Scaffold material for cell culture and cell culture container

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 scaffold for cell culture used for culturing cells. The present invention also relates to a cell culture vessel using the scaffold for cell culture. [Background technology]

[0002] In recent years, cell medicine and next-generation medical treatments using stem cells have been attracting attention. In particular, human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), as well as differentiated cells derived from them, are expected to be applied to drug discovery and regenerative medicine. To achieve such applications, it is necessary to safely and reproducibly culture and proliferate pluripotent stem cells and differentiated cells.

[0003] These stem cells can form cell masses called spheroids by aggregation and adhesion of the stem cell population. Spheroids are thought to be able to be cultured and maintained in a state closer to the three-dimensional structure in vivo, and are known to show superior characteristics compared to conventional flat adherent cultures. In fact, spheroids are used for anticancer drug screening using cancer cells.

[0004] For example, Patent Document 1 listed below discloses a cell culture substrate having multiple microwells formed on one culture surface, which is capable of suppressing the migration of spheroids from the microwells and forming spheroids of uniform size without making the microwells too deep. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2018 / 123663 Summary of the Invention [Problem to be solved by the invention]

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

[0007] An object of the present invention is to provide a scaffold for cell culture and a vessel for cell culture which enable easy and efficient formation of cell masses. [Means for solving the problem]

[0008] The scaffold material for cell culture according to the present invention comprises a base material and convex portions formed by patterning the base material in a dot or line shape, the convex portions containing a synthetic resin, the synthetic resin containing at least a polyvinyl alcohol derivative or a poly(meth)acrylic resin, the convex portions having cell adhesive properties, and the average height of a portion of the convex portions located at the center of the convex portions in a planar view is 10 nm or more and 10 μm or less.

[0009] In a specific aspect of the cell culture scaffold material of the present invention, the ratio of the protein adsorption amount of the convex portions to the portion where the convex portions are not provided (convex portions / portions where the convex portions are not provided) is 2 or more.

[0010] In another specific aspect of the cell culture scaffold according to the present invention, the protrusions are formed by patterning the substrate in a dot shape, and the protrusions have an average bottom area of ​​0.005 mm 2 More than 5mm 2 The following is the result.

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

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

[0013] In a further specific aspect of the scaffold 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 cell culture scaffold according to the present invention, the water swelling rate of the protrusions is 50% or less.

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

[0016] The cell culture vessel according to the present invention includes a cell culture scaffold material constructed according to the present invention in at least a part of a cell culture region. Effect of the Invention

[0017] According to the present invention, it is possible to provide a scaffold for cell culture and a vessel for cell culture, which are capable of easily and efficiently forming cell masses. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 is a schematic plan view showing a scaffold for cell culture according to one embodiment of the present invention. [Diagram 2] FIG. 2(a) is a schematic plan view showing an enlarged view of the periphery of a convex portion having a wall portion on its peripheral portion, and FIG. 2(b) is a schematic front cross-sectional view showing an enlarged view of the periphery of a convex portion having a wall portion on its peripheral portion. [Diagram 3] FIG. 3 is a schematic front cross-sectional view showing a cell culture vessel according to one embodiment of the present invention. [Figure 4] FIG. 4 is a photograph showing the pattern of protrusions in the cell culture scaffold prepared in Example 1. [Diagram 5]FIG. 5 is a photograph showing the pattern of protrusions in the cell culture scaffold prepared in Example 2. [Figure 6] FIG. 6 is a micrograph of cells seeded in the cell culture vessel prepared in Example 1. As shown in FIG. [Figure 7] FIG. 7 is a micrograph of cells cultured in the cell culture vessel prepared in Example 1 for 5 days. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention will be clarified below by describing specific embodiments of the present invention with reference to the drawings.

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

[0021] Since the scaffold for cell culture of the present invention has the above-mentioned configuration, it is possible to easily and efficiently form cell aggregates.

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

[0023] The inventors focused on protrusions having cell adhesive properties, and found that cell masses can be formed easily and efficiently, particularly by making the average height of the protrusions at specific positions 10 nm or more and 10 μm or less.

[0024] Specifically, in the cell culture scaffold of the present invention, the seeded cells gather on the convex portions to form cell clusters. At this time, the cell clusters can be formed along the shape of the convex portions. Therefore, by controlling the size and shape of the convex portions, the shape and size of the cell clusters can be easily controlled.

[0025] Furthermore, in the cell culture scaffold of the present invention, cells can be seeded both on the convex portions and on the portions where no convex portions are provided. In particular, for cells seeded on the portions where no convex portions are provided, only cells with good migration properties can be gathered on the convex portions. Therefore, only cells with good quality can be selected, and a cell mass with good quality can be obtained efficiently. Furthermore, since it is not necessary to selectively seed cells only on the convex portions, a cell mass can be formed easily and efficiently.

[0026] Therefore, according to the cell culture scaffold of the present invention, good quality cell aggregates can be formed easily and efficiently, and the shape and size of the cell aggregates can be easily controlled.

[0027] Furthermore, since the present invention can use synthetic resins, it is easier to handle, less expensive, has less variation between lots, and is safer than scaffold materials using natural polymeric materials.

[0028] Hereinafter, with reference to FIG. 1, a schematic plan view will be described as an example of a scaffold for cell culture having protrusions configured by dot patterning.

[0029] A cell culture scaffold 1 shown in Fig. 1 includes a base material 2 and a plurality of protrusions 3 provided on the base material 2. The plurality of protrusions 3 contain a synthetic resin and have cell adhesive properties. In addition, a portion 4 where no protrusions are provided is also provided on the base material 2. Note that, in this embodiment, the number of cells that adhere per unit area to the plurality of protrusions 3 is greater than that to the portion 4 where no protrusions are provided.

