Cell culture scaffolds and cell culture vessels

Abstract

To provide a scaffold material for a cell culture capable of allowing cell masses to be formed easily and efficiently.SOLUTION: A scaffold material for a cell culture according to the present invention includes: a substrate; and protrusions formed by patterning in dots or lines on the substrate, wherein the protrusions contain a synthetic resin, the synthetic resin contains at least a polyvinyl alcohol derivative or a poly(meth)acrylic resin, the protrusion has cell adhesion properties, and the central portions of the protrusions in plan view have an average height of 10 nm or more and 10 μm or less.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a cell culture scaffold material used for culturing cells. The present invention also relates to a cell culture container using the above-mentioned cell culture scaffold material. [Background technology]

[0002] In recent years, next-generation medicines using cell therapy and stem cells have attracted attention. In particular, human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), or differentiated cells derived from them, are expected to have applications in drug discovery and regenerative medicine. To achieve such applications, it is necessary to safely and reliably culture and proliferate pluripotent stem cells and differentiated cells.

[0003] These stem cells can form cell aggregates called spheroids through aggregation and adhesion of stem cell populations. Spheroids are thought to be able to be cultured or maintained in a state closer to the three-dimensional structure in vivo, and this is known to exhibit superior characteristics compared to conventional planar adherent culture. In fact, spheroids are being used in anticancer drug screening using cancer cells, among other applications.

[0004] For example, Patent Document 1 below discloses a cell culture substrate in which multiple microwells are formed on a single culture surface, which can suppress the migration of spheroids from the microwells and form spheroids of uniform size without making the microwells too deep. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2018 / 123663 [Overview of the project] [Problems that the invention aims to solve]

[0006] However, the cell culture substrate described in Patent Document 1 has the problem of low cell culture efficiency because the culture surface is a low cell adhesion surface (a surface on which cells do not adhere or adhere poorly). In addition, there is the problem that cell aggregates are easily detached from the culture surface due to vibrations such as those caused by changing the culture medium, making it difficult to control the shape and size of the cell aggregates.

[0007] The object of the present invention is to provide a cell culture scaffold material and a cell culture container that can easily and efficiently form cell aggregates. [Means for solving the problem]

[0008] The cell culture scaffold material according to the present invention comprises a base material and protrusions formed by patterning the base material in a dot-like or line-like manner, wherein the protrusions contain a synthetic resin, the synthetic resin contains at least a polyvinyl alcohol derivative or a poly(meth)acrylic resin, the protrusions have cell adhesion properties, and the average height of the portion located in the center of the protrusions in a plan view is 10 nm or more and 10 μm or less.

[0009] In a specific surface of the cell culture scaffold material according to the present invention, the ratio of the amount of protein adsorbed by the protrusions to the amount adsorbed by the portion without the protrusions (protrusions / portion without protrusions) is 2 or more.

[0010] In another specific aspect of the cell culture scaffold material according to the present invention, the protrusions are formed by dot-like patterning on the substrate, and the protrusions have an average base area of ​​0.005 mm². 2 Above 5mm 2 The following applies:

[0011] In yet another specific aspect of the cell culture scaffold material according to the present invention, the convex portion has a wall portion at its periphery.

[0012] In still another specific aspect of the scaffold material for cell culture according to the present invention, the elastic modulus of the convex portion at 25° C. at the center of the convex portion in plan view, measured by nanoindentation method, is 1 GPa or more.

[0013] In a further specific aspect of the scaffold material for cell culture according to the present invention, the synthetic resin contains 0.2 mol % or more and 20 mol % or less of a Bronsted basic group in the constitutional unit.

[0014] In a further specific aspect of the scaffold material for cell culture according to the present invention, the water swelling rate of the convex portion is 50% or less.

[0015] In a further specific aspect of the scaffold material for cell culture according to the present invention, the convex portion has a peptide portion on its surface.

[0016] The cell culture container according to the present invention includes a scaffold material for cell culture configured according to the present invention in at least a part of the cell culture region.

Advantages of the Invention

[0017] According to the present invention, it is possible to provide a scaffold material for cell culture and a cell culture container that can easily and efficiently form cell aggregates.

Brief Description of the Drawings

[0018] [Figure 1] FIG. 1 is a schematic plan view showing a scaffold material for cell culture according to an embodiment of the present invention. [Figure 2] FIG. 2(a) is a schematic plan view showing an enlarged periphery of a convex portion having a wall portion at the peripheral edge, and FIG. 2(b) is a schematic front cross-sectional view showing an enlarged periphery of a convex portion having a wall portion at the peripheral edge. [Figure 3] FIG. 3 is a schematic front cross-sectional view showing a cell culture container according to an embodiment of the present invention. [Figure 4] FIG. 4 is a photograph showing a pattern of convex portions in the scaffold material for cell culture produced in Example 1. [Figure 5]FIG. 5 is a photograph showing the pattern of the convex portions in the scaffold for cell culture produced in Example 2. [Figure 6] FIG. 6 is a microscopic photograph when cells are seeded in the cell culture container produced in Example 1. [Figure 7] FIG. 7 is a microscopic photograph after culturing cells for 5 days in the cell culture container produced in Example 1.

Mode for Carrying Out the Invention

[0019] Hereinafter, the present invention will be clarified by describing specific embodiments of the present invention while referring to the drawings.

[0020] The scaffold for cell culture of the present invention is a scaffold that includes a base material and convex portions formed by being patterned in dot or line form on the base material. In the scaffold for cell culture of the present invention, the convex portions contain a synthetic resin, and the synthetic resin contains at least a polyvinyl alcohol derivative or a poly(meth)acrylic resin. In the scaffold for cell culture of the present invention, the convex portions have cell adhesiveness. In the scaffold for cell culture of the present invention, in a plan view, the average height of the portion located at the center of the convex portion is 10 nm or more and 10 μm or less.

[0021] Since the scaffold for cell culture of the present invention has the above-described configuration, cell aggregates can be formed easily and efficiently.

[0022] In a cell culture space provided in a recess patterned by a conventional microfabrication technique, there is a problem that the culture efficiency of cells is low and it is difficult to form cell aggregates easily and efficiently.

[0023] The present inventors focused on the convex portions having cell adhesiveness, and particularly found that cell aggregates can be formed easily and efficiently by setting the average height at a specific position of the convex portions to 10 nm or more and 10 μm or less.

[0024] Specifically, in the cell culture scaffold material of the present invention, seeded cells gather on the protrusions, forming cell aggregates. In this process, the cell aggregates can be formed along the shape of the protrusions. Therefore, by controlling the size and shape of the protrusions, the shape and size of the cell aggregates can be easily controlled.

[0025] Furthermore, in the cell culture scaffold material of the present invention, cells can be seeded on both the protruding and non-protruding sections. In particular, cells seeded on the non-protruding sections can be gathered onto the protruding sections, allowing only cells with good migratory ability to be collected. Therefore, only high-quality cells can be selected, and high-quality cell aggregates can be efficiently obtained. In addition, since it is not necessary to selectively seed cells only on the protruding sections, cell aggregates can be formed easily and efficiently.

[0026] Therefore, the cell culture scaffold material of the present invention makes it possible to easily and efficiently form high-quality cell aggregates, and moreover, to easily control the shape and size of the cell aggregates.

[0027] Furthermore, since synthetic resins can be used in this invention, compared to scaffolding materials made from natural polymer materials, it offers better operability, lower cost, less variation between lots, and superior safety.

[0028] The following is a schematic plan view of an example of a cell culture scaffold material, which has protrusions formed by dot-like patterning, with reference to Figure 1.

[0029] The cell culture scaffold material 1 shown in Figure 1 comprises a base material 2 and a plurality of protrusions 3 provided on the base material 2. The plurality of protrusions 3 contain synthetic resin and have cell adhesion properties. In addition, the base material 2 also has a portion 4 where no protrusions are provided. In this embodiment, the plurality of protrusions 3 adhere to a larger number of cells per unit area compared to the portion 4 where no protrusions are provided.

[0030] Furthermore, in the cell culture scaffold material 1, the protrusions 3 are formed by patterning in a dot shape. The planar shape of the protrusions 3 is approximately circular. Also, in a planar view, the average height of the central part of the protrusions 3 is between 10 nm and 10 μm.

[0031] Therefore, with the cell culture scaffold material 1, high-quality cell aggregates can be formed easily and efficiently, and the shape and size of the cell aggregates can be easily controlled.

[0032] In the above embodiment, the planar shape of the protrusion was approximately circular, but the planar shape of the protrusion may also be approximately rectangular or triangular, and is not particularly limited. Furthermore, some or all of the multiple protrusions may be formed by a series of adjacent dots.

[0033] The details of the cell culture scaffold material of the present invention will be further described below.

[0034] [Convex part] The cell culture scaffold material of the present invention comprises protrusions arranged on a substrate. The cell culture scaffold material of the present invention comprises a substrate and dot-shaped or line-shaped protrusions provided on the substrate. The protrusions are formed by patterning the substrate in a dot-like or line-like manner. By arranging the protrusions on the substrate in a dot-like or line-like manner, it is possible to form high-quality cell aggregates more easily and efficiently, and the shape and size of the cell aggregates can be controlled more easily. The protrusions may be formed by patterning the substrate in a dot-like manner, by patterning the substrate in a line-like manner, or by patterning the substrate in both a dot-like and line-like manner.

[0035] From the viewpoint of more easily controlling the shape and size of the cell aggregate, it is preferable that the protrusions are constructed by patterning in a dot shape. The cell culture scaffold material described above preferably comprises at least protrusions constructed by patterning in a dot shape.

[0036] The number of protrusions may be singular or multiple. For example, the number of protrusions can be from 1 to 1500. When the protrusions are formed by dot-shaped patterning, the number of protrusions is preferably 1 or more, more preferably 5 or more, even more preferably 10 or more, preferably 1500 or less, more preferably 1000 or less, and even more preferably 500 or less. When the number of protrusions is above the lower limit and below the upper limit, it becomes easier and more efficient to form high-quality cell aggregates, and the shape and size of the cell aggregates can be controlled more easily. When the protrusions are formed by line-shaped patterning, the number of protrusions may be 1 or 2 or more.

[0037] The above-mentioned protrusions have an average height of 10 nm or more and 10 μm or less at the center of the protrusion when viewed in plan. Preferably, the above-mentioned protrusions have an average height of 50 nm or more, more preferably 100 nm or more, preferably 5 μm or less, more preferably 2 μm or less, and even more preferably 1 μm or less. When the above-mentioned average height is within the above range, high-quality cell aggregates can be formed more easily and efficiently, and the shape and size of the cell aggregates can be controlled more easily.

[0038] The height of the protrusion at the center of the protrusion in a plan view can be measured using a hybrid laser microscope or the like. The average height of the protrusion at the center of the protrusion in a plan view is the average value of the height of the protrusion at the center of the protrusion in a plan view. The average height of the protrusion at the center of the protrusion in a plan view is the average height from the surface (top surface) of the substrate to the center of the protrusion in a plan view. The above average height is the average height at the center of the planar shape when the dot-shaped protrusion is viewed from above. When the planar shape when the dot-shaped protrusion is viewed from above is circular or elliptical, the center of the protrusion in a plan view means the center of the circle or ellipse. When the planar shape when the dot-shaped protrusion is viewed from above is polygonal or the like, the center of the protrusion in a plan view means the centroid of the polygon or the like. The above average height is the average height at the center of the line width of the line-shaped protrusion when the protrusion is line-shaped. If the protrusion is formed by dot patterning and there is only one such protrusion, the above average height refers to the height at the center of the protrusion in a plan view. If the protrusion is formed by dot patterning and there are fewer than 10 such protrusions, the above average height refers to the average of the heights at the center of these protrusions in a plan view. If the protrusion is formed by dot patterning and there are 10 or more such protrusions, the above average height refers to the average of the heights at the center of any 10 protrusions in a plan view. Furthermore, if the protrusion is formed by line patterning, the above average height refers to the average of the heights at the center of any 10 line widths that are at least 10 μm apart from each other. Note that the center does not have to be the exact center, may be slightly off-center within the range of measurement error, or may be approximately in the center.

[0039] If the protrusions are formed by dot patterning, the average base area of ​​the protrusions is preferably 0.005 mm². 2 More preferably 0.02 mm2 More preferably 0.1 mm 2 The above is preferable to 5 mm 2 More preferably 3mm 2 More preferably 1.5 mm 2 The following is true: In this case, high-quality cell aggregates can be formed more easily and efficiently, and the shape and size of the cell aggregates can be controlled more easily.

[0040] The average base area of ​​a convex part refers to the base area of ​​the single convex part if there is only one convex part. If there are fewer than 10 convex parts, the average base area of ​​the convex parts refers to the average value of the base areas of those convex parts. If there are 10 or more convex parts, the average base area of ​​a convex part refers to the average value of the base areas of any 10 convex parts.

[0041] Furthermore, the protrusions may be formed by patterning in a linear shape. In this case, the protrusions may be formed in a straight line, a bent line, or a curved shape, or they may be formed in a circular or spiral shape.

[0042] When the protrusions are formed by patterning in a linear fashion, the average line width of the protrusions is preferably 50 μm or more, more preferably 100 μm or more, more preferably 1.5 mm or less, and more preferably 0.5 mm or less. In this case, high-quality cell aggregates can be formed more easily and efficiently, and the shape and size of the cell aggregates can be controlled more easily.

[0043] The protrusions have cell adhesion properties. In this invention, "protrusions having cell adhesion properties" means that the number of cells that adhere to a unit area of ​​the protrusions is greater than that of the parts without protrusions. Therefore, it is preferable that the protrusions have higher cell adhesion properties than the substrate.

[0044] In the present invention, the ratio of protein adsorption amount of the protrusions to the portion without protrusions (protrusions / portion without protrusions) is preferably 1.5 or more, more preferably 2 or more, preferably 15 or less, and more preferably 10 or less. In this case, the cell adhesion of the protrusions can be further enhanced, and cell aggregates can be formed more easily and efficiently.

[0045] The above protein adsorption amount can be measured, for example, by the following method.

[0046] For cell culture scaffolding, 40 μL of FITC-labeled bovine serum albumin (Cosmo Bio Co., Ltd.) at 0.1 mg / mL was cast and allowed to stand at 37°C for 1 hour. After that, the cell culture scaffolding was washed with pure water and dried in an oven at 45°C for 1 hour. In addition, for calibration of adsorbed protein amount, 1 μL of FITC-labeled bovine serum albumin at concentrations of 0.005 mg / mL, 0.02 mg / mL, and 0.05 mg / mL was dispensed onto polystyrene substrates and dried in an oven at 45°C for 1 hour. The cell culture scaffolding was photographed using a fluorescence microscope, and the fluorescence intensity of the convex areas and the areas without convex areas was determined. The amount of adsorbed protein was converted from the linear approximation of the fluorescence intensity obtained using the polystyrene substrates for calibration.

[0047] Furthermore, the ratio of protein adsorption can be increased by increasing the content of cationic functional groups such as amino groups and anionic functional groups such as carboxyl groups in the synthetic resin contained in the protrusions.

[0048] In the present invention, the elastic modulus of the convex portion at 25°C at the central position of the convex portion in a plan view, as measured by nanoindentation, is preferably 1 GPa or more, more preferably 2 GPa or more, preferably 8 GPa or less, and more preferably 6 GPa or less. When the elastic modulus of the convex portion is within the above range, the cell adhesion of the convex portion can be further enhanced, and cell aggregates can be formed more easily and efficiently.

[0049] The above-mentioned elastic modulus (surface modulus) can be measured, for example, using a nanoindenter (Hysitron, Triboindenter, manufactured by Bruker). A Berkovich (triangular pyramidal) indenter with a tip diameter R of 100 nm can be used as the indenter, and the measurement can be performed by single indentation measurement under air conditions at 25°C. The indentation depth can be 50 nm. Note that the center position does not have to be exactly in the center; it may be slightly off-center within the range of measurement error, or it may be approximately in the center.

[0050] The above surface modulus can be increased by methods such as increasing the molecular weight of the synthetic resin contained in the protrusions, increasing the degree of crosslinking of the synthetic resin contained in the protrusions, or introducing a crystalline molecular structure into the synthetic resin contained in the protrusions.

[0051] In the present invention, the water swelling rate of the protrusions is preferably 50% or less, more preferably 40% or less. In this case, the cell adhesion of the protrusions can be further enhanced, and cell aggregates can be formed more easily and efficiently. The lower limit of the water swelling rate is not particularly limited, but for example, it can be 0.5%. The water swelling rate can be measured, for example, as follows.

[0052] Prepare the substrate and cell culture scaffold material separately and measure their respective weights. Subtract the weight of the substrate from the weight of the cell culture scaffold material to obtain the "sample weight before immersion". Immerse the substrate and cell culture scaffold material separately in 25°C water for 24 hours. After immersion, remove the water adhering to the substrate and cell culture scaffold material and measure their respective weights. Subtract the weight of the substrate after immersion from the weight of the cell culture scaffold material after immersion to obtain the "sample weight after immersion". Calculate the water swelling rate = (sample weight after immersion - sample weight before immersion) / (sample weight before immersion) × 100 (%).

[0053] The above water swelling rate can be reduced, for example, by increasing the number of hydrophobic functional groups in the synthetic resin contained in the protrusions, or by lowering the number-average molecular weight.

[0054] It is preferable that the convex portion has a wall portion at its periphery. In this case, it is possible to form high-quality cell aggregates more easily and efficiently, and the shape and size of the cell aggregates can be controlled more easily. Note that the wall portion is part of the convex portion. Therefore, when the convex portion has a wall portion, the base area and line width of the convex portion described above are the base area and line width of the convex portion including the wall portion.

[0055] Figure 2(a) is a schematic plan view showing an enlarged view of the area around a protrusion with a wall at its periphery, and Figure 2(b) is a schematic front cross-sectional view showing an enlarged view of the area around a protrusion with a wall at its periphery. Figure 2(b) is a cross-sectional view along line II in Figure 2(a). In Figure 2, one protrusion is shown in enlargement.

[0056] In Figure 2, a protrusion 3A is positioned on the surface of the substrate 2A. The protrusion 3A has a wall portion 31A at its periphery. The protrusion 3A has a wall portion 31A and a portion without a wall portion 31A. In plan view, the protrusion 3A is circular. In plan view, the center of the protrusion 3A is the center of the circle. The upper surface of the protrusion 3A is composed of the upper surface of the wall portion 31A and the upper surface of the portion without a wall portion 31A. The height Hb of the wall portion 31A is higher than the height of the portion without a wall portion 31A. The height Hb of the wall portion 31A is higher than the height Ha of the protrusion 3A at the center of the protrusion 3A in plan view. The height Hb of the wall portion is the height of the protrusion at the center of the wall portion in the width direction in plan view, and the height of the protrusion at the center of the wall portion in the thickness direction. The height Hb of the wall portion is the distance from the surface (top surface) of the base material 2A to the center of the top surface of the wall portion 31A in the width direction. The protrusion 3A has a wall portion 31A with a thickness Tb. The thickness Tb of the wall portion corresponds to the width of the wall portion in a plan view. The portion with the wall portion 31A and the portion without the wall portion 31A are formed of the same material. The portion without the wall portion 31A is surrounded by the wall portion 31A.

[0057] The average height of the wall portion is preferably 100 nm or more, more preferably 300 nm or more, preferably 50 μm or less, and more preferably 20 μm or less. When the average height of the wall portion is above the lower limit and below the upper limit, high-quality cell aggregates can be formed more easily and efficiently, and the shape and size of the cell aggregates can be controlled more easily.

[0058] The average thickness of the wall portion is preferably 50 nm or more, more preferably 100 nm or more, preferably 5 μm or less, and more preferably 1 μm or less. When the average thickness of the wall portion is above the lower limit and below the upper limit, high-quality cell aggregates can be formed more easily and efficiently, and the shape and size of the cell aggregates can be controlled more easily.

[0059] The average height of the wall portion is preferably higher than the average height of the convex portion located in the center of the convex portion in a plan view. The ratio of the average height of the wall portion to the average height of the convex portion located in the center of the convex portion in a plan view (average height of the wall portion / average height of the convex portion located in the center of the convex portion in a plan view) is preferably 2 or more, more preferably 4 or more, preferably 100 or less, and more preferably 50 or less. When the above ratio is above the lower limit and below the upper limit, high-quality cell aggregates can be formed more easily and efficiently, and the shape and size of the cell aggregates can be controlled more easily.

[0060] The average height and average thickness of the wall sections refer to the height and thickness of the wall section at a specific protrusion if there is only one protrusion with a wall section. If there are fewer than 10 protrusions with wall sections, the average height and average thickness of the wall sections at those protrusions refer to the average values ​​of the wall sections at those protrusions. If there are 10 or more protrusions with wall sections, the average height and average thickness of the wall sections at any 10 protrusions refer to the average values ​​of the wall sections at those protrusions.

[0061] The method for forming the wall portion is not particularly limited, but examples include methods using transfer molding with a mold, methods using precision machine cutting, and methods using chemical etching.

[0062] A simple method for manufacturing a protrusion with a wall is as follows: First, a solution containing a synthetic resin for forming the protrusion is prepared. Next, minute droplets of the solution are placed on a substrate and the solvent is gradually evaporated. When minute droplets of a solution containing a low concentration of synthetic resin are gradually dried, a protrusion with a wall at its periphery is formed on the substrate.

[0063] (Synthetic resin) The protrusions of the cell culture scaffold material of the present invention contain a synthetic resin (hereinafter sometimes referred to as synthetic resin X). Synthetic resin X is the synthetic resin contained in the protrusions. In this specification, "structural unit" refers to the repeating unit of the monomer constituting the synthetic resin. If the synthetic resin has a graft chain, it includes the repeating unit of the monomer constituting that graft chain.

[0064] The synthetic resin X preferably contains 0.2 mol% or more of Brønsted basic groups in its constituent units, more preferably 2 mol% or more, more preferably 30 mol% or less, more preferably 20 mol% or less, and even more preferably 15 mol% or less. In this case, the effects of the present invention can be exhibited even more effectively. Brønsted basic groups and the like will be described later.

[0065] The synthetic resin X comprises at least a polyvinyl alcohol derivative or a poly(meth)acrylic resin. The inclusion of a polyvinyl alcohol derivative or poly(meth)acrylic resin in the synthetic resin X enhances adhesion to cells and further suppresses swelling of the cell culture scaffold material in liquid culture medium. The synthetic resin X may contain a polyvinyl alcohol derivative, a poly(meth)acrylic resin, or both. The polyvinyl alcohol derivative and poly(meth)acrylic resin may be used individually or in combination of two or more types.

[0066] The polyvinyl alcohol derivative is preferably a synthetic resin having a polyvinyl acetal skeleton. The poly(meth)acrylic resin is preferably a synthetic resin having a poly(meth)acrylic acid ester skeleton.

[0067] <Synthetic resin with a polyvinyl acetal backbone> The protrusions of the cell culture scaffold material preferably contain a synthetic resin having a polyvinyl acetal skeleton. The synthetic resin X preferably contains a synthetic resin having a polyvinyl acetal skeleton.

[0068] In this specification, "synthetic resin having a polyvinyl acetal skeleton" may be referred to as "polyvinyl acetal resin X".

[0069] The polyvinyl acetal resin X preferably has acetal groups, acetyl groups, and hydroxyl groups in its side chains. However, the polyvinyl acetal resin X may not have acetyl groups, for example. For example, all of the acetyl groups of the polyvinyl acetal resin X may be bonded to a linker described later, so that the polyvinyl acetal resin X does not have acetyl groups.

[0070] The synthesis of polyvinyl acetal resin X includes at least a step of acetalizing polyvinyl alcohol with an aldehyde.

[0071] The aldehyde used in the acetalization of polyvinyl alcohol to obtain polyvinyl acetal resin X is not particularly limited. Examples of aldehydes include those having 1 to 10 carbon atoms. The aldehyde may have a linear aliphatic group, a cyclic aliphatic group, or an aromatic group. The aldehyde may be a linear aldehyde or a cyclic aldehyde.

[0072] Examples of the above-mentioned aldehydes include formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, pentanal, hexanal, heptanal, octanal, nonanal, decanal, acrolein, benzaldehyde, cinnamaldehyde, perillaldehyde, formylpyridine, formylimidazole, formylpyrrole, formylpiperidine, formyltriazole, formyltetrazole, formylindole, formylisoindole, formylpurine, formylbenzimidazole, formylbenzotriazole, formylquinoline, formylisoquinoline, formylquinoxaline, formylcinnoline, formylpteridine, formylfuran, formyloxolane, formyloxane, formylthiophene, formylthiolane, formylthian, formyladenine, formylguanine, formylcytosine, formylthymine, and formyluracil. The above aldehydes may be used individually or in combination of two or more.

[0073] The aldehyde is preferably formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, or pentanal, and more preferably butyraldehyde. Therefore, the polyvinyl acetal skeleton is preferably a polyvinyl butyral skeleton. The polyvinyl acetal resin X is preferably a synthetic resin having a polyvinyl butyral skeleton, and more preferably a polyvinyl butyral resin.

[0074] The polyvinyl acetal resin X may be copolymerized with a vinyl compound. That is, the polyvinyl acetal resin X may be a copolymer of the structural units of the polyvinyl acetal resin and a vinyl compound. In this invention, a polyvinyl acetal resin copolymerized with a vinyl compound is also referred to as a polyvinyl acetal resin.

[0075] Vinyl compounds are compounds that have a vinyl group (H2C=CH-). Vinyl compounds may also be polymers that have structural units containing a vinyl group.

[0076] The copolymer may be a block copolymer of polyvinyl acetal resin and a vinyl compound, or a graft copolymer in which a vinyl compound is grafted onto polyvinyl acetal resin. The copolymer is preferably a graft copolymer.

[0077] The above copolymer can be synthesized, for example, by the following methods (1) to (3): (1) A method for synthesizing polyvinyl acetal resin using polyvinyl alcohol copolymerized with a vinyl compound. (2) A method for synthesizing polyvinyl acetal resin using polyvinyl alcohol and polyvinyl alcohol copolymerized with a vinyl compound. (3) A method for graft copolymerizing a vinyl compound onto polyvinyl acetal resin before graft copolymerization.

[0078] Examples of vinyl compounds include ethylene, allylamine, vinylpyrrolidone, vinylimidazole, maleic anhydride, maleimide, itaconic acid, (meth)acrylic acid, vinylamine, and / or (meth)acrylic acid esters. These vinyl compounds may be used individually or in combination of two or more.

[0079] From the viewpoint of further enhancing cell adhesion, the polyvinyl acetal resin X preferably has Brønsted basic groups or Brønsted acidic groups, and more preferably has Brønsted basic groups. That is, it is preferable that a portion of the polyvinyl acetal resin X is modified with Brønsted basic groups or Brønsted acidic groups, and more preferably that a portion of the polyvinyl acetal resin X is modified with Brønsted basic groups.

[0080] The polyvinyl acetal resin X preferably has a portion of Brønsted basic groups or Brønsted acidic groups, and more preferably has Brønsted basic groups. In this case, cell adhesion can be further enhanced. In this case, monomers having Brønsted basic groups or Brønsted acidic groups may be copolymerized or graft copolymerized. The degree of modification of the Brønsted basic groups is preferably 0.2 mol% or more, more preferably 2 mol% or more, preferably 30 mol% or less, more preferably 20 mol% or less, and even more preferably 15 mol% or less. If the degree of modification due to the Brønsted basic groups is within the above specific range, cell adhesion can be further enhanced.

[0081] Brønsted basic groups are a general term for functional groups that can accept hydrogen ions (H+) from other substances. Examples of Brønsted basic groups include amine-based basic groups such as substituents having an imine structure, substituents having an imide structure, substituents having an amine structure, and substituents having an amide structure. Brønsted basic groups are not particularly limited, but include hydroxyamino groups, urea groups, conjugated amine functional groups such as guanidine and biguanide, piperazine, piperidine, pyrrolidine, 1,4-diazabicyclo[2.2.2]octane, hexamethylenetetraamine, morpholine, pyridine, pyridazine, pyrimidine, pyrazine, pyrrole, azatropyridene, pyridone, imidazole, benzimidazole, benzotriazole, pyrazole, oxazole, imidazoline, triazole, thiazole, thiazine, tetrazole, indole, isoindole, purine, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline, pteridine, carbazole, acridine, adenine, guanine, cytosine, thymine, uracil, melamine, heterocyclic amino functional groups, porphyrin, chlorine, choline, and cyclic pyrrole functional groups, and their derivatives.

[0082] Examples of Brønsted acidic groups include carboxyl groups, sulfonic acid groups, maleic acid groups, sulfinic acid groups, sulfenic acid groups, phosphate groups, phosphonic acid groups, or salts thereof. A carboxyl group is preferred as the Brønsted acidic group.

[0083] The polyvinyl acetal resin X preferably has structural units having an imine structure, an imide structure, an amine structure, or an amide structure. In this case, it may have only one of these structural units, or it may have two or more.

[0084] The polyvinyl acetal resin X may have structural units having an imine structure. An imine structure is a structure having a C=N bond. In particular, it is preferable that the polyvinyl acetal resin X has an imine structure in its side chains.

[0085] The polyvinyl acetal resin X may have a structural unit having an imide structure. The structural unit having an imide structure is preferably a structural unit having an imino group (=NH).

[0086] The polyvinyl acetal resin X preferably has an imino group in the side chain. In this case, the imino group may be directly bonded to a carbon atom constituting the main chain of the polyvinyl acetal resin X, or may be bonded to the main chain via a linking group such as an alkylene group.

[0087] The polyvinyl acetal resin X may have a structural unit having an amine structure. The amine group in the above amine structure may be a primary amine group, a secondary amine group, a tertiary amine group, or a quaternary amine group.

[0088] The structural unit having an amine structure may be a structural unit having an amide structure. The above amide structure refers to a structure having -C(=O)-NH-.

[0089] The polyvinyl acetal resin X preferably has an amine structure or an amide structure in the side chain. In this case, the amine structure or the amide structure may be directly bonded to a carbon atom constituting the main chain of the polyvinyl acetal resin X, or may be bonded to the main chain via a linking group such as an alkylene group.

[0090] In addition, the content of the structural unit having an imide structure, the content of the structural unit having an imide structure, the content of the structural unit having an amine structure, and the content of the structural unit having an amide structure can be measured by 1 H-NMR (nuclear magnetic resonance spectrum).

[0091] <Synthetic resin having a poly(meth)acrylate ester skeleton> The protrusions of the cell culture scaffold material of the present invention preferably contain a synthetic resin having a poly(meth)acrylic acid ester skeleton. The synthetic resin X preferably contains a synthetic resin having a polyvinyl acetal skeleton.

[0092] In this specification, "synthetic resin having a poly(meth)acrylic acid ester skeleton" may be referred to as "poly(meth)acrylic acid ester resin X".

[0093] Therefore, poly(meth)acrylic acid ester resin X is a resin having a poly(meth)acrylic acid ester skeleton.

[0094] Poly(meth)acrylic acid ester resin X is obtained by polymerization of (meth)acrylic acid ester, or by copolymerization of (meth)acrylic acid ester with other monomers.

[0095] Examples of (meth)acrylic acid esters include alkyl (meth)acrylates, cyclic alkyl (meth)acrylates, aryl (meth)acrylates, (meth)acrylamides, polyethylene glycol (meth)acrylates, and phosphorylcholine (meth)acrylate.

[0096] Examples of alkyl (meth)acrylates include methyl (meth)acrylate, ethyl (meth)acrylate, n-propyl (meth)acrylate, isopropyl (meth)acrylate, n-butyl (meth)acrylate, isobutyl (meth)acrylate, t-butyl (meth)acrylate, n-octyl (meth)acrylate, isooctyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, nonyl (meth)acrylate, isononyl (meth)acrylate, decyl (meth)acrylate, isodecyl (meth)acrylate, lauryl (meth)acrylate, stearyl (meth)acrylate, and isotetradecyl (meth)acrylate.

[0097] Furthermore, the alkyl (meth)acrylate may be substituted with substituents such as alkoxy groups having 1 to 3 carbon atoms and tetrahydrofurfuryl groups. Examples of such alkyl (meth)acrylates include methoxyethyl acrylate and tetrahydrofurfuryl acrylate.

[0098] Examples of cyclic alkyl (meth)acrylates include cyclohexyl (meth)acrylate and isobornyl (meth)acrylate.

[0099] Examples of aryl (meth)acrylates include phenyl (meth)acrylate and benzyl (meth)acrylate.

[0100] Examples of (meth)acrylamides include (meth)acrylamide, N-isopropyl(meth)acrylamide, N-tert-butyl(meth)acrylamide, N,N'-dimethyl(meth)acrylamide, (3-(meth)acrylamidopropyl)trimethylammonium chloride, 4-(meth)acryloylmorpholine, 3-(meth)acryloyl-2-oxazolidinone, N-[3-(dimethylamino)propyl](meth)acrylamide, N-(2-hydroxyethyl)(meth)acrylamide, N-methylol(meth)acrylamide, and 6-(meth)acrylamidohexanoic acid.

[0101] Examples of polyethylene glycol (meth)acrylates include methoxy-polyethylene glycol (meth)acrylate, ethoxy-polyethylene glycol (meth)acrylate, hydroxy-polyethylene glycol (meth)acrylate, methoxy-diethylene glycol (meth)acrylate, ethoxy-diethylene glycol (meth)acrylate, hydroxy-diethylene glycol (meth)acrylate, methoxy-triethylene glycol (meth)acrylate, ethoxy-triethylene glycol (meth)acrylate, and hydroxy-triethylene glycol (meth)acrylate.

[0102] Examples of (meth)acrylic acid phosphorylcholine include 2-(meth)acryloyloxyethyl phosphorylcholine.

[0103] Vinyl compounds are preferably used as other monomers copolymerized with (meth)acrylic acid esters. Examples of vinyl compounds include ethylene, allylamine, vinylpyrrolidone, maleic anhydride, maleimide, itaconic acid, (meth)acrylic acid, vinylamine, or (meth)acrylic acid esters. Only one vinyl compound may be used, or two or more may be used in combination.

[0104] In this specification, "(meth)acrylic" means "acrylic" or "methacrylic," and "(meth)acrylate" means "acrylate" or "methacrylate."

[0105] The poly(meth)acrylic acid ester resin X described above preferably has a portion of a Brønsted basic group or a Brønsted acidic group, similar to the polyvinyl acetal resin X. In this case, cell adhesion can be further enhanced. The Brønsted basic group only needs to be present in a portion of the poly(meth)acrylic acid ester resin X, and in this case, monomers having the Brønsted basic group may be copolymerized or graft copolymerized. The degree of modification of the Brønsted basic group is preferably 0.2 mol% or more, more preferably 2 mol% or more, preferably 30 mol% or less, more preferably 20 mol% or less, and even more preferably 15 mol% or less. If the degree of modification due to the Brønsted basic group is within the above specific range, cell adhesion can be further enhanced.

[0106] <Synthetic resin having a peptide portion> The protrusions of the cell culture scaffold material of the present invention preferably contain a synthetic resin having a peptide portion. The synthetic resin X preferably contains a synthetic resin having a peptide portion. By using a synthetic resin having a peptide portion as the material for the protrusions of the cell culture scaffold material, it is possible to obtain protrusions having a peptide portion on their surface. That is, it is preferable that the protrusions have a peptide portion on their surface. In this case, the adhesion to cells after seeding can be further improved, and the cell proliferation rate can be further increased. In addition, in this case, it is possible to form high-quality cell aggregates more easily and efficiently, and the shape and size of the cell aggregates can be controlled more easily.

[0107] A synthetic resin having a peptide portion can be obtained by reacting a synthetic resin with a linker and a peptide. The synthetic resin having a peptide portion is preferably a peptide-containing polyvinyl acetal resin having a polyvinyl acetal resin portion, a linker portion, and a peptide portion, and more preferably a peptide-containing polyvinyl butyral resin having a polyvinyl butyral resin portion, a linker portion, and a peptide portion. Therefore, the above-mentioned synthetic resin having a polyvinyl acetal skeleton (polyvinyl acetal resin X) is preferably a peptide-containing polyvinyl acetal resin, and more preferably a peptide-containing polyvinyl butyral resin. Only one type of synthetic resin having a peptide portion may be used, or two or more types may be used in combination.

[0108] The peptide portion is preferably composed of three or more amino acids, more preferably of four or more amino acids, even more preferably of five or more amino acids, preferably of ten or fewer amino acids, and more preferably of six or fewer amino acids. When the number of amino acids constituting the peptide portion is above the lower limit and below the upper limit, the adhesion to cells after seeding can be further enhanced, and the cell proliferation rate can be further enhanced.

[0109] The peptide portion described above preferably has a cell-adherent amino acid sequence. A cell-adherent amino acid sequence refers to an amino acid sequence whose cell-adherent activity has been confirmed by phage display, Sepharose bead method, or plate-coating method. For example, the phage display method described in "The Journal of Cell Biology, Volume 130, Number 5, September 1995 1189-1196" can be used. For example, the Sepharose bead method described in "Proteins, Nucleic Acids, Enzymes Vol. 45 No. 15 (2000) 2477" can be used. For example, the plate-coating method described in "Proteins, Nucleic Acids, Enzymes Vol. 45 No. 15 (2000) 2477" can be used.

[0110] Examples of the above-mentioned cell-adhering amino acid sequences include RGD sequence (Arg-Gly-Asp), YIGSR sequence (Tyr-Ile-Gly-Ser-Arg), PDSGR sequence (Pro-Asp-Ser-Gly-Arg), HAV sequence (His-Ala-Val), ADT sequence (Ala-Asp-Thr), QAV sequence (Gln-Ala-Val), LDV sequence (Leu-Asp-Val), IDS sequence (Ile-Asp-Ser), REDV sequence (Arg-Glu-Asp-Val), IDAPS sequence (Ile-Asp-Ala-Pro-Ser), KQAGDV sequence (Lys-Gln-Ala-Gly-Asp-Val), and TDE sequence (Thr-Asp-Glu). Furthermore, examples of the above-mentioned cell-adherent amino acid sequences include those described in "Pathophysiology, Vol. 9, No. 7, pp. 527-535, 1990" and "Osaka Prefectural Maternal and Child Health Center Journal, Vol. 8, No. 1, pp. 58-66, 1992." The above-mentioned peptide portion may have only one of the above-mentioned cell-adherent amino acid sequences, or it may have two or more.

[0111] The above-mentioned cell-adherent amino acid sequence preferably has at least one of the above-mentioned cell-adherent amino acid sequences, more preferably has at least an RGD sequence, a YIGSR sequence, or a PDSGR sequence, and even more preferably has at least an RGD sequence represented by the following formula (1). In this case, adhesion to cells after seeding can be further enhanced, and the cell proliferation rate can be further increased.

[0112] Arg-Gly-Asp-X...Formula (1)

[0113] In the above formula (1), X represents Gly, Ala, Val, Ser, Thr, Phe, Met, Pro, or Asn.

[0114] The peptide portion may be linear or may have a cyclic peptide skeleton. The cyclic peptide skeleton is a cyclic skeleton composed of multiple amino acids. From the viewpoint of more effectively exhibiting the effects of the present invention, the cyclic peptide skeleton is preferably composed of 4 or more amino acids, preferably of 5 or more amino acids, and preferably of 10 or fewer amino acids.

[0115] In a synthetic resin having a peptide portion, the content of the peptide portion is preferably 0.1 mol% or more, more preferably 1 mol% or more, even more preferably 5 mol% or more, and particularly preferably 10 mol% or more. In a synthetic resin having a peptide portion, the content of the peptide portion is preferably 60 mol% or less, more preferably 50 mol% or less, even more preferably 35 mol% or less, and particularly preferably 25 mol% or less. When the content of the peptide portion is above the lower limit, the phase separation structure can be formed more easily. When the content of the peptide portion is above the lower limit, the adhesion to cells after seeding can be further improved, and the cell proliferation rate can be further improved. Furthermore, when the content of the peptide portion is below the upper limit, the manufacturing cost can be reduced. Note that the content of the peptide portion (mol%) is the amount of substance of the peptide portion relative to the sum of the amounts of substance of each structural unit constituting the synthetic resin having a peptide portion.

[0116] The content of the peptide portion can be measured by FT-IR or LC-MS.

[0117] In a synthetic resin having a peptide portion, it is preferable that the synthetic resin portion and the peptide portion are linked via a linker. That is, it is preferable that the synthetic resin having a peptide portion is a synthetic resin having a peptide portion and a linker portion. The linker may be of one type only, or two or more types may be used in combination.

[0118] The linker described above is preferably a compound having a functional group that can condense with the carboxyl group or amino group of the peptide. Examples of functional groups that can condense with the carboxyl group or amino group of the peptide include carboxyl groups, thiol groups, and amino groups. From the viewpoint of good reaction with the peptide, the linker is preferably a compound having a carboxyl group. The vinyl compound described above can also be used as the linker.

[0119] Examples of linkers having the carboxyl group mentioned above include (meth)acrylic acid and carboxyl group-containing acrylamide. By using a polymerizable unsaturated carboxylic acid (carboxylic acid monomer) as the linker having the carboxyl group, the carboxylic acid monomer can be polymerized by graft polymerization when the linker is reacted with the synthetic resin, thereby increasing the number of carboxyl groups that can react with the peptide.

[0120] <Other resins> The protrusions of the cell culture scaffold material may contain polymers other than the synthetic resins mentioned above. Examples of such polymers include polyvinylpyrrolidone, polystyrene, ethylene-vinyl acetate copolymer, polyolefin resin, polyether resin, polyvinyl alcohol resin, polyester, epoxy resin, polyamide resin, polyimide resin, polyurethane resin, and polycarbonate resin.

[0121] [Base material] The cell culture scaffold material of the present invention comprises a base material. A portion of the surface of the base material constitutes a portion without protrusions. Therefore, it is preferable that the base material is made of a material to which fewer cells adhere per unit area than the number of cells adhering per unit area compared to the protrusions.

[0122] Examples of base material materials include resins, metals, and inorganic materials. Examples of resins include polystyrene, polyethylene, polypropylene, polycarbonate, polyester, polyisoprene, cycloolefin polymer, polyimide, polyamide, polyamide-imide, (meth)acrylic resin, epoxy resin, and silicone. Examples of metals include stainless steel, copper, iron, nickel, aluminum, titanium, gold, silver, and platinum. Examples of inorganic materials include silicon dioxide (glass), aluminum oxide, titanium dioxide, zirconium oxide, iron oxide, and silicon nitride.

[0123] [More details on cell culture scaffolding materials] The protrusions of the cell culture scaffold material according to the present invention contain the synthetic resin X. From the viewpoint of effectively exhibiting the effects of the present invention and increasing productivity, the content of the synthetic resin X in 100% by weight of the protrusions of the cell culture scaffold material is preferably 90% by weight or more, more preferably 95% by weight or more, even more preferably 97.5% by weight or more, particularly preferably 99% by weight or more, and most preferably 100% by weight (total amount). Therefore, it is most preferable that the protrusions of the cell culture scaffold material be the synthetic resin X. If the content of the synthetic resin X is above the lower limit, the effects of the present invention can be exhibited even more effectively.

[0124] When the synthetic resin X contained in the protrusions contains a polyvinyl alcohol derivative, the content of the polyvinyl alcohol derivative in 100% by weight of the synthetic resin X is preferably 90% by weight or more, more preferably 95% by weight or more, even more preferably 97.5% by weight or more, particularly preferably 99% by weight or more, and most preferably 100% by weight (total amount). Therefore, it is most preferable that the synthetic resin X contained in the protrusions is a polyvinyl alcohol derivative. When the content of the polyvinyl alcohol derivative is above the lower limit mentioned above, the effects of the present invention can be exhibited even more effectively.

[0125] When the synthetic resin X contained in the protrusions contains poly(meth)acrylic resin, the content of poly(meth)acrylic resin in 100% by weight of synthetic resin X is preferably 90% by weight or more, more preferably 95% by weight or more, even more preferably 97.5% by weight or more, particularly preferably 99% by weight or more, and most preferably 100% by weight (total amount). Therefore, it is most preferable that the synthetic resin X contained in the protrusions is poly(meth)acrylic resin. When the content of poly(meth)acrylic resin is above the lower limit mentioned above, the effects of the present invention can be exhibited even more effectively.

[0126] The protrusions of the cell culture scaffold material described above may contain components other than the synthetic resin X. Examples of components other than the synthetic resin X include polysaccharides, cellulose, and synthetic peptides.

[0127] From the viewpoint of effectively exhibiting the effects of the present invention, it is preferable that the content of components other than the synthetic resin X is as low as possible. In 100% by weight of the protrusions of the cell culture scaffold material, the content of the component is preferably 10% by weight or less, more preferably 5% by weight or less, even more preferably 2.5% by weight or less, particularly preferably 1% by weight or less, and most preferably 0% by weight (not contained). Therefore, it is most preferable that the protrusions of the cell culture scaffold material do not contain any components other than the synthetic resin X.

[0128] The above-mentioned cell culture scaffold material preferably does not contain animal-derived raw materials. By not containing animal-derived raw materials, it is possible to provide a cell culture scaffold material that is highly safe and has little variation in quality during manufacturing. "Substantially free of animal-derived raw materials" means that the amount of animal-derived raw materials in the cell culture scaffold material is 3% by weight or less. The above-mentioned cell culture scaffold material preferably contains 1% by weight or less of animal-derived raw materials, and most preferably 0% by weight. In other words, it is most preferable that the cell culture scaffold material of the present invention does not contain any animal-derived raw materials at all.

[0129] The protrusions of the cell culture scaffold material described above preferably contain 3% by weight or less, more preferably 1% by weight or less, and most preferably 0% by weight. In other words, it is most preferable that the protrusions of the cell culture scaffold material contain no animal-derived materials at all.

[0130] (Cell culture using cell culture scaffolding) The cell culture scaffold material according to the present invention is used for culturing cells. The cell culture scaffold material according to the present invention is used as a scaffold for cells when culturing them.

[0131] Examples of the cells mentioned above include animal cells from humans, mice, rats, pigs, cattle, and monkeys. Other examples of the cells include somatic cells, such as stem cells, progenitor cells, and mature cells. These somatic cells may also be cancer cells.

[0132] Examples of the above-mentioned stem cells include mesenchymal stem cells (MSCs), iPS cells, ES cells, Muse cells, embryonic cancer cells, embryonic germ stem cells, and mGS cells.

[0133] Examples of the mature cells mentioned above include nerve cells, cardiomyocytes, retinal cells, and hepatocytes.

[0134] [Cell culture container] The present invention also relates to a cell culture container equipped with the above-mentioned cell culture scaffold material in at least a portion of the cell culture area. Figure 3 is a schematic front cross-sectional view showing a cell culture container according to one embodiment of the present invention.

[0135] The cell culture container 11 comprises a container body 12. The bottom of the container body 12 is made of the base material 2 of the cell culture scaffold material 1. Therefore, the protrusion 3 is provided on the surface 12a of the container body 12.

[0136] By adding liquid culture medium to the cell culture container 11 and seeding cells onto the surface of the cell culture scaffold material 1, cells can be cultured in a planar manner.

[0137] The container body may include a second container body, such as a cover glass, on the bottom surface of the first container body. The first container body and the second container body may be separable. In this case, at least a part of the second container body may be the base material for the cell culture scaffold material.

[0138] Conventional known container bodies (containers) can be used as the container body. The shape and size of the container body are not particularly limited.

[0139] Examples of the container body include cell culture plates and cell culture flasks having one or more wells (holes). The number of wells in the plate is not particularly limited. Examples of the number of wells include 2, 4, 6, 12, 24, 48, 96, 384, etc. Examples of the shape of the wells are not particularly limited, but include circles, ellipses, triangles, squares, rectangles, pentagons, etc. Examples of the shape of the bottom surface of the wells are not particularly limited, but include flat bottoms, round bottoms, etc.

[0140] The material of the container body is not particularly limited, but examples include resins, metals, and inorganic materials. Examples of resins include polystyrene, polyethylene, polypropylene, polycarbonate, polyester, polyisoprene, cycloolefin polymer, polyimide, polyamide, polyamide-imide, (meth)acrylic resin, epoxy resin, and silicone. Examples of metals include stainless steel, copper, iron, nickel, aluminum, titanium, gold, silver, and platinum. Examples of inorganic materials include silicon dioxide (glass), aluminum oxide, titanium dioxide, zirconium oxide, iron oxide, and silicon nitride. In this case, the base material of the cell culture scaffold also becomes part of the container body, so the same material as the material constituting the container body can be used. [Examples]

[0141] Next, the present invention will be clarified by giving specific examples and comparative examples of the present invention. However, the present invention is not limited to the following examples.

[0142] The following synthetic resins X1 to X9 were synthesized as raw materials for cell culture scaffolds. The structural unit content in the obtained synthetic resins was determined after dissolving the synthetic resins in DMSO-D6 (dimethyl sulfoxide). 1 The results were measured using 1H-NMR (nuclear magnetic resonance spectroscopy).

[0143] <Synthetic resin X1> In a reactor equipped with a stirring device, 2700 mL of deionized water and 300 parts by weight of amine-modified polyvinyl alcohol with an average degree of polymerization of 800, a degree of amine modification of 1 mol%, and a degree of saponification of 99 mol% were added, and the mixture was heated and dissolved while stirring to obtain a solution. To the obtained solution, 35% by weight hydrochloric acid was added as a catalyst to achieve a hydrochloric acid concentration of 0.2% by weight. Next, the temperature was adjusted to 15°C, and 22 parts by weight of n-butyraldehyde was added while stirring. Then, 148 parts by weight of n-butyraldehyde was added to precipitate white particulate polyvinyl butyral resin. 15 minutes after precipitation, 35% by weight hydrochloric acid was added to achieve a hydrochloric acid concentration of 1.8% by weight, and the mixture was heated to 50°C and held at 50°C for 2 hours. Next, the solution was cooled and neutralized, and the polyvinyl butyral resin was washed with water and dried to obtain synthetic resin X1, which is polyvinyl butyral resin.

[0144] The obtained polyvinyl butyral resin (synthetic resin X1) had an average degree of polymerization of 800, a hydroxyl group content of 20 mol%, an amine group content of 1 mol%, a degree of acetylation of 1 mol%, and a degree of acetalization (butyralization) of 78 mol%.

[0145] <Synthetic resin X2> A polyvinyl butyral resin was obtained in the same manner as synthetic resin X1, except that polyvinyl alcohol with an average degree of polymerization of 300 and a degree of saponification of 99 mol% was used instead of amine-modified polyvinyl alcohol. 20 parts by weight of the obtained polyvinyl butyral resin was dissolved in 80 parts by weight of tetrahydrofuran (THF), and 2 parts by weight of dimethylaminoacrylamide was added. Next, 0.1 parts by weight of Irgacure 184 (manufactured by IGM Resins BV) was dissolved as an initiator, and the mixture was irradiated with UV light for 20 minutes in a UV polymerization tester to obtain synthetic resin X2 having the composition shown in Table 1.

[0146] <Synthetic resin X3> Except for changing the amount of dimethylaminoacrylamide added from 2 parts by weight to 8 parts by weight, a polyvinyl butyral resin (synthetic resin X3) having the configuration shown in Table 1 was obtained in the same manner as synthetic resin X2.

[0147] <Synthetic resin X4> Except for using polyvinyl alcohol with an average degree of polymerization of 250 and a degree of saponification of 99 mol%, and using 10 parts by weight of diethylaminoacrylamide instead of dimethylaminoacrylamide, a polyvinyl butyral resin (synthetic resin X4) having the composition shown in Table 1 was obtained in the same manner as synthetic resin X2.

[0148] <Synthetic resin X5> Except for using 5 parts by weight of vinylimidazole instead of diethylaminoacrylamide and setting its content to 13 mol%, a polyvinyl butyral resin (synthetic resin X5) having the composition shown in Table 1 was obtained in the same manner as synthetic resin X4.

[0149] <Synthetic resin X6> A polyvinyl butyral resin having the configuration shown in Table 1 was used.

[0150] <Synthetic resin X7> A polyvinyl acetal resin (polyvinyl butyral resin) was obtained in the same manner as synthetic resin X1, except that polyvinyl alcohol with an average degree of polymerization of 250 and a degree of saponification of 98 mol% was used instead of amine-modified polyvinyl alcohol.

[0151] Linker installation: The obtained polyvinyl acetal resin (90 parts by weight) and acrylic acid (linker) (15 parts by weight) were dissolved in 300 parts by weight of THF, and the mixture was reacted for 20 minutes under ultraviolet irradiation in the presence of a photoradical polymerization initiator to introduce the linker by graft copolymerization of the polyvinyl acetal resin and acrylic acid.

[0152] Formation of the peptide portion: A linear peptide having the amino acid sequence Gly-Arg-Gly-Asp-Ser (5 amino acid residues, indicated as GRGDS in the table) was prepared as the peptide. 10 parts by weight of this peptide and 10 parts by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (condensing agent) were added to 80 parts by weight of methanol that does not contain either calcium or magnesium to prepare a peptide-containing solution. 100 parts by weight of this peptide-containing solution and 80 parts by weight of polyvinyl acetal resin with a linker introduced were added to 300 parts by weight of methanol and reacted to dehydrate condensation of the carboxyl group of the linker and the amino group of the peptide. After the reaction was complete, vacuum drying was performed at 60°C for 1 hour to volatilize the methanol. In this way, a peptide-containing polyvinyl acetal resin (synthetic resin X7) having a polyvinyl acetal resin portion, a linker portion, and a peptide portion was prepared.

[0153] <Synthetic resin X8> A polyvinyl butyral resin with a linker introduced was obtained in the same manner as with synthetic resin X7. The following peptide moieties were formed using this resin.

[0154] Formation of the peptide portion: A cyclic peptide having the amino acid sequence Arg-Gly-Asp-Phe-Lys (5 amino acid residues, with Arg and Lys forming a cyclic skeleton, Phe being the D-isomer, indicated as c-RGDfK in the table) was prepared. Ten parts by weight of this peptide and ten parts by weight of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (condensing agent) were added to 80 parts by weight of methanol that does not contain either calcium or magnesium to prepare a peptide-containing solution. 100 parts by weight of this peptide-containing solution and 80 parts by weight of polyvinyl acetal resin with a linker introduced were added to 300 parts by weight of methanol and reacted to dehydrate condensation of the carboxyl group of the linker and the amino group of the peptide. After the reaction was complete, vacuum drying was performed at 60°C for 1 hour to volatilize the methanol. In this way, a peptide-containing polyvinyl acetal resin (synthetic resin X8) having a polyvinyl acetal resin portion, a linker portion, and a cyclic peptide portion was prepared.

[0155] <Synthetic resin X9> 23.4 parts by weight of butyl methacrylate and 76.6 parts by weight of methoxymethyl acrylate were mixed to obtain a (meth)acrylic monomer solution. Next, 0.5 parts by weight of Irgacure 184 (manufactured by BASF) was dissolved in the obtained (meth)acrylic monomer solution and coated onto a PET film. The coated material was exposed to light at a wavelength of 365 nm with an integrated light intensity of 2000 mJ / cm² using an I-Graphics UV conveyor system "ECS301G1" at 25°C. 2 By irradiating with [a specific method], a poly(meth)acrylic acid ester resin (synthetic resin X9) was obtained.

[0156] (Examples 1-13 and Comparative Examples 1-2) In Examples 1, 2, 8-10 and Comparative Example 2, synthetic resin X1 was used. In Examples 3-7, synthetic resins X2-X6 were used, respectively. In Examples 11-13, synthetic resins X7-9 were used, respectively.

[0157] Preparation of cell culture vessels; In Examples 1-7 and 13, each synthetic resin was dissolved in butanol at a concentration of 5 wt%. The resulting solution was then patterned in a dot or line pattern on a 35 mm diameter polystyrene dish using a dispenser, and then dried in a 45°C oven for 6 hours to create a cell culture container with a bottom composed of a cell culture scaffold material having multiple protrusions. As shown in Figure 4, a dot pattern was created in the cell culture scaffold material prepared in Example 1. Similarly, a dot pattern was prepared in Examples 3-7. Furthermore, as shown in Figure 5, a spiral pattern was created as a line pattern in the cell culture scaffold material prepared in Example 2.

[0158] In Examples 8-12, each synthetic resin was dissolved in butanol at a concentration of 0.5 wt%. The resulting solution was then patterned in a dot pattern on a 35 mm diameter polystyrene dish using a dispenser, left to stand at room temperature for 30 minutes, and dried in a 45°C oven for 6 hours to produce a cell culture container with a bottom composed of a cell culture scaffold material having multiple protrusions. In Examples 8-12, protrusions with walls at the periphery were obtained.

[0159] In Comparative Example 1, the polystyrene dish itself was used as a cell culture container.

[0160] In Comparative Example 2, synthetic resin X1 was dissolved in butanol at a concentration of 5 wt%. The resulting 40 μL was cast onto a polystyrene dish and dried in an oven at 45°C for 6 hours to form a flat film.

[0161] (evaluation) (1) Pattern shape; Image observation was performed using a hybrid laser microscope OPTELICS HYBRID (Lasertec Corporation, objective lens for measuring the average height of convex parts: CFI T Plan EPI SLWD 20x, measurement mode: surface shape, resolution: 0.45 μm), and the following measurements were taken.

[0162] If it is dot-shaped, the average base area of ​​the convex parts If it is linear, the average line width of the convex part In a plan view, the average height of the convex portion located at the center of the convex portion. Average height of the wall

[0163] The average base area of ​​the convex portion is the average value of the base areas of any 10 convex portions. The average line width of the convex portion is the average value of the line widths at any 10 locations separated by 10 μm or more. The average height of the portion of the convex portion located in the center in a plan view is the average value of the heights of the convex portion at the center of any 10 convex portions in a plan view if the convex portion is dot-shaped, or the average value of the heights of the convex portion at the center of 10 line widths separated by 10 μm or more if the convex portion is line-shaped. The average height of the wall portion is the average value of the wall portion heights of any 10 convex portions.

[0164] (2) Ratio of protein adsorption amount; For each cell culture vessel, 40 μL of FITC-labeled bovine serum albumin (Cosmo Bio Co., Ltd.) at 0.1 mg / mL was cast and allowed to stand at 37°C for 1 hour. The polystyrene dishes were then washed with pure water and dried in a 45°C oven for 1 hour. In addition, 1 μL of FITC-labeled bovine serum albumin at concentrations of 0.005 mg / mL, 0.02 mg / mL, and 0.05 mg / mL was dispensed into polystyrene dishes for calibration of adsorbed protein, and dried in a 45°C oven for 1 hour. Each cell culture vessel was photographed using a fluorescence microscope, and the fluorescence intensity of the convex and non-convex areas was determined. The amount of adsorbed protein was converted from the linear approximation of the fluorescence intensity obtained using the calibration polystyrene dishes. From the amount of protein adsorbed on the protruding portion and the amount of protein adsorbed on the portion without the protruding portion, the ratio of the amount of protein adsorbed on the protruding portion to the amount of protein adsorbed on the portion without the protruding portion (protruding portion / portion without protruding portion) was calculated.

[0165] Water swelling rate of the convex portion; Each cell culture vessel was immersed in 25°C water for 24 hours. After immersion, the cell culture vessels were placed with the opening facing downwards on a Kimwipe for 10 minutes to remove any water adhering to them. Next, the weight of the sample before immersion was calculated by subtracting the weight of the polystyrene dish from the weight of the cell culture vessel before immersion. The weight of the sample after immersion was calculated by subtracting the weight of the polystyrene dish from the weight of the cell culture vessel after immersion. The water swelling rate was calculated from the weight of the sample before immersion and the weight of the sample after immersion as follows: Water swelling rate = (weight of sample after immersion - weight of sample before immersion) / (weight of sample before immersion) × 100 (%). Note that the weight of the polystyrene dish itself did not change before and after immersion.

[0166] (3) The elastic modulus of the convex portion at the central position of the convex portion in a plan view; The surface modulus at 25°C was determined at the center of the upper surface of the protrusions of each cell culture scaffold material using a nanoindenter (Hysitron, Triboindenter, Bruker). A Berkovich (triangular pyramidal) indenter with a tip diameter of R 100 nm was used, and single-indentation measurements were performed under air conditions at 25°C. The indentation depth was 50 nm. For Comparative Example 2, the surface modulus of a flat film was measured.

[0167] (4) Seeding and culturing of cells; The following liquid culture media and ROCK (Rho-binding kinase) specific inhibitors were prepared.

[0168] TeSR E8 medium (manufactured by STEM CELL) ROCK-Inhibitor (Y27632)

[0169] One mL of phosphate-buffered saline was added to the resulting cell culture vessel and left to stand in a 37°C incubator for one hour. After that, the phosphate-buffered saline was removed from the cell culture vessel.

[0170] Colonies of confluent h-iPS cells 253G1 were placed in a φ35 mm dish, 1 mL of 0.5 mM ethylenediamine / phosphate buffer solution was added, and the dish was allowed to stand at room temperature for 2 minutes. After removing the ethylenediamine / phosphate buffer solution, cell aggregates of 50 μm to 200 μm were obtained by pipetting with 1 mL of TeSR E8 medium. The obtained cell aggregates (cell count 0.2 × 10⁶) 5 The cells were clamp-seed into the cell culture vessels described above.

[0171] At the time of seeding, 1.5 mL of liquid medium and a ROCK-specific inhibitor to a final concentration of 10 μM were added to the cell culture vessel, and the cells were cultured in an incubator at 37°C with a CO2 concentration of 5%. Subsequently, the liquid in the cell culture vessel was replaced with 1.5 mL of fresh liquid medium every 24 hours, and this process was repeated for 5 days. During this time, any cells that had detached from the scaffold or were suspended were collected and discarded by pipetting.

[0172] After cell culture, the cell culture vessels were observed using a hybrid microscope to evaluate the uniformity of the cell aggregates formed on the scaffold material. Cell adhesion was also evaluated by observing the presence or absence of cell aggregate adhesion using a phase-contrast microscope.

[0173] Figure 6 shows a micrograph of cells seeded in the cell culture vessel prepared in Example 1. Figure 7 shows a micrograph of cells cultured for 5 days in the cell culture vessel prepared in Example 1.

[0174] As is clear from Figure 7, in Example 1, cell aggregates corresponding to the scaffold pattern can be formed easily and efficiently. Similarly, observations were made in Examples 2 to 13, and it was confirmed that cell aggregates corresponding to the scaffold pattern can be formed easily and efficiently.

[0175] Furthermore, observations were made regarding Examples 1-13 and Comparative Examples 1-2, and they were evaluated according to the following evaluation criteria.

[0176] [Evaluation Criteria] (Uniformity of cell aggregates) After culturing for 5 days, the cells in the wells were observed using a phase-contrast microscope, and 10 arbitrary cell clusters were selected. The area of ​​the cell clusters when viewed from above under the microscope was calculated, and the degree of dispersion of the cell cluster area was calculated using the following formula (A).

[0177]

number

[0178] D: Dispersion of the surface area of ​​the cell aggregate n: Number of data points Xi: Planar area of ​​cell mass X: Average surface area of ​​cell masses

[0179] [Criteria for determining the uniformity of cell aggregates] A...Spread is less than 0.001 B...Variance of 0.001 or greater, and less than 0.002. C...Variance is 0.002 or higher, but less than 0.003. D...Spread of 0.003 or higher

[0180] (Adhesion of cell aggregates) After 5 days of culture, the cell aggregates in the wells were observed using a phase-contrast microscope, and the adhesion of the formed cell aggregates was evaluated according to the following criteria.

[0181] [Criteria for determining the adhesion of cell aggregates] A... All cell clusters are adhered together. B... Some cell clumps have detached. C...All cell clusters have detached.

[0182] The results are shown in Tables 1 and 2 below.

[0183] [Table 1]

[0184] [Table 2] [Explanation of symbols]

[0185] 1…Scaffold material for cell culture 2,2A…Base material 3,3A…Convex part 4…Parts where no protrusions are provided 11...Cell culture container 12…Container body 12a…Surface 31A…Wall part Ha...Height of the convex at the central position of the convex in a plan view. Hb...Height of the wall Tb...Thickness of the wall

Claims

1. Substrate and The substrate comprises a protrusion formed by patterning in a dot-like or line-like manner, The material of the base material is a resin, metal, or inorganic material. The aforementioned resin is polystyrene, polyethylene, polypropylene, polycarbonate, polyester, polyisoprene, cycloolefin polymer, polyimide, polyamide, polyamideimide, (meth)acrylic resin, epoxy resin, or silicone. The aforementioned protrusion contains synthetic resin, The synthetic resin includes a synthetic resin having a polyvinyl acetal skeleton, and the synthetic resin having a polyvinyl acetal skeleton has structural units having an imine structure, structural units having an imide structure, structural units having an amine structure, or structural units having an amide structure. The aforementioned protrusions have cell adhesion properties, The aforementioned protrusion is a cell culture scaffold material in which the average height of the portion located in the center of the protrusion in a plan view is 50 nm or more and 1 μm or less.

2. The cell culture scaffold material according to claim 1, wherein the ratio of the amount of protein adsorbed by the protrusions to the portion without the protrusions (protrusions / portion without protrusions) is 2 or more.

3. The aforementioned protrusions are formed by patterning the substrate in a dot-like manner. The aforementioned protrusion has an average base area of ​​0.005 mm². ２ Above 5 mm ２ The cell culture scaffold material according to claim 1 or 2, which is as follows:

4. The cell culture scaffold material according to any one of claims 1 to 3, wherein the convex portion has a wall portion at its peripheral edge.

5. A cell culture scaffold material according to any one of claims 1 to 4, wherein the elastic modulus of the protrusion at 25°C at the central position of the protrusion in a plan view, as measured by nanoindentation, is 1 GPa or more.

6. The aforementioned synthetic resin contains 0.2 mol% to 20 mol% of Brønsted basic groups in its constituent units, the cell culture scaffold material according to any one of claims 1 to 5.

7. The cell culture scaffold material according to any one of claims 1 to 6, wherein the water swelling rate of the convex portion is 50% or less.

8. The cell culture scaffold material according to any one of claims 1 to 7, wherein the protrusion has a peptide portion on its surface.

9. A cell culture container comprising a cell culture scaffold material according to any one of claims 1 to 8 in at least a portion of the cell culture area.

Images