Scaffold substrate for pluripotent stem cells, cell culture vessel, and method for culturing pluripotent stem cells

Abstract

To provide protein- and peptide-free pluripotent stem cell culture substrates made of artificially prepared materials with chemically known components.SOLUTION: A scaffold substrate for pluripotent stem cells is formed as a fibrous structure in which fibers are arranged three-dimensionally, and the fibers have a three-dimensional nanostructure on a surface of the fibers in which metal-containing particles composed of at least one of a metal and a metal compound and having a particle size of 1 nm or more and 60 nm or less are arranged three-dimensionally.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a scaffold substrate for pluripotent stem cells, a cell culture vessel, and a method for culturing pluripotent stem cells. [Background technology]

[0002] In the field of regenerative medicine and various academic fields, undifferentiated pluripotent stem cells (such as ES cells and iPS cells) are used, and there is a demand for techniques for culturing pluripotent stem cells of higher quality. Conventional culture methods for maintaining pluripotent stem cells include on-feeder culture using mouse embryonic fibroblasts as feeder cells, and feeder-free culture in which extracellular matrix (ECM) molecules are coated on a plastic culture substrate (see, for example, Patent Documents 1 and 2). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 7101377 [Patent Document 2] Japanese Patent Publication No. 2022-77840 Summary of the Invention [Problem to be solved by the invention]

[0004] However, the above-mentioned on-feeder culture is a method of mixed culture with heterologous cells, while feeder-free culture is a culture method that includes heterologous proteins derived from organisms other than the animal from which the pluripotent stem cells are derived. Thus, both of the above-mentioned methods involve the risk of contamination with unknown viruses and heterologous proteins due to the use of heterologous cells and heterologous proteins. However, particularly in regenerative medicine settings, there is a demand for xeno-free culture methods that do not contain xenogeneic components. Specifically, particularly for medical purposes, it is desirable that the constituent components of the materials used are chemically defined, and it is desirable to minimize contamination with unknown components derived from heterologous organisms.

[0005] Pluripotent stem cells display integrin molecules, which function as ECM receptors, on their surfaces. For example, in the feeder-free culture described above, pluripotent stem cells can recognize and adhere to the ECM molecules coated on the culture substrate. This integrin-ECM binding signaling the cells to "survival," allowing pluripotent stem cells to avoid anoikis, a type of anchorage-dependent cell death. This integrin-ECM binding is a protein-protein interaction achieved by integrin recognition of the RGD amino acid sequence, a characteristic amino acid sequence of ECM. Because this interaction between integrin and ECM is mediated by the specific three-dimensional structure of both proteins and electrostatic interactions between them, there was previously no known method for creating protein- or peptide-free pluripotent stem cell culture substrates using artificially engineered materials with known chemical components. [Means for solving the problem]