[0030] In the cell culture scaffold 1, the protrusions 3 are configured by patterning in a dot shape. The planar shape of the protrusions 3 is substantially circular. The average height of the portion located at the center of the protrusions 3 in plan view is 10 nm or more and 10 μm or less.

[0031] Therefore, with the cell culture scaffold 1, good quality cell clusters can be formed easily and efficiently, and the shape and size of the cell clusters can be easily controlled.

[0032] In the above embodiment, the planar shape of the protrusions is substantially circular, but the planar shape of the protrusions may be substantially rectangular or triangular, and is not particularly limited. In addition, some or all of the multiple protrusions may be formed by adjacent dots connected together.

[0033] The scaffold for cell culture of the present invention will be described in further detail below.

[0034] [Convex] The scaffold for cell culture of the present invention comprises a convex portion arranged on a substrate. The scaffold for cell culture of the present invention comprises a substrate and a dot-shaped convex portion or a line-shaped convex portion provided on the substrate. The convex portion is configured by patterning the substrate in a dot-shaped or line-shaped form. By arranging the convex portions on the substrate in a dot-shaped or line-shaped form, it is possible to more easily and efficiently form a cell aggregate having good quality, and furthermore, it is possible to more easily control the shape and size of the cell aggregate. The convex portion may be configured by patterning the substrate in a dot-shaped form, may be configured by patterning the substrate in a line-shaped form, or may be configured by patterning the substrate in a dot-shaped and line-shaped form.

[0035] From the viewpoint of easier control of the shape and size of the cell aggregate, the convex portions are preferably configured by patterning in a dot shape. The above-mentioned cell culture scaffold material preferably includes at least the convex portions configured by patterning in a dot shape.

[0036] The number of the convex parts may be one or more. The number of the convex parts may be, for example, 1 to 1500. When the convex parts are configured by patterning in a dot shape, the number of the convex parts 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 the convex parts is equal to or more than the lower limit and equal to or less than the upper limit, a cell aggregate having good quality can be formed more easily and efficiently, and the shape and size of the cell aggregate can be controlled more easily. When the convex parts are configured by patterning in a line shape, the number of the convex parts may be 1 or more.

[0037] The convex portion has an average height of 10 nm or more and 10 μm or less at the center of the convex portion in plan view. The convex portion has 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 at the center of the convex portion in plan view. When the average height is within the above range, it is possible to more easily and efficiently form high-quality cell clusters, and moreover, it is possible to more easily control the shape and size of the cell clusters.

[0038] The height of the convex part located at the center of the convex part in plan view can be measured by a hybrid laser microscope or the like. The average height of the convex part located at the center of the convex part in plan view is the average value of the height of the convex part at the center position of the convex part in plan view. The average height of the convex part located at the center of the convex part in plan view is the average height from the surface (upper surface) of the substrate to the center position of the convex part in plan view. When the convex part is dot-shaped, the average height is the average height at the center position of the planar shape of the dot-shaped convex part when viewed in plan. When the planar shape of the dot-shaped convex part when viewed in plan is a circle or an ellipse, the center position of the convex part in plan view means the center of the circle or the ellipse. When the planar shape of the dot-shaped convex part when viewed in plan is a polygonal shape or the like, the center position of the convex part in plan view means the center of the polygon or the like. When the convex part is line-shaped, the average height is the average height at the center position of the line width of the line-shaped convex part. In addition, when the convex portion is a convex portion formed by patterning in a dot shape and the number of the convex portion is one, the above average height means the height at the center position of the convex portion in a planar view. When the convex portion is a convex portion formed by patterning in a dot shape and the number of the convex portions is less than 10, the above average height means the average value of the heights at the center positions of these convex portions in a planar view. When the convex portion is a convex portion formed by patterning in a dot shape and the number of the convex portions is 10 or more, the above average height means the average value of the heights at the center positions of any 10 convex portions in a planar view. In addition, when the convex portion is a convex portion formed by patterning in a line shape, the above average height means the average value of the heights at the center positions of any 10 line widths that are 10 μm or more apart from each other. In addition, the center position does not have to be exactly the center position, and may be away from the center position within the range of measurement error, or may be approximately the center position.

[0039] When the convex portions are formed by patterning in a dot shape, the average bottom area of ​​the convex portions is preferably 0.005 mm 2 More than 0.02mm, preferably 0.02mm2 More preferably, 0.1 mm 2 More than 5 mm, preferably 2 Less than 3mm, more preferably 2 Less than 1.5 mm, more preferably 2 In this case, it is possible to more easily and efficiently form cell clusters with good quality, and it is also possible to more easily control the shape and size of the cell clusters.

[0040] The average base area of ​​the protrusions means the base area of ​​the protrusion when there is one protrusion. When there are less than 10 protrusions, the average base area of ​​the protrusions means the average value of the base areas of these protrusions. When there are 10 or more protrusions, the average base area of ​​the protrusions means the average value of the base areas of any 10 protrusions.

[0041] The convex portion may be configured by being patterned into a line shape. In this case, the convex portion may be configured to have a straight line shape, a broken line shape, or a curved line shape, or may be configured to have a circular shape or a spiral shape.

[0042] When the convex portions are configured by line patterning, the average line width of the convex portions is preferably 50 μm or more, more preferably 100 μm or more, and preferably 1.5 mm or less, more preferably 0.5 mm or less. In this case, cell clusters of good quality can be formed more easily and efficiently, and the shape and size of the cell clusters can be controlled more easily.

[0043] The convex portions have cell adhesiveness. In the present invention, the term "convex portions having cell adhesiveness" means that a greater number of cells adhere to the convex portions per unit area compared to a portion where the convex portions are not provided. Therefore, it is preferable that the convex portions have higher cell adhesiveness than the substrate.

[0044] In the present invention, the ratio of the protein adsorption amount of the convex parts to the parts where the convex parts are not provided (convex parts / parts where the convex parts are not provided) is preferably 1.5 or more, more preferably 2 or more, and preferably 15 or less, more preferably 10 or less. In this case, the cell adhesiveness of the convex parts can be further increased, and cell aggregates can be formed more easily and efficiently.

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

[0046] 40μL of 0.1mg / mL FITC-labeled bovine serum albumin (Cosmo Bio) was cast onto the cell culture scaffold and left to stand at 37℃ for 1 hour, then the cell culture scaffold was washed with pure water and dried in an oven at 45℃ for 1 hour. In addition, 1μL of 0.005mg / mL, 0.02mg / mL, and 0.05mg / mL FITC-labeled bovine serum albumin was dispensed onto the polystyrene substrate for calibration of the amount of adsorbed protein, and dried in an oven at 45℃ for 1 hour. The cell culture scaffold was photographed using a fluorescence microscope, and the fluorescence intensity of the convex part and the part without the convex part were measured. The linear approximation of the fluorescence intensity obtained using the polystyrene substrate for the calibration curve was converted into the amount of protein adsorption.

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

[0048] In the present invention, the elastic modulus of the convex portion at the center of the convex portion in a planar view measured by nanoindentation at 25° C. is preferably 1 GPa or more, more preferably 2 GPa or more, and preferably 8 GPa or less, more preferably 6 GPa or less. When the elastic modulus of the convex portion is within the above range, the cell adhesiveness of the convex portion can be further increased, and cell masses can be formed more easily and efficiently.

[0049] The elastic modulus (surface elastic modulus) can be measured, for example, using a nanoindenter (Hysitron, Triboindenter, manufactured by Bruker). A Berkovich (triangular pyramid) indenter with a tip diameter R of 100 nm is used as the indenter, and a single indentation measurement can be performed at 25° C. under atmospheric pressure. The indentation depth can be 50 nm. The central position does not have to be exactly the central position, and may be away from the central position within the range of measurement error, or may be approximately the central position.

[0050] The surface elastic modulus can be increased, for example, by increasing the molecular weight of the synthetic resin contained in the convex portions, increasing the degree of cross-linking of the synthetic resin contained in the convex portions, introducing a crystalline molecular structure into the synthetic resin contained in the convex portions, or the like.

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

[0052] A substrate and a scaffold for cell culture are prepared and their weights are measured. The weight of the substrate is subtracted from the weight of the scaffold for cell culture to obtain the "sample weight before immersion". The substrate and the scaffold for cell culture are then immersed in water at 25°C for 24 hours. After immersion, the water adhering to the substrate and the scaffold for cell culture are removed and their weights are measured. The weight of the substrate after immersion is subtracted from the weight of the scaffold for cell culture after immersion to obtain the "sample weight after immersion". The water swelling rate is calculated as (sample weight after immersion - sample weight before immersion) / (sample weight before immersion) x 100 (%).

[0053] The water swelling rate can be reduced, for example, by increasing the hydrophobic functional groups of the synthetic resin contained in the convex portions, decreasing the number average molecular weight, or the like.

[0054] It is preferable that the convex portion has a wall portion at the periphery. In this case, it is possible to more easily and efficiently form a cell mass of good quality, and moreover, it is possible to more easily control the shape and size of the cell mass. The wall portion is a part of the convex portion. Therefore, when the convex portion has a wall portion, the base area, line width, etc. of the convex portion described above are the base area, line width, etc. of the convex portion including the wall portion.

[0055] Fig. 2(a) is a schematic plan view showing an enlarged view of the periphery of a protrusion having a wall on the periphery, and Fig. 2(b) is a schematic front cross-sectional view showing an enlarged view of the periphery of a protrusion having a wall on the periphery. Fig. 2(b) is a cross-sectional view taken along line II in Fig. 2(a). Fig. 2 shows an enlarged view of one protrusion.

[0056] In FIG. 2, the protrusion 3A is disposed on the surface of the substrate 2A. The protrusion 3A has a wall 31A at its periphery. The protrusion 3A has a wall 31A and a portion that does not have the wall 31A. The protrusion 3A is circular in plan view. The central position of the protrusion 3A in plan view is the center of the circle. The upper surface of the wall 31A and the upper surface of the portion that does not have the wall 31A form the upper surface of the protrusion 3A. The height Hb of the wall 31A is higher than the height of the portion that does not have the wall 31A. The height Hb of the wall 31A is higher than the height Ha of the protrusion 3A at the central position of the protrusion 3A in plan view. The height Hb of the wall is the height of the protrusion at the central position in the width direction of the wall in plan view, and is the height of the protrusion at the central position in the thickness direction of the wall. The wall height Hb is the distance from the surface (top surface) of the base material 2A to the center position in the width direction of the top surface of the wall 31A. The protrusion 3A has a wall 31A with a thickness Tb. The wall thickness Tb corresponds to the width of the wall in a plan view. The wall 31A and the portion not having the wall 31A are formed of the same material. The portion not having the wall 31A is surrounded by the wall 31A.

[0057] The average height of the wall is preferably 100 nm or more, more preferably 300 nm or more, and preferably 50 μm or less, more preferably 20 μm or less. When the average height of the wall is equal to or more than the above lower limit and equal to or less than the above upper limit, cell clusters of good quality can be formed more easily and efficiently, and the shape and size of the cell clusters can be controlled more easily.

[0058] The average thickness of the wall is preferably 50 nm or more, more preferably 100 nm or more, and preferably 5 μm or less, more preferably 1 μm or less. When the average thickness of the wall is equal to or more than the above lower limit and equal to or less than the above upper limit, cell clusters of good quality can be formed more easily and efficiently, and the shape and size of the cell clusters can be controlled more easily.

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

[0060] The average wall height and the average wall thickness respectively refer to the wall height and wall thickness of the protrusion when the number of protrusions having a wall is 1. When the number of protrusions having a wall is less than 10, the average wall height and the average wall thickness respectively refer to the average values ​​of the wall height and wall thickness of these protrusions. When the number of protrusions having a wall is 10 or more, the average wall height and the average wall thickness respectively refer to the average values ​​of the wall height and wall thickness of any 10 protrusions.

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

[0062] A simple method for manufacturing a protrusion having a wall can be, for example, the following method. 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 the minute droplets of the solution containing a low concentration of synthetic resin are gradually dried, a protrusion having a wall on the periphery is formed on the substrate.

[0063] (Synthetic resin) The convex portions of the cell culture scaffold of the present invention contain a synthetic resin (hereinafter, may be referred to as synthetic resin X). Synthetic resin X is the synthetic resin contained in the convex portions. In this specification, the term "structural unit" refers to a repeating unit of a monomer constituting the synthetic resin. In addition, when the synthetic resin has a graft chain, it includes a repeating unit of a monomer constituting the graft chain.

[0064] The synthetic resin X preferably contains 0.2 mol% or more of the Bronsted basic group in the structural unit, 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 effect of the present invention can be more effectively exhibited. The Bronsted basic group and the like will be described later.

[0065] The synthetic resin X contains at least a polyvinyl alcohol derivative or a poly(meth)acrylic resin. By containing a polyvinyl alcohol derivative or a poly(meth)acrylic resin, the adhesiveness to cells can be increased, and the swelling of the scaffold for cell culture in a liquid medium can be further suppressed. The synthetic resin X may contain a polyvinyl alcohol derivative, may contain a poly(meth)acrylic resin, or may contain both a polyvinyl alcohol derivative and a poly(meth)acrylic resin. The above-mentioned polyvinyl alcohol derivative and poly(meth)acrylic resin may each be used alone or in combination of two or more kinds.

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

[0067] <Synthetic resin having a polyvinyl acetal skeleton> The protrusions of the cell culture scaffold 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, the "synthetic resin having a polyvinyl acetal skeleton" may be referred to as "polyvinyl acetal resin X."

[0069] The polyvinyl acetal resin X preferably has an acetal group, an acetyl group, and a hydroxyl group in the side chain. However, the polyvinyl acetal resin X may not have an acetyl group, for example. For example, the polyvinyl acetal resin X may not have an acetyl group by bonding all of the acetyl groups of the polyvinyl acetal resin X to a linker described later.

[0070] The synthesis of the 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 the polyvinyl acetal resin X is not particularly limited. The aldehyde may be, for example, an aldehyde having 1 to 10 carbon atoms. The aldehyde may have a chain aliphatic group, a cyclic aliphatic group, or an aromatic group. The aldehyde may be a chain aldehyde or a cyclic aldehyde.

[0072] Examples of the aldehyde 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, formylthiane, formyladenine, formylguanine, formylcytosine, formylthymine, and formyluracil. The aldehydes may be used alone or in combination of two or more.

[0073] The aldehyde is preferably formaldehyde, acetaldehyde, propionaldehyde, butylaldehyde, or pentanal, and more preferably butylaldehyde. 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] A vinyl compound may be copolymerized in the polyvinyl acetal resin X. That is, the polyvinyl acetal resin X may be a copolymer of a structural unit of the polyvinyl acetal resin and a vinyl compound. In the present invention, the polyvinyl acetal resin copolymerized with a vinyl compound is also referred to as the polyvinyl acetal resin.

[0075] The vinyl compound is a compound having a vinyl group (H2C=CH-). The vinyl compound may be a polymer having a structural unit having a vinyl group.

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

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

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

[0079] From the viewpoint of further enhancing the adhesiveness of cells, the polyvinyl acetal resin X preferably has a Brønsted basic group or a Brønsted acidic group, more preferably has a Brønsted basic group. That is, it is preferable that a part of the polyvinyl acetal resin X is modified with a Brønsted basic group or a Brønsted acidic group, and more preferably that a part of the polyvinyl acetal resin X is modified with a Brønsted basic group.

[0080] The polyvinyl acetal resin X preferably has a Brønsted basic group or a Brønsted acidic group in a part thereof, and more preferably has a Brønsted basic group. In that case, the adhesiveness of cells can be further improved. In that case, a monomer having a Brønsted basic group or a Brønsted acidic group may be copolymerized or graft copolymerized. The degree of modification in 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 by the Brønsted basic group is within the above specific range, the adhesiveness of cells can be further improved.

[0081] The Bronsted basic group is a general term for a functional group that can receive a hydrogen ion H+ from another substance. Examples of the Bronsted basic group include amine-based basic groups such as a substituent having an imine structure, a substituent having an imide structure, a substituent having an amine structure, and a substituent having an amide structure. Examples of the Bronsted basic group include, but are not limited to, conjugated amine functional groups such as hydroxyamino group, urea group, guanidine, and biguanide; heterocyclic amino functional groups such as 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, and melamine; cyclic pyrrole functional groups such as porphyrin, chlorine, and choline, and derivatives thereof.

[0082] Examples of the Bronsted acid group include a carboxyl group, a sulfonic acid group, a maleic acid group, a sulfinic acid group, a sulfenic acid group, a phosphoric acid group, a phosphonic acid group, or salts thereof. The Bronsted acid group is preferably a carboxyl group.

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

[0084] The polyvinyl acetal resin X may have a structural unit having an imine structure. The imine structure refers to a structure having a C=N bond. In particular, the polyvinyl acetal resin X preferably has an imine structure in a side chain.

[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 a 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 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, which refers to a structure having -C(=O)-NH-.

[0089] The polyvinyl acetal resin X preferably has an amine structure or an amide structure in a 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] The content of the structural unit having an imine 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 are 1 It can be measured by H-NMR (nuclear magnetic resonance spectroscopy).

[0091] <Synthetic resin having poly(meth)acrylic acid ester skeleton> The convex portions of the cell culture scaffold 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, the "synthetic resin having a poly(meth)acrylic acid ester skeleton" may be referred to as "poly(meth)acrylic acid ester resin X".

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

[0094] The poly(meth)acrylic acid ester resin X can be obtained by polymerization of a (meth)acrylic acid ester or by copolymerization of a (meth)acrylic acid ester with another monomer.

[0095] Examples of the (meth)acrylic acid ester include (meth)acrylic acid alkyl esters, (meth)acrylic acid cyclic alkyl esters, (meth)acrylic acid aryl esters, (meth)acrylamides, (meth)acrylic acid polyethylene glycols, and (meth)acrylic acid phosphorylcholine.

[0096] Examples of (meth)acrylic acid alkyl esters 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] The (meth)acrylic acid alkyl ester may be substituted with a substituent such as an alkoxy group having 1 to 3 carbon atoms and a tetrahydrofurfuryl group. Examples of such (meth)acrylic acid alkyl ester include methoxyethyl acrylate and tetrahydrofurfuryl acrylate.

[0098] Examples of the (meth)acrylic acid cyclic alkyl ester include cyclohexyl (meth)acrylate and isobornyl (meth)acrylate.

[0099] Examples of the (meth)acrylic acid aryl ester 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 the (meth)acrylate phosphorylcholine include 2-(meth)acryloyloxyethyl phosphorylcholine.

[0103] As another monomer to be copolymerized with the (meth)acrylic acid ester, a vinyl compound is preferably used. Examples of the vinyl compound include ethylene, allylamine, vinylpyrrolidone, maleic anhydride, maleimide, itaconic acid, (meth)acrylic acid, vinylamine, and (meth)acrylic acid ester. Only one type of vinyl compound may be used, or two or more types 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 preferably has a Brønsted basic group or a Brønsted acidic group in part, similar to the polyvinyl acetal resin X. In that case, the adhesiveness of cells can be further improved. The Brønsted basic group may be part of the poly(meth)acrylic acid ester resin X, and in that case, a monomer having a Brønsted basic group may be copolymerized or graft copolymerized. The degree of modification in 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 by the Brønsted basic group is within the above specific range, the adhesiveness of cells can be further improved.

[0106] <Synthetic resin having a peptide portion> The convex portion of the cell culture scaffold of the present invention preferably contains 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 convex portion of the cell culture scaffold, a convex portion having a peptide portion on the surface can be obtained. That is, the convex portion preferably has a peptide portion on the surface. In this case, the adhesiveness with the cells after seeding can be further increased, and the cell proliferation rate can be further increased. In addition, in this case, a cell mass with good quality can be formed more easily and efficiently, and the shape and size of the cell mass can be controlled more easily.

[0107] The synthetic resin having a peptide portion can be obtained by reacting a synthetic resin, 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 synthetic resin having the 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 3 or more amino acids, more preferably 4 or more amino acids, even more preferably 5 or more amino acids, preferably 10 or less amino acids, and more preferably 6 or less amino acids. When the number of amino acids constituting the peptide portion is equal to or more than the above lower limit and equal to or less than the above upper limit, the adhesiveness to cells after seeding can be further increased, and the proliferation rate of the cells can be further increased.

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

[0110] Examples of the cell adhesive amino acid sequences include the 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). Further, examples of the cell adhesive amino acid sequence include the sequences described in "Pathophysiology, Vol. 9, No. 7, pp. 527-535, 1990" and "Osaka Prefectural Maternal and Child Medical Center Journal, Vol. 8, No. 1, pp. 58-66, 1992." The peptide portion may have only one type of cell adhesive amino acid sequence, or two or more types.

[0111] The cell adhesive amino acid sequence preferably has at least any one of the above-mentioned cell adhesive 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, the adhesiveness to cells after seeding can be further increased, 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 have a cyclic peptide backbone. The cyclic peptide backbone is a cyclic backbone composed of a plurality of amino acids. From the viewpoint of more effectively exerting the effects of the present invention, the cyclic peptide backbone is preferably composed of 4 or more amino acids, more preferably composed of 5 or more amino acids, and more preferably composed of 10 or less amino acids.

[0115] In the 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 the 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 equal to or more than the lower limit, the phase separation structure can be formed more easily. When the content of the peptide portion is equal to or more than the lower limit, the adhesiveness to the cells after seeding can be further improved, and the cell proliferation rate can be further improved. In addition, when the content of the peptide portion is equal to or less than the upper limit, the manufacturing cost can be reduced. The content of the peptide portion (mol%) is the amount of substance of the peptide portion relative to the sum of the amounts of substances of each structural unit constituting the synthetic resin having a peptide portion.

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

[0117] In the synthetic resin having a peptide portion, the synthetic resin portion and the peptide portion are preferably bonded via a linker. That is, the synthetic resin having a peptide portion is preferably a synthetic resin having a peptide portion and a linker portion. The above linkers may be used alone or in combination of two or more types.

[0118] The linker is preferably a compound having a functional group capable of condensing with the carboxyl group or amino group of the peptide. Examples of the functional group capable of condensing with the carboxyl group or amino group of the peptide include a carboxyl group, a thiol group, and an amino group. From the viewpoint of reacting well with the peptide, the linker is preferably a compound having a carboxyl group. The above-mentioned vinyl compound can also be used as the linker.

[0119] Examples of the linker having a carboxyl group include (meth)acrylic acid and acrylamide containing a carboxyl group, etc. By using a carboxylic acid (carboxylic acid monomer) having a polymerizable unsaturated group as the linker having a carboxyl group, the carboxylic acid monomer can be polymerized by graft polymerization when reacting the linker with a synthetic resin, so that the number of carboxyl groups that can be reacted with a peptide can be increased.

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

[0121] [Base material] The cell culture scaffold of the present invention includes a substrate. A part of the surface of the substrate constitutes a portion on which no protrusions are provided. Therefore, the substrate is preferably made of a material that adheres to a smaller number of cells per unit area than the protrusions.

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

[0123] [Details of other cell culture scaffolds] The convex portions of the scaffold for cell culture according to the present invention contain the synthetic resin X. From the viewpoint of effectively exerting the effects of the present invention and increasing productivity, the content of the synthetic resin X in 100% by weight of the convex portions of the scaffold for cell culture 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 convex portions of the scaffold for cell culture are the synthetic resin X. When the content of the synthetic resin X is equal to or more than the lower limit, the effects of the present invention can be exerted even more effectively.

[0124] When the synthetic resin X contained in the convex portion 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 convex portion is a polyvinyl alcohol derivative. When the content of the polyvinyl alcohol derivative is equal to or more than the lower limit, the effect of the present invention can be more effectively exhibited.

[0125] When the synthetic resin X contained in the convex portion contains a poly(meth)acrylic resin, the content of the poly(meth)acrylic resin 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, the synthetic resin X contained in the convex portion is most preferably a poly(meth)acrylic resin. When the content of the poly(meth)acrylic resin is equal to or more than the lower limit, the effect of the present invention can be more effectively exhibited.

[0126] The convex portions of the above-mentioned cell culture scaffold may contain components other than the above-mentioned synthetic resin X. Examples of the components other than the above-mentioned synthetic resin X include polysaccharides, cellulose, synthetic peptides, and the like.

[0127] From the viewpoint of effectively exerting the effects of the present invention, the content of components other than the synthetic resin X is preferably as small as possible. In 100% by weight of the convex portions of the scaffold for cell culture, the content of the components 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 convex portions of the scaffold for cell culture do not contain components other than the synthetic resin X.

[0128] The above-mentioned cell culture scaffold preferably does not contain any animal-derived raw materials. By not containing any animal-derived raw materials, it is possible to provide a cell culture scaffold that is highly safe and has little variation in quality during production. Here, "substantially free of animal-derived raw materials" means that the animal-derived raw materials in the cell culture scaffold are 3% by weight or less. The above-mentioned cell culture scaffold 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 of the present invention does not contain any animal-derived raw materials at all.

[0129] The convex portions of the scaffold for cell culture preferably contain 3% by weight or less of animal-derived raw materials, more preferably 1% by weight or less, and most preferably 0% by weight. That is, it is most preferable that the convex portions of the scaffold for cell culture do not contain any animal-derived raw materials at all.

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

[0131] The above cells include animal cells such as human, mouse, rat, pig, cow, and monkey cells. The above cells also include somatic cells, such as stem cells, progenitor cells, and mature cells. The above somatic cells may be cancer cells.

[0132] Examples of the 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 include nerve cells, cardiomyocytes, retinal cells, and hepatic cells.

[0134] [Cell culture container] The present invention also relates to a cell culture vessel comprising the above-mentioned cell culture scaffold in at least a part of a cell culture region. Fig. 3 is a schematic front cross-sectional view showing a cell culture vessel according to one embodiment of the present invention.

[0135] The cell culture vessel 11 includes a vessel body 12. The bottom of the vessel body 12 serves as the base material 2 of the cell culture scaffold 1. Accordingly, the protrusions 3 are provided on a surface 12a of the vessel body 12.

[0136] A liquid medium is added to the cell culture vessel 11, and the cells are seeded on the surface of the scaffold 1 for cell culture, whereby the cells can be cultured on a surface.

[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 a base material for the above-mentioned cell culture scaffold material.

[0138] The container body may be a conventionally known container body (container). The shape and size of the container body are not particularly limited.

[0139] The container body includes a cell culture plate and a cell culture flask having one or more wells (holes). The number of wells in the plate is not particularly limited. The number of wells is not particularly limited, but may be, for example, 2, 4, 6, 12, 24, 48, 96, 384, etc. The shape of the well is not particularly limited, but may be a perfect circle, an ellipse, a triangle, a square, a rectangle, a pentagon, etc. The shape of the bottom of the well is not particularly limited, but may be a flat bottom, a round bottom, etc.

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

[0141] The present invention will now be described in more detail with reference to specific examples and comparative examples, although 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 content of structural units in the obtained synthetic resins was measured by dissolving the synthetic resins in DMSO-D6 (dimethyl sulfoxide) and then 1 The measurements were performed using H-NMR (nuclear magnetic resonance spectroscopy).

[0143] <Synthetic resin X1> In a reactor equipped with a stirrer, 2700 mL of ion-exchanged water and 300 parts by weight of amine-modified polyvinyl alcohol with an average degree of polymerization of 800, an amine modification degree of 1 mol%, and a saponification degree of 99 mol% were added, and the mixture was heated and dissolved while stirring to obtain a solution. 35% by weight hydrochloric acid was added as a catalyst to the obtained solution so that the hydrochloric acid concentration was 0.2% by weight. Next, the temperature was adjusted to 15°C, and 22 parts by weight of n-butylaldehyde were added while stirring. Next, 148 parts by weight of n-butylaldehyde were added to precipitate a white particulate polyvinyl butyral resin. 15 minutes after the precipitation, 35% by weight hydrochloric acid was added so that the hydrochloric acid concentration was 1.8% by weight, and then 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 a synthetic resin X1, which is a polyvinyl butyral resin.

[0144] The obtained polyvinyl butyral resin (synthetic resin X1) had an average degree of polymerization of 800, a hydroxyl group amount of 20 mol %, an amine group amount of 1 mol %, an acetylation degree of 1 mol %, and an acetalization degree (butyralization degree) 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 polymerization degree of 300 and a saponification degree 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 UV irradiation was performed for 20 minutes using a UV polymerization tester to obtain synthetic resin X2 having the composition shown in Table 1.

[0146] <Synthetic resin X3> A polyvinyl butyral resin (synthetic resin X3) having the composition shown in Table 1 was obtained in the same manner as synthetic resin X2, except that the amount of dimethylaminoacrylamide added was changed from 2 parts by weight to 8 parts by weight.

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

[0148] <Synthetic resin X5> A polyvinyl butyral resin (synthetic resin X5) having the composition shown in Table 1 was obtained in the same manner as synthetic resin X4, except that 5 parts by weight of vinyl imidazole was used instead of diethylaminoacrylamide and its content was 13 mol %.

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

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

[0151] Introducing the linker: 90 parts by weight of the obtained polyvinyl acetal resin and 15 parts by weight of acrylic acid (linker) were dissolved in 300 parts by weight of THF, and reacted for 20 minutes under ultraviolet irradiation in the presence of a photoradical polymerization initiator, thereby introducing the linker by graft copolymerizing the polyvinyl acetal resin and acrylic acid.

[0152] Formation of the peptide part: A linear peptide having an amino acid sequence of Gly-Arg-Gly-Asp-Ser (5 amino acid residues, indicated as GRGDS in the table) was prepared as a 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 not containing either calcium or magnesium to prepare a peptide-containing liquid. 100 parts by weight of this peptide-containing liquid and 80 parts by weight of a polyvinyl acetal resin to which a linker had been introduced were added to 300 parts by weight of methanol, and the reaction was carried out to dehydrate and condense the carboxyl group of the linker part and the amino group of the peptide. After the reaction was completed, the mixture was vacuum dried 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 part, a linker part, and a peptide part was prepared.

[0153] <Synthetic resin X8> A polyvinyl butyral resin with a linker introduced therein was obtained in the same manner as for synthetic resin X7. This resin was used to form the following peptide portion.

[0154] Formation of the peptide part: A cyclic peptide having an amino acid sequence of Arg-Gly-Asp-Phe-Lys (5 amino acid residues, Arg and Lys are bonded to form a cyclic skeleton, Phe is D-form, and is indicated as c-RGDfK in the table) was prepared. 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 containing neither calcium nor magnesium to prepare a peptide-containing liquid. 100 parts by weight of this peptide-containing liquid and 80 parts by weight of a polyvinyl acetal resin to which a linker had been introduced were added to 300 parts by weight of methanol, and the reaction was carried out to dehydrate and condense the carboxyl group of the linker part and the amino group of the peptide. After the reaction was completed, the mixture was vacuum dried 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 part, a linker part, and a cyclic peptide part 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 applied onto a PET film. The applied material was exposed to light of 365 nm wavelength at an integrated light intensity of 2000 mJ / cm using a UV conveyor device "ECS301G1" manufactured by iGraphics Co., Ltd. at 25°C. 2 A poly(meth)acrylic acid ester resin (Synthetic Resin X9) was obtained by irradiating with light at 400 nm.

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

[0157] Fabrication of cell culture vessels; In Examples 1 to 7 and 13, each synthetic resin was dissolved in butanol at a concentration of 5 wt%. The obtained solution was patterned in dots or lines on a polystyrene dish with a diameter of 35 mm using a dispenser, and then dried in an oven at 45°C for 6 hours to prepare a cell culture vessel having a bottom made of a cell culture scaffold having a plurality of convex portions. As shown in FIG. 4, a dot pattern was prepared in the cell culture scaffold prepared in Example 1. Similarly, dot patterns were prepared in Examples 3 to 7. As shown in FIG. 5, a spiral pattern was prepared as a line pattern in the cell culture scaffold prepared in Example 2.

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

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

[0160] In Comparative Example 2, synthetic resin X1 was dissolved in butanol at a concentration of 5 wt %. 40 μL of the resulting solution was cast onto a polystyrene dish and then 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 (manufactured by Lasertec Corporation, objective lens when measuring the average height of the convex parts: CFI T Plan EPI SLWD 20x, measurement mode: surface profile, resolution: 0.45 μm), and the following were measured.

[0162] In the case of dots, the average base area of ​​the protrusions If it is linear, the average line width of the protruding part The average height of the convex part located at the center of the convex part in a plan view Average wall height

[0163] The average bottom area of ​​the convex portion is the average bottom area of ​​10 arbitrary convex portions. The average line width of the convex portion is the average value of the line widths of 10 arbitrary points that are 10 μm or more apart from each other. The average height of the convex portion at the center of the convex portion in plan view is the average value of the height of the convex portion at the center position of 10 arbitrary convex portions in plan view when the convex portion is dot-shaped, and is the average value of the height of the convex portion at the center position of 10 line widths that are 10 μm or more apart from each other when the convex portion is line-shaped. The average height of the wall portion is the average value of the height of the wall portion of 10 arbitrary convex portions.

[0164] (2) protein adsorption ratio; For each cell culture vessel, 40 μL of 0.1 mg / mL FITC-labeled bovine serum albumin (Cosmo Bio) was cast and left to stand at 37°C for 1 hour, after which the polystyrene dish was washed with pure water and dried in an oven at 45°C for 1 hour. In addition, 1 μL of 0.005 mg / mL, 0.02 mg / mL, and 0.05 mg / mL FITC-labeled bovine serum albumin was dispensed on the polystyrene dish for calibration of the amount of adsorbed protein, and dried in an oven at 45°C for 1 hour. Each cell culture vessel was photographed using a fluorescent microscope, and the fluorescence intensity of the above-mentioned convex part and the part without the convex part was obtained. The linear approximation of the fluorescence intensity obtained using the polystyrene dish for the calibration curve was converted into the amount of protein adsorption. From the obtained protein adsorption amount of the convex parts and the protein adsorption amount of the part where no convex parts were provided, the ratio of the protein adsorption amount of the convex parts to the protein adsorption amount of the part where no convex parts were provided (convex parts / part where no convex parts were provided) was calculated.

[0165] Water swelling rate of the convex part; Each cell culture vessel was immersed in water at 25°C for 24 hours. The cell culture vessel after immersion was placed on a Kimtowel with the opening of the cell culture vessel facing down for 10 minutes to remove water adhering to the cell culture vessel. Next, the weight of the polystyrene dish was subtracted from the weight of the cell culture vessel before immersion to obtain the weight of the sample before immersion. The weight of the polystyrene dish was subtracted from the weight of the cell culture vessel after immersion to obtain the weight of the sample after immersion. The water swelling rate = (sample weight after immersion - sample weight before immersion) / (sample weight before immersion) x 100 (%) was calculated from the weight of the sample before immersion and the weight of the sample after immersion. Note that the weight of the polystyrene dish itself does not change before and after immersion.

[0166] (3) Elastic modulus of the convex portion at the center position of the convex portion when viewed in a plane; The surface elastic modulus at 25°C was determined at the center of the upper surface of the protrusion of each cell culture scaffold using a nanoindenter (Hysitron, Triboindenter, manufactured by Bruker). A Berkovich (triangular pyramid) indenter with a tip diameter R of 100 nm was used, and a single indentation measurement was performed in air at 25°C. The indentation depth was 50 nm. For Comparative Example 2, the surface elastic modulus of a flat film was measured.

[0167] (4) cell seeding and culture; The following liquid medium and ROCK (Rho-associated kinase) specific inhibitor were prepared.

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

[0169] 1 mL of phosphate buffered saline was added to the obtained cell culture vessel, and the vessel was allowed to stand in an incubator at 37° C. for 1 hour, after which the phosphate buffered saline was removed from the cell culture vessel.

[0170] A colony of h-iPS cells 253G1 that had reached a confluent state was placed in a φ35 mm dish, 1 mL of 0.5 mM ethylenediamine / phosphate buffer solution was added, and the dish was left to stand at room temperature for 2 minutes. After removing the ethylenediamine / phosphate buffer solution, cell clusters were obtained that were broken into pieces of 50 μm to 200 μm in size by pipetting with 1 mL of TeSR E8 medium. The resulting cell clusters (cell count 0.2 × 10 5 The cells were clamp-seeded in the above cell culture vessel.

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

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

[0173] Fig. 6 is a micrograph showing cells seeded in the cell culture vessel prepared in Example 1. Fig. 7 is a micrograph showing cells cultured for 5 days in the cell culture vessel prepared in Example 1.

[0174] As is clear from Fig. 7, it is understood that cell clusters corresponding to the scaffold pattern can be easily and efficiently formed in Example 1. Similarly, observation was also performed for Examples 2 to 13, and it was confirmed that cell clusters corresponding to the scaffold pattern can be easily and efficiently formed.

[0175] Moreover, Examples 1 to 13 and Comparative Examples 1 and 2 were observed and 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 random cell clusters were selected. The area (planar area) of the cell clusters when viewed in a plane under the microscope was calculated, and the dispersity of the planar area of ​​the cell clusters was calculated using the following formula (A).

[0177]

number

[0178] D: Dispersion degree of the plane area of ​​cell aggregates n: number of data Xi: Planar area of ​​cell cluster X: Average surface area of ​​cell clusters

[0179] [Criteria for determining uniformity of cell aggregates] A: Dispersion degree is less than 0.001 B: Dispersity is 0.001 or more and less than 0.002 C: Dispersion degree is 0.002 or more and less than 0.003 D: Dispersion degree is 0.003 or more

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

[0181] [Criteria for determining adhesiveness of cell aggregates] A: All cell clusters are attached B: Some cell clusters have detached. C: All cell clusters are detached.

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

[0183] [Table 1]

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

[0185] 1. Scaffold for cell culture 2,2A…Base material 3,3A…Convex part 4…Area where no protrusion is provided 11...Cell culture container 12...Container body 12a…Surface 31A…Wall part Ha: Height of the convex portion at the center position of the convex portion in a plan view Hb: Height of wall Tb: wall thickness

Claims

1. A substrate; A convex portion formed by patterning the substrate in a dot or line shape, the protrusion includes a synthetic resin, the synthetic resin contains at least a polyvinyl alcohol derivative or a poly(meth)acrylic resin; The convex portion has cell adhesiveness, A scaffold for cell culture, wherein the average height of the protrusions at the center of the protrusions in a planar view is 10 nm or more and 10 μm or less.

2. 2. The scaffold for cell culture according to claim 1, wherein a ratio of protein adsorption amount of the convex portions to a portion where the convex portions are not provided (convex portions / portion where the convex portions are not provided) is 2 or more.

3. the protrusions are configured by patterning the base material in a dot shape, The convex portion has an average bottom area of ​​0.005 mm ２ More than 5 mm ２ 3. The scaffold for cell culture according to claim 1 or 2, which is as follows:

4. The scaffold for cell culture according to any one of claims 1 to 3, wherein the convex portion has a wall portion on the periphery.

5. 5. The scaffold for cell culture according to claim 1, wherein the elastic modulus of the convex portions at 25° C. is 1 GPa or more at the center position of the convex portions in a planar view, as measured by a nanoindentation method.

6. The cell culture scaffold according to any one of claims 1 to 5, wherein the synthetic resin contains 0.2 mol % or more and 20 mol % or less of Bronsted basic groups in its constituent units.

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

8. The cell culture scaffold according to any one of claims 1 to 7, wherein the convex portions have peptide portions on the surface.

9. A cell culture vessel comprising the scaffold for cell culture according to any one of claims 1 to 8 in at least a part of a cell culture region.

Images