the same as or different from the area of ​​the opening. The area of ​​the bottom surface of the (A) region is, for example, 0.001 mm 2 More than 6mm 2 The area may be 0.001 mm or less. 2 More than 5mm 2 Preferably less than 0.005mm 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:

[0043] 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 region (A) is 80% or less, more preferably 50% or less, even more preferably 20% or less, and most preferably 5% or less.

[0044] Furthermore, since this is suitable for increasing the oxygen concentration around the cells and increasing the survival rate of the cell aggregates, the minimum distance between the (A) regions 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.

[0045] Region (B) is adjacent to region (A) and 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 uniformizing 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.

[0046] 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).

[0047] The area ratio of region (A) to region (B) is not particularly limited, but since this is suitable for increasing the number of cell aggregates that can be produced per unit area of ​​cell culture substrate, the area of ​​region (A) 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 ​​region (A) and region (B). Furthermore, since this is suitable for providing a sufficient distance between multiple (A) regions and preventing the cell aggregates of multiple (A) regions from fusing together to form a non-uniform shape, the area of ​​region (B) 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 ​​region (A) and region (B).

[0048] The manufacturing method according to this embodiment does not include a surface modification treatment step for the recessed portions of the cell culture substrate. In this specification, the term "surface modification treatment" refers to a treatment for chemically modifying the surface. Examples of chemical surface modification treatments include plasma treatment, corona discharge treatment, and UV treatment.

[0049] As described above, in the manufacturing method according to the present embodiment, in the patterning step, a laser is irradiated onto a portion of the surface of the substrate, thereby forming a finely textured surface of the recesses formed in the substrate, to which cells can adequately adhere, and these recesses become regions (A) that have cell adhesive properties and cell proliferation properties. Therefore, the manufacturing method according to the present embodiment does not require a surface modification treatment, nor does it require the preparation of other components (such as a mask or film) for the surface modification treatment. This allows for easy manufacturing of cell culture substrates.

[0050] In addition to the patterning step, the manufacturing method according to this embodiment may further include, as necessary, one or more steps selected from the group consisting of a hydrophilic polymer-containing layer-forming step, a laminating step, a temperature-responsive layer-forming step, a biological substance-containing layer-forming step, and a sterilization step.

[0051] The hydrophilic polymer-containing layer-forming step is a step of forming a layer containing a hydrophilic polymer on the surface of region (B). By forming a layer containing a hydrophilic polymer on the surface of region (B), a region that is less susceptible to cell adhesion and cell proliferation can be formed in the cell culture substrate. The method for forming the hydrophilic polymer-containing layer is not particularly limited, and examples include a method of forming the layer by applying a composition containing a hydrophilic polymer to at least a portion of the surface of region (B). Examples of methods for applying the composition containing a 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. Another example is a method in which a laser is first applied to a portion of the surface of the hydrophilic polymer-containing layer after at least a portion of the surface of the substrate is coated with a composition containing a hydrophilic polymer, and then the patterning step is performed.

[0052] 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 makes it easier for cells to adhere and proliferate only in region (A). Furthermore, cells are more likely to migrate to region (A), 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 region (A)). 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 distance at 10 randomly selected points, and averaging the measured distances. For thicknesses of 10 nm or less, the distance can be measured using an ellipsometer. Because this is suitable for inhibiting cell adhesion to region (B), a thickness of 10 nm or more is more preferable, and 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.

[0053] The hydrophilic polymer contains a phosphorylcholine group or a hydroxyl group. By including a phosphorylcholine group or a hydroxyl group in the hydrophilic polymer, the area coated with the hydrophilic polymer can be made into an area to which cells do not adhere. Other than the inclusion of a phosphorylcholine group or a hydroxyl group, the type of hydrophilic polymer is not particularly limited. Examples of commercially available hydrophilic polymers include Lipidure® CM5206 (manufactured by NOF Corporation), Lipidure® CM2001 (manufactured by NOF Corporation), and BIOSURFINE®-AWP (manufactured by Toyo Gosei Co., Ltd.). Examples of commercially available substrates coated with a hydrophilic polymer include PrimeSurface® (manufactured by Sumitomo Bakelite Co., Ltd.), EZ-BindShut® (manufactured by AGC Technoglass Co., Ltd.), and EZ-BindShutII® (manufactured by AGC Technoglass Co., Ltd.).

[0054] 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).

[0055] [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.

[0056] [ka] [In general formula (2), R 4 , R 5 and R 6 each independently represents a hydrogen atom or a methyl group, and R 7 represents a hydrogen atom or an arbitrary organic group, and x, y, and z each independently represent a positive integer.

[0057] [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.

[0058] 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 (A) region. 3 , R 7 , and R 10are 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.

[0059] When the hydrophilic polymer is reactive to active energy rays, the hydrophilic polymer-containing layer-forming 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.

[0060] 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 deformation of 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 region (A) formed in the patterning step described below can be maintained.

[0061] When the method includes a hydrophilic polymer-containing layer forming step, a step of washing the hydrophilic polymer with a solvent to dissolve and remove the hydrophilic polymer not immobilized on the surface from the surface of the cell culture substrate may be performed. The solvent used preferably contains water and an alcohol, as this is suitable for removing compounds by-produced by the self-reaction of the hydrophilic polymer reactive to active energy rays, such as compounds containing nitrenes derived from azide groups. The alcohol is preferably a lower alcohol, such as methanol, ethanol, 2-propanol, t-butanol, isobutanol, pentanol, or hexanol, with methanol or ethanol being more preferred. The alcohol content is preferably 50 to 95%, more preferably 50 to 90%, particularly preferably 60 to 90%, and most preferably 70 to 90%.

[0062] The lamination process is carried out after the patterning process, with the cross-sectional area in the in-plane direction being 0.05 cm 2 More than 100cm 2 This is a process of bonding a plate with the following through-holes to a substrate on the side of the substrate where the recesses are present. 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. 2 is a schematic diagram (perspective view) of the cell culture substrate after the lamination process. The cell culture substrate 11 shown in FIG. 2 is, for example, a cell culture substrate 10 shown in FIG. 1 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.

[0063] The temperature-responsive layer formation step is a step in which the surface of the cell culture substrate after the patterning step is coated with a composition containing a temperature-responsive polymer to form a layer containing the temperature-responsive polymer. In this case, by forming the layer containing the temperature-responsive polymer to a thickness of 100 nm or less, the surface of the formed region (A) is easily coated with sparse molecular chains of the temperature-responsive polymer, which is suitable for imparting temperature responsiveness while maintaining the functions of region (A) (cell adhesiveness and cell proliferation). Furthermore, by forming the layer containing the temperature-responsive polymer to a thickness of 1 nm or more, sufficient temperature responsiveness can be imparted, making it possible to produce a cell culture substrate capable of rapid formation of cell aggregates, which is also suitable.

[0064] Region (A) may be temperature-responsive, as it is suitable for detaching cultured cell aggregates. When region (A) is temperature-responsive, the response temperature is preferably 50°C or lower, more preferably 35°C or lower, as this allows cells to be cultured at temperatures close to body temperature when culturing cells on the cell culture substrate. 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 operations at temperatures that do not damage the cells.

[0065] When region (A) is temperature-responsive, for example, the surface of the cell culture substrate (the surface including regions (A) and (B)) may further comprise a layer containing a temperature-responsive polymer and having a layer thickness of 1 nm to 100 nm. 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 region (A) without impairing the respective properties of regions (A) and (B) formed on the surface of the cell culture substrate. To be suitable for imparting temperature responsiveness to region (A) without impairing the respective properties of regions (A) and (B), the layer thickness of the layer containing the temperature-responsive polymer is preferably 3 nm to 50 nm, 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 imparting temperature responsiveness to region (A) without impairing cell proliferation and for 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.

[0066] 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.

[0067] 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 described above, even if the entire surface of the cell culture substrate is coated with the temperature-responsive polymer, the properties of the above-mentioned regions (A) and (B) are less impaired.

[0068] 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.

[0069] 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.

[0070] 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;

[0071] 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.

[0072] 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, the mass productivity of the cell culture substrate can be improved by using a commonly used coating method without patterning. Furthermore, by coating the entire surface of the cell culture substrate with a composition containing a temperature-responsive polymer, temperature responsiveness is imparted to region (A), and the temperature-responsive polymer is also coated in region (B). Coating region (B) with a temperature-responsive polymer can reduce the cell adhesiveness of region (B).

[0073] The biological substance-containing layer forming step is a step of coating the surface of the cell culture substrate after the patterning 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 patterning step, or may be present only on the surface of region (A). The biological substance is not particularly limited, but examples include matrigel, laminin, fibronectin, vitronectin, collagen, etc.

[0074] As a method for coating with a composition containing a biological substance, the same method as the above-mentioned method for applying a composition containing a hydrophilic polymer can be suitably used.

[0075] 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.

[0076] 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.

[0077] 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 Matrixome, Inc.) are suitable for use due to their ease of availability.

[0078] 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 that can be used include human plasma-derived vitronectin (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), Synthemax (manufactured by Corning Incorporated), and Vitronectin (VTN-N) (manufactured by Thermo Fisher Scientific) due to their availability.

[0079] Fibronectin may be a natural product, artificially synthesized by genetic engineering or the like, 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 Fujifilm Wako Pure Chemical Industries, Ltd.), and Retronectin (manufactured by Takara Bio Inc.) can be preferably used.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] The sterilization step is a step of sterilizing the cell culture substrate. As the sterilization method, the above-mentioned methods can be suitably used. 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. [Example]

[0084] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0085] [Example 1] A polycarbonate film (trade name: Panlite (registered trademark), manufactured by Teijin Limited, thickness: 0.18 mm) (substrate) was irradiated with a femtosecond laser (manufactured by esi, trade name: LodeStone) to form region (A) in the area irradiated with the laser by ablation processing. Region (B) was also formed in the area not irradiated with the laser. The femtosecond laser irradiation was performed under the conditions of 0.1 W output, 100 kHz, 1.250 MW peak power, and 800 fs pulse width.

[0086] [Comparative Example 1] A metal mask (spot center distance 800 μm) with multiple 0.2 mm diameter circular holes (spots) was placed on the substrate, and plasma treatment was performed on top of the metal mask using a plasma irradiation device (manufactured by Samco Inc., product name: Aqua Plasma (registered trademark) Cleaner AQ-500) to form region (A) in the plasma-treated area and region (B) in the area masked by the metal mask. A cell culture substrate was fabricated in the same manner as in Example 1. The plasma treatment was performed under conditions of 250 W power, 10 Pa pressure, and 120 seconds treatment time.

[0087] <Evaluation of cell culture substrate> (A) Evaluation of the shape and visibility of the area The shape and visibility of region (A) were evaluated by observation using a phase-contrast microscope (manufactured by Olympus Corporation, trade name: IX73). The results are shown in Figures 3 and 4.

[0088] As shown in Figure 4, the shape of region (A) of the cell culture substrate of Comparative Example 1 was good, but many scratches were observed in region (A) due to the use of a mask. On the other hand, as shown in Figure 3, the shape and visibility of region (A) of the cell culture substrate of Example 1 was good.

[0089] (Cell culture evaluation) The cell culture substrate was attached to the bottom of a bottomless 6-well plate, which was then sterilized and used as a culture vessel. Human iPS cell line 201B7 was used, with a density of 15,000 cells / cm. 2Cells were seeded in area (A) so that the cells were 10 μM thick and cultured in AK02N medium (Ajinomoto Co., Inc.) (2 mL / well) at 37°C with 5% CO2. Until 24 hours after seeding, Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd.) (10 μM) and a cell culture protein substrate (Matrixome Co., Ltd., product name: iMatrix-511) (1.25 μg / mL) were added to the medium. The medium was changed 1, 3, and 5 days after the start of culture. After 6 days of culture, cell nuclei were stained with DAPI, and the cell adhesion and proliferation to the cell culture substrate were evaluated using a fluorescence microscope and a phase-contrast microscope. The results are shown in Figures 5 and 6.

[0090] As shown in Figure 6, cell adhesion and proliferation were confirmed on the cell culture substrate of Comparative Example 1. This is believed to be due to the surface of the cell culture substrate being chemically modified by the plasma treatment. Furthermore, as shown in Figure 5, cell adhesion and proliferation were confirmed on the cell culture substrate of Example 1. This is believed to be due to the ablation phenomenon caused by femtosecond laser irradiation, which changed the shape of the surface (bottom of the recess) of the cell culture substrate. Regarding the method for producing the cell culture substrate of Comparative Example 1, it was necessary to create and manage a metal mask, and it was necessary to accurately position the metal mask in a predetermined position on the substrate during patterning, which required a lot of effort. In contrast, in Example 1, patterning was possible by directly irradiating the substrate surface with a laser without using other components such as a mask, and the cell culture substrate could be easily produced. [Explanation of symbols]

[0091] A...area (A), B...area (B), H...maximum depth of area (A), 1...substrate, 10, 11...cell culture substrate, 20...partition plate.

Claims

1. A method for producing a cell culture substrate, comprising: The cell culture substrate 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 region (A) is a recess formed in the cell culture substrate, a patterning step of irradiating a part of the surface of the substrate with a laser to form the region (A), A method for producing a cell culture substrate, which does not include a surface modification treatment step for the recesses.

2. The manufacturing method according to claim 1 , wherein the patterning step is a step of forming the region (A) by ablation processing.

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

4. The method according to claim 1 or 2, wherein the peak power of the laser is 1.250 MW or more.

5. The manufacturing method according to claim 1 or 2, wherein the peak wavelength of the laser is in the range of 300 nm to 700 nm.

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