[0006] The present disclosure can be realized in the following forms. (1) According to one embodiment of the present disclosure, there is provided a scaffold for pluripotent stem cells, which is formed as a fibrous structure in which fibers are arranged three-dimensionally, and the fibers have a three-dimensional nanostructure on the surface of the fibers in which metal-containing particles composed of at least one of a metal and a metal compound and having a particle size of 1 nm to 60 nm are arranged three-dimensionally. In this form of scaffold for pluripotent stem cells, the fibers that make up the fiber structure have a three-dimensional nanostructure on their surface, with metal-containing particles with a particle size of 1 nm to 60 nm arranged three-dimensionally. Therefore, by using a protein- and peptide-free cell culture scaffold made of chemically known, artificially prepared materials, it is possible to culture pluripotent stem cells while suppressing the introduction of unknown components such as unknown viruses. (2) In the scaffold for pluripotent stem cells of the above embodiment, the three-dimensional nanostructure may have a primary structure composed of the metal-containing particles having a particle size of 5 nm to 10 nm, and a secondary structure composed of clusters of the metal-containing particles having a diameter of 30 nm to 100 nm. This configuration can improve the ability to maintain the undifferentiated state of pluripotent stem cells and enable their cultivation. (3) In the scaffold for pluripotent stem cells of the above embodiment, the fibers may have a cross-sectional diameter of 100 nm to 700 nm. This configuration facilitates ensuring a contact area between the fibers constituting the scaffold for pluripotent stem cells and the pluripotent stem cells, and allows the pluripotent stem cells to recognize the nano-level microstructure, thereby increasing their adhesiveness to the scaffold for pluripotent stem cells and enabling successful proliferation of the pluripotent stem cells. (4) In the scaffold for pluripotent stem cells of the above embodiment, the fiber structure may be formed by folding two or more layers of the fibers. This configuration can increase the efficiency of supplying oxygen, nutrients, and the like to pluripotent stem cells cultured on the scaffold for pluripotent stem cells, thereby improving the growth of the pluripotent stem cells. (5) In the scaffold for pluripotent stem cells of the above embodiment, the spacing between the fibers constituting the fiber structure may be 0.5 μm or more and 10 μm or less. With this configuration, it is possible to reliably retain each individual pluripotent stem cell on the scaffold for pluripotent stem cells. (6) In the scaffold for pluripotent stem cells of the above embodiment, the fibers may be formed in a tubular or semi-tubular shape, which increases the flexibility of the entire fiber structure and makes the scaffold for pluripotent stem cells easier to handle. (7) In the scaffold for pluripotent stem cells of the above embodiment, the metal-containing particles may be composed of at least one of a metal and a metal oxide, which can improve the ability to maintain the undifferentiated state of pluripotent stem cells and enable their cultivation. (8) In the scaffold for pluripotent stem cells of the above embodiment, the metal element constituting the metal-containing particles may include at least one of titanium (Ti), zirconium (Zr), aluminum (Al), and platinum (Pt). This configuration improves the ability to maintain the undifferentiated state of pluripotent stem cells and enable their cultivation. (9) In the scaffold for pluripotent stem cells of the above embodiment, the metal element constituting the metal-containing particles may include at least one of titanium (Ti) and aluminum (Al), which can improve the ability to maintain the undifferentiated state of pluripotent stem cells and enable their cultivation. (10) In the scaffold for pluripotent stem cells of the above embodiment, the metal element constituting the metal-containing particles may include titanium (Ti), which can improve the ability to maintain the undifferentiated state of pluripotent stem cells while culturing them, and in particular, improve the ability to enable subculture while maintaining the undifferentiated state. The present disclosure can be realized in various forms other than those described above, such as a cell culture vessel equipped with a scaffold substrate for pluripotent stem cells, a method for culturing pluripotent stem cells, and a method for manufacturing a scaffold substrate for pluripotent stem cells. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 is an explanatory diagram showing a schematic diagram of the structure of a scaffold substrate for pluripotent stem cells. [Figure 2] 1 is a flowchart showing an example of a method for producing a scaffold material for pluripotent stem cells. [Figure 3] FIG. 1 is a perspective view schematically showing the appearance of a cell culture vessel. [Figure 4] A cross-sectional schematic diagram showing the process of culturing pluripotent stem cells using a cell culture vessel. [Figure 5] FIG. 1 is an explanatory diagram showing a schematic diagram of a method for culturing pluripotent stem cells. [Figure 6] Schematic representation of the putative mechanism that enables the cultivation of pluripotent stem cells. [Figure 7] FIG. 1 is an explanatory diagram showing surface images of each sample observed using an SEM. [Figure 8] FIG. 1 is an explanatory diagram showing the fibers of sample S1 observed using STEM. [Figure 9] FIG. 1 is an explanatory diagram showing the results of EDX analysis of element distribution on the surface of NST. [Figure 10] FIG. 1 is an explanatory diagram showing the results of EDX analysis of element distribution on the surface of NST. [Figure 11] FIG. 1 is an explanatory diagram showing the results of EDX analysis of element distribution on the surface of NST. [Figure 12] FIG. 1 is an explanatory diagram showing the results of EDX analysis of element distribution on the surface of NST. [Figure 13] An explanatory diagram showing the results of culturing iPS cells and confirming that they maintain an undifferentiated state. [Figure 14] An explanatory diagram showing the results of maintaining an undifferentiated state after reseeding on "substrate (I)". [Figure 15] FIG. 1 is an explanatory diagram showing the results of maintaining an undifferentiated state after reseeding on the same substrate as in the first passage. [Figure 16] FIG. 1 is an explanatory diagram showing the results of adherent growth and undifferentiated state at each passage number. DETAILED DESCRIPTION OF THE INVENTION

[0008] A. Composition of scaffold substrate for pluripotent stem cells: 1 is an explanatory diagram schematically illustrating the configuration of a scaffold 10 for pluripotent stem cells according to an embodiment of the present disclosure. The scaffold 10 for pluripotent stem cells of this embodiment is a scaffold used for culturing pluripotent stem cells, which are animal cells that are in an undifferentiated state and have the pluripotency to differentiate into various types of cells, such as ES cells and iPS cells.

[0009] The pluripotent stem cell scaffold 10 of this embodiment is formed as a fiber structure 20 in which fibers 22 are arranged three-dimensionally. The fibers 22 have a three-dimensional nanostructure on their surface in which metal-containing particles 24 composed of at least one of a metal and a metal compound and having a particle size of 1 nm to 60 nm are arranged three-dimensionally. The particle size of the metal-containing particles 24 can be calculated as the relative value between the size of a scale bar and the pixel distance from one end of the observed object (metal-containing particle 24) in an image captured using a scanning electron microscope (SEM) or a transmission electron microscope (TEM). The fibers 22 may have the above-described three-dimensional nanostructure on their surface. For example, the entire fibers 22 may be composed of an aggregate of metal-containing particles 24, or the three-dimensional nanostructure may be formed on a matrix composed of a material different from that of the metal-containing particles 24. The fiber structure 20 is preferably formed in the form of a fabric, such as a nonwoven fabric, in which the fibers 22 are connected three-dimensionally. In the following description, the textile structure 20 consisting of fibers 22 having a three-dimensional nanostructure on its surface composed of metal-containing particles 24 (hereinafter also referred to as "nanoparticles") will also be referred to as a nanostructured textile (NST).

[0010] The three-dimensional nanostructure on the surface of the fiber 22 may have a primary structure formed of metal-containing particles 24 having a particle size of 5 nm to 10 nm, and a secondary structure formed of clusters having a diameter of 30 nm to 100 nm, which are aggregates of such metal-containing particles 24. Figure 1 shows an enlarged view of the surface of the fiber 22, illustrating how the three-dimensional nanostructure having the above-mentioned primary and secondary structures is formed.

[0011] The metal-containing particles 24 constituting the pluripotent stem cell scaffold 10 may be composed of a single metal element, an alloy, a metal compound, or a mixture thereof. The metal-containing particles 24 may be crystalline or amorphous. The metal compound constituting the metal-containing particles 24 may be, for example, at least one of metal oxide, metal sulfide, metal nitride, metal carbide, metal phosphide, and metal iodide, with metal oxide being preferred.

[0012] The metal element constituting the metal-containing particles 24 may be, for example, a titanium group (Group 4) element such as titanium (Ti) and zirconium (Zr), or a transition metal element such as vanadium (V), chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), yttrium (Y), niobium (Nb), molybdenum (Mo), etc. The metal element constituting the metal-containing particles 24 may also be a platinum group element such as platinum (Pt), ruthenium (Ru), rhodium (Rh), palladium (Pd), osmium (Os), iridium (Ir), or a precious metal element such as gold (Au), silver (Ag), etc. The metal element constituting the metal-containing particles 24 may be a typical metal element such as aluminum (Al), magnesium (Mg), silicon (Si), gallium (Ga), germanium (Ge), arsenic (As), selenium (Se), indium (In), tin (Sn), antimony (Sb), or tellurium (Te). The metal element constituting the metal-containing particles 24 preferably includes at least one of titanium (Ti), zirconium (Zr), aluminum (Al), and platinum (Pt), and more preferably includes at least one of titanium (Ti) and aluminum (Al). From the perspective of successfully subculturing pluripotent stem cells using the pluripotent stem cell scaffold 10, the metal element constituting the metal-containing particles 24 particularly preferably includes titanium (Ti).

[0013] The fibers 22 can be fabricated by, for example, using a fibrous base material made of a material different from the constituent material of the metal-containing particles 24 and providing a layer of metal-containing particles 24 having a three-dimensional nanostructure on the surface of the fibrous base material. The layer of metal-containing particles 24 on the base material can be formed by, for example, physical vapor deposition (PVD), chemical vapor deposition (CVD), or atomic layer deposition (ALD), with physical vapor deposition being preferred. The substrate (base material) on which the layer is formed can be, for example, a cloth-like material such as a nonwoven fabric made of a fibrous base material made of a material different from the constituent material of the metal-containing particles 24. Specifically, a resin fibrous base material can be used. Using a resin base material allows for relatively easy nucleation and grain growth of nanoparticles on the surface of the base material when forming a layer of metal-containing particles 24 by physical vapor deposition or the like. For example, the presence of oxygen in addition to the metal element during vapor deposition can provide a metal layer or a metal oxide layer depending on the type of metal element used. When fabricating fibers 22 in this manner, the deposition material wraps around during deposition, allowing a tubular or semi-tubular layer to be formed on the surface of the base fiber as a layer of metal-containing particles 24 with a three-dimensional nanostructure. By forming layers on both sides of the base fiber, it becomes possible to form a tubular layer with high precision.

[0014] For example, when the constituent material of the metal-containing particles 24 is physically vapor-deposited on the surface of a fibrous base material, numerous nanoparticle nuclei are generated on the surface of the base material, and the particles grow. Further physical vapor deposition repeatedly generates nanoparticle nuclei and particle growth on the surface of the fibrous base material. As a result, clusters 26, which are aggregates of the metal-containing particles 24, are formed on the surface of the fibrous base material, forming a three-dimensional nanostructure having the primary and secondary structures described above. The clusters 26 are formed in the form of protrusions with a pyramidal or conical shape, for example. The diameter of such clusters 26 can be, for example, 30 nm to 100 nm. The "diameter of the cluster 26" refers to the maximum diameter of the protrusions (e.g., the diameter of the base in the case of a cone). The diameter of the clusters 26 can be calculated using images captured by SEM or TEM in the same manner as for the particle size of the metal-containing particles 24 described above. The diameter and number of the protrusions can be controlled by adjusting the vapor deposition conditions.

[0015] After forming a layer having a three-dimensional nanostructure on the surface of the fibrous base material, the base material may be completely removed, partially left, or entirely left. For example, if the material constituting the fibrous base material is insoluble in cell culture medium or is non-cytotoxic, at least a portion of the fibrous base material can be left in the scaffold substrate 10 for pluripotent stem cells. Figure 1 shows an example of a fiber structure 20 obtained by forming a semi-tubular layer as a layer having a three-dimensional nanostructure on the surface of the fibrous base material, followed by removing the base material. The semi-tubular fibers 22 in the fiber structure 20 shown in Figure 1 have a base space 25 formed after the base material is removed. By removing the base material and replacing the fibers 22 with a metal or alloy as described above, the flexibility of the entire fiber structure 20 can be increased, making it easier to handle.

[0016] The fibers 22 constituting the pluripotent stem cell scaffold 10 of this embodiment preferably have a cross-sectional diameter (average diameter) of 100 nm or more, more preferably 150 nm or more, and even more preferably 200 nm or more. The cross-sectional diameter (average diameter) of the fibers 22 is preferably 700 nm or less, more preferably 650 nm or less, and even more preferably 600 nm or less. The diameter of the fibers 22 can be calculated using images captured by SEM or TEM in the same manner as for the particle size of the metal-containing particles 24 described above. The diameter of the fibers 22 can be adjusted, for example, by the diameter of the fibrous body that serves as the base material for forming a layer having a three-dimensional nanostructure or the layer formation conditions using the fibrous body as the base material. When the fibers 22 constituting the fiber structure 20 are formed in a semi-tubular shape (i.e., when the cross section is partially missing, such as a crescent-shaped cross section), the diameter of the fibers 22 refers to the diameter of a pseudo-circle whose cross section is circular, including the missing portion (see diameter D in FIG. 1). The average diameter of the fibers 22 can be determined by taking an image of the fiber structure 20 using a scanning electron microscope (SEM), observing a sufficient number of pre-set fields of view (e.g., five fields of view), determining the diameter of the fibers 22 selected for each field of view, and calculating the average value of the diameter values measured in each field of view.

[0017] Furthermore, the fiber structure 20, which is the scaffold 10 for pluripotent stem cells, may have, for example, one or more layers of fibers 22. The layers of fibers 22 may be formed by overlapping multiple layers in the thickness direction, i.e., two or more layers of fibers 22 may be formed by folding over each other. The number of layers of fibers 22 constituting the fiber structure 20 may be, for example, 300 or less, or may be 200 or less, or may be 100 or less. In such a fiber structure 20, the spacing between the fibers 22 constituting the fiber structure 20 may be, for example, 0.5 μm or more and 10 μm or less. Here, the "spacing between fibers" is defined as the length of the long side (longest side) of a mesh structure in which three fibers 22 intersect to form a triangle, when the shape of the fiber structure 20 is understood as a shape stacked in the thickness direction with the minimum unit being a mesh structure.

[0018] B. Method for producing scaffold substrate for pluripotent stem cells: Figure 2 is a flowchart showing an example of a method for manufacturing a scaffold 10 for pluripotent stem cells. Below, based on Figure 2, a method for manufacturing a scaffold 10 for pluripotent stem cells will be described, in which a layer having a three-dimensional nanostructure is formed on the surface of a fibrous base material by sputtering, which is a physical vapor deposition method.

[0019] The process for producing the pluripotent stem cell scaffold 10 begins with the preparation of a fibrous base material (step T100). The fibrous base can be prepared, for example, by electrospinning. Electrospinning is a well-known technique for producing nanofibers, capable of spinning various materials, including resins (polymers), into nanofibers. It can also be used to directly produce nonwoven fabrics. Specifically, a polymer solution or molten polymer is placed in a syringe, and the polymer solution is injected under high voltage to generate a jet of charged polymer. This jet is then collected to form ultrafine polymer fibers. The power supply voltage can be adjusted appropriately depending on the type of polymer solution to be charged and the desired thickness of the polymer fibers. The fiber diameter of the resulting fibrous base can be adjusted, for example, by the polymer concentration of the electrospinning solution, the electric field, the solution supply rate, and the like.

[0020] Various polymers can be used as the base material, such as polyethersulfone (PES), polyvinylidene fluoride (PVDF), polyvinylpyrrolidone (PVP), polyethylene (PE), polypropylene (PP), polyester, polyvinyl alcohol (PVA), polyacrylonitrile (PAN), polyethylene oxide (PEO), polyacrylate (ACM), and polypropylene oxide (PPO).

[0021] After the fibrous body is prepared in step T100, a layer having a three-dimensional nanostructure is then formed on the surface of the prepared fibrous body (step T110). As previously mentioned, various methods can be used to form the layer having a three-dimensional nanostructure. However, physical vapor deposition, such as sputtering or pulsed laser deposition (PLD), is preferred, with sputtering being particularly preferred. In this step, the layer may be formed in a reduced pressure inert gas or in an oxygen-containing gas phase. The various metal elements previously described as constituent materials of the metal-containing particles 24 can be used as the material for the layer having a three-dimensional nanostructure. The conditions for forming the layer having a three-dimensional nanostructure are not particularly limited and can be adjusted appropriately depending on the purpose. In physical vapor deposition, the longer the deposition time, the thicker the layer formed. Furthermore, because physical vapor deposition allows for atomic-level control of the deposition amount, optimizing the deposition conditions allows for the formation of clusters 26 of the desired size. Furthermore, as described above, by performing vapor deposition from one or both sides of the base material, a semi-tubular or tubular layer can be formed as a layer of metal-containing particles 24 having a three-dimensional nanostructure.

[0022] After forming the layer having a three-dimensional nanostructure in step T110, the base material is removed to produce the fiber structure 20 (step T120). The method for removing the base material is not particularly limited, but it can be performed by, for example, calcination. Calcination can be performed at a temperature at which the base material (resin material) is decomposed and removed. Depending on the material of the metal-containing particles 24, this calcination process can further adjust the shape of the clusters 26. Furthermore, when a solvent-soluble polymer is used as the base material, the base material can be removed by dissolving it in a solvent. Examples of solvents that can dissolve various polymers include dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), a sodium borohydride (NaBH4) solution in a 1:1 mixture of water and ethanol, chloroform, acetone, alcohols such as methanol and ethanol, water, 2-methyltetrahydrofuran, dioxane, dimethyl sulfoxide, sulfolane, and nitromethane. However, in step T120, as described above, only a portion of the base material may be removed, or the base material may be left to produce the fiber structure 20 without performing step T120.

[0023] After step T120, the obtained fiber structure 20 is disinfected or sterilized (step T130) to complete the scaffold 10 for pluripotent stem cells.

[0024] In this way, when a scaffold 10 for pluripotent stem cells comprising metal-containing particles 24 is produced by a gas phase method such as physical vapor deposition, processes such as collecting, washing, and drying the nanoparticles are not required, and equipment for safely handling the nanoparticles, as is required when synthesizing nanoparticles in a liquid phase, is not required, making it easier to produce a scaffold 10 for pluripotent stem cells, which is a structure comprising nanoparticles. Furthermore, the above production method makes it easy to produce a large-area scaffold 10 for pluripotent stem cells.

[0025] C. Cell culture vessels: Fig. 3 is a perspective view schematically showing the appearance of a cell culture vessel 30 of this embodiment. Fig. 4 is a cross-sectional schematic diagram showing the culturing of pluripotent stem cells 40 using the cell culture vessel 30. The cell culture vessel 30 is provided with the pluripotent stem cell scaffold 10 of this embodiment in at least a part of the culturable region in the vessel portion 32 that constitutes the cell culture vessel 30. Such a cell culture vessel 30 can be suitably used for culturing pluripotent stem cells.

[0026] The shape of the vessel portion 32 constituting the cell culture vessel 30 is not particularly limited, but may be, for example, a bottomed cylindrical shape with a bottom surface 34 serving as a substrate portion that forms a cultureable area for placing the pluripotent stem cell scaffold 10. The vessel portion 32 shown in FIG. 3 is a bottomed cylindrical shape that is circular in top view, but may have a different shape. Alternatively, the vessel portion 32 may be in the shape of a plate having multiple bottomed cylindrical wells, with the pluripotent stem cell scaffold 10 placed on the bottom surface of each well. The number of wells, the size of each well, and the shape of each well in top view can be appropriately set depending on the purpose of the culture, etc.

[0027] The material of the container 32 is not particularly limited, and examples thereof include resin materials, inorganic materials, and metal materials. Examples of resin materials that can be used include polystyrene, polyethylene, polycarbonate, polypropylene, acrylic resin, and silicone resin. Examples of inorganic materials that can be used include glass and titanium oxide. Examples of metal materials that can be used include stainless steel, nickel, and titanium.

[0028] D. Pluripotent Stem Cell Culture Methods: 5 is an explanatory diagram schematically illustrating an example of a method for culturing pluripotent stem cells 40 using a cell culture vessel 30 equipped with a scaffold 10 for pluripotent stem cells according to the present embodiment. In FIG. 5, when culturing pluripotent stem cells 40 using the cell culture vessel 30, the pluripotent stem cells 40 are first prepared by culturing them in a general undifferentiated cell medium 37 containing ECM without using the scaffold 10 for pluripotent stem cells (Step A). The prepared pluripotent stem cells 40 are then dispersed into single cells or cell clusters (Step B), and the individual pluripotent stem cells 40 are seeded on the scaffold 10 for pluripotent stem cells in the cell culture vessel 30 (Step C). The cells are then cultured in an ECM-free medium 38 that is substantially free of ECM until confluent (Step D). The operation of dispersing the pluripotent stem cells 40 in step B can be carried out by, for example, detaching the pluripotent stem cells 40 from the scaffold 10 for pluripotent stem cells using an enzyme treatment or a calcium chelating agent such as EDTA.

[0029] The pluripotent stem cells 40 cultured in step D are cultured in ECM-free medium 38. Therefore, the constituent components are chemically known, and the cells are cultured in an artificially adjusted environment, making them suitable for various experiments. Furthermore, if the culture in ECM-free medium 38 is to be continued for a longer period of time, the pluripotent stem cells 40 that have grown sufficiently in step D can be dispersed again and seeded (reseeded) in a new cell culture vessel 30 (step E). Whether the cells cultured in step D maintain an undifferentiated state can be confirmed, for example, by seeding the cells cultured in step D in a culture plate (a plate for confirming an undifferentiated state) containing a general undifferentiated cell medium 37 containing ECM (step F), followed by staining with an undifferentiated state marker.

[0030] In the pluripotent stem cell scaffold 10 of this embodiment configured as described above, the fibers 22 constituting the fiber structure 20 have a three-dimensional nanostructure on their surfaces, in which metal-containing particles 24 with particle diameters of 1 nm to 60 nm are three-dimensionally arranged. Therefore, by using a protein- and peptide-free cell culture scaffold composed of chemically known, artificially prepared materials, it becomes possible to culture pluripotent stem cells 40 while suppressing the inclusion of unknown components. Specifically, it becomes possible to proliferate pluripotent stem cells 40 using a cell culture scaffold composed of chemically known, artificially prepared materials that does not contain any endogenous ECM proteins such as laminin, fibronectin, or vitronectin, or their partial sequence peptides.

[0031] Figure 6 is an explanatory diagram showing a presumed mechanism by which the scaffold 10 for pluripotent stem cells enables the cultivation of pluripotent stem cells 40. Figure 6 shows the state in which pluripotent stem cells 40 are cultivated on the scaffold 10 for pluripotent stem cells, with pseudopodia 42 extending between fibers 22. Figure 6 also shows an enlarged view of the contact area between the pluripotent stem cells 40 and the fibers 22, thereby showing a presumed mechanism for the interaction between the pluripotent stem cells 40 and the fibers 22.

[0032] Pluripotent stem cells, such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), typically recognize and adhere to ECM molecules via integrin molecules on their surface, specifically the electrostatic distribution and nanostructures (approximately 5-10 nm in size containing RGD sequences) of specific amino acid sequences. This allows them to avoid anchorage-dependent cell death (anoikis). In the pluripotent stem cell scaffold 10 of the present embodiment, the three-dimensional nanostructure on the surface of the fibers 22, which are made of artificially engineered materials, mimics the above-described cell scaffold microenvironment, presumably allowing pluripotent stem cells 40 to mistakenly recognize the surface structure of the fibers 22 as endogenous ECM molecules, thereby avoiding anoikis and allowing them to adhere and proliferate as normal undifferentiated pluripotent stem cells.

[0033] In particular, when the pluripotent stem cell scaffold 10 of this embodiment is formed into a mesh structure in which multiple fibers 22 are folded in the thickness direction and pluripotent stem cells 40 are cultured on such a scaffold 10, the growth of the pluripotent stem cells 40 is improved. That is, when the pluripotent stem cell scaffold 10 is used, as shown in FIG. 4 , the pluripotent stem cells 40 can receive oxygen, nutrients in the medium, and proteinaceous growth factors not only from above but also from below the pluripotent stem cells 40 through the spaces within the mesh structure that constitutes the scaffold, thereby improving their growth. In FIG. 4 , the supply of oxygen and nutrients to the pluripotent stem cells 40 is indicated by dashed arrows. From the viewpoint of ensuring efficient supply of oxygen, nutrients, and the like from below the pluripotent stem cells 40, the number of layers in which the fibers 22 are folded in the pluripotent stem cell scaffold 10 is not particularly limited, and neither is the thickness of the pluripotent stem cell scaffold 10.

[0034] In such a scaffold 10 for pluripotent stem cells, the spacing between the fibers 22 constituting the fiber structure 20 is preferably 0.5 μm or more and 10 μm or less. This allows each pluripotent stem cell 40 to be reliably held on the surface of the fiber structure 20. Furthermore, by sufficiently reducing the cross-sectional diameter of the fibers 22 to, for example, 100 nm or more and 700 nm or less, it becomes easier for the pluripotent stem cells 40 to extend their pseudopodia 42 and ensure a sufficient contact area with the scaffold 10 for pluripotent stem cells, thereby increasing adhesion to the scaffold 10 for pluripotent stem cells and enabling good proliferation. [Example]

[0035] <Preparation of scaffold material for pluripotent stem cells (NST)> (Preparation of the base fiber) A nonwoven fabric (PVP nonwoven fabric) composed of polyvinylpyrrolidone (PVP) was fabricated as a fibrous base material for nanostructured nonwoven fabrics (NST). The PVP nonwoven fabric was fabricated by electrospinning on a conductive metal substrate such as titanium (Ti). Electrospinning was performed using a methanol solution containing 8 wt% PVP loaded into a syringe under the following conditions: applied voltage: 1 kV / cm, flow rate: 1 mL / h, total flow volume: 0.4 mL. The size of the resulting fibers 22 and the size of the gaps between the fibers 22 could be controlled by changing the type of fiber and electrospinning conditions (solution concentration, applied voltage, distance from the flow port to the substrate, and flow volume).

[0036] (Fabrication of NST with 3D nanostructure) NSTs were fabricated by forming a 3D nanostructure on a PVP nonwoven fabric base material. Four types of NSTs were fabricated: "Sample S1" containing titanium (Ti), "Sample S2" containing zirconium (Zr), "Sample S3" containing aluminum (Al), and "Sample S4" containing platinum (Pt) as the metals contained in the metal-containing particles 24 that make up the 3D nanostructure. "Sample S1" is also called "NST-Ti," "Sample S2" is also called "NST-Zr," "Sample S3" is also called "NST-Al," and "Sample S4" is also called "NST-Pt." The 3D nanostructures of each sample were fabricated using sputtering, a physical vapor deposition process.

[0037] To form the 3D nanostructure of sample S1, a PVP nonwoven fabric was used as the base material and sputtered in a 0.56% oxygen / 99.44% argon atmosphere with Ti as the target element. Specifically, after evacuating the metal substrate coated with the PVP nonwoven fabric, argon at a flow rate of 30 sccm and oxygen at a flow rate of 0.17 sccm were introduced, and the chamber pressure was adjusted to 10.0 Pa for sputtering. The 3D nanostructures of samples S2 to S4 were also formed in the same manner, using the corresponding metal element as the target element. By changing the target element and adjusting the gas species appropriately, 3D nanostructures made of other metals can be formed.

[0038] After sputtering, the fibers with the 3D nanostructure formed on their surfaces were punched out to a diameter of 12 mm and dropped into a container of water. The fibers with the 3D nanostructure formed were washed in water to obtain a fiber structure with the PVP matrix removed. The resulting fiber structure was then scooped up with a Ti mesh and attached to a glass substrate with the side not touching the mesh. The fiber structure was then transferred to the glass substrate. After drying for 5 minutes in a 70°C incubator, it was then heat-treated in a baking oven at 500°C in air for 30 minutes to complete the NST samples with the PVP removed. The primary and secondary structures of the 3D nanostructure of the NST can be controlled by the type of metal (target type) deposited on the PVP nonwoven fabric, the sputtering conditions (atmospheric gas composition, pressure, sputtering deposition rate), and the heat treatment conditions.

[0039] (Confirmation of the fine structure of NST) Figure 7 shows surface images of each sample obtained by SEM (scanning electron microscope). As shown in Figure 7, each sample (NST) produced by electrospinning was confirmed to have a dense fiber structure in the vertical direction (thickness direction) regardless of the target element. The constituent fibers were folded over each other, forming a mesh structure in which three fibers intersect to form a triangle at the smallest unit (the long side length, i.e., the spacing between fibers, was 3 to 5 μm), and these fibers were folded over in a complex three-dimensional manner in the vertical direction. The diameter of a single fiber in each sample was approximately 300 to 500 nm. The roughness of the particles formed on the fiber surface by sputtering after electrospinning varied depending on the metal species; however, particles of approximately 10 to 20 nm were formed in samples S1 to S3, and particles of approximately 20 to 500 nm were formed in sample S4. The aspect ratio of each particle in the depth direction is approximately 1:1, but in the case of sample S4 (NST-Pt), multiple particles form clusters and are fixed in a fibrous shape, and deep crevasse-like valleys (approximately 60 nm) were observed to be formed.

[0040] FIG. 8 is an explanatory diagram showing a state where the fibers constituting the sample S1 (NST-Ti) are observed at a larger magnification using a scanning transmission electron microscope (STEM). In FIG. 8, on the right side of the figure, an image obtained by further magnifying a part of the surface of the fiber is shown. In the SEM image of FIG. 7, the particles observed were found to be composed of clusters 26 formed by aggregation of primary particles (metal-containing particles 24) having a particle size of about 3 to 10 nm.

[0041] (Confirmation of Element Distribution on the Surface of NST by EDX) FIGS. 9 to 12 are explanatory diagrams showing the results of examining the element distribution on the surface of the NST of each sample by EDX. FIG. 9 shows the results of sample S1, FIG. 10 shows the results of sample S2, FIG. 11 shows the results of sample S3, and FIG. 12 shows the results of sample S4. In each figure, the upper part shows the qualitative analysis chart of EDX, and the lower part shows the quantitative calculation results. In the element column of the quantitative calculation results, the type of characteristic X-ray is shown together with the element.

[0042] As shown in FIGS. 9 to 12, when EDX analysis (energy-dispersive fluorescence X-ray analysis) was performed on any fiber of each sample at a spot, a metal element corresponding to the target element species of sputtering was detected in any sample. Since a large amount of oxygen was detected in the NST of samples S1 to S3, it was inferred that in these samples, the metal elements exist in the form of oxides. In sample S4 using platinum (Pt) as the target element species, since the detected amount of oxygen is very small, it is considered that platinum is sputtered as particles in a metallic state.

[0043] (Seeding and Evaluation of iPS Cells on NST) (Pretreatment and Disinfection of NST) In order to disinfect the bacteria present on the surface of each prepared sample (NST), each sample was placed in a well of a 24-well multi-well plate and treated with 70% ethanol for 15 minutes. Then, the ethanol was removed, the surface was rinsed with sterile water to completely remove the washing solution, and then air-dried.

[0044] (Seeding of iPS cells and confirmation of maintenance of undifferentiated state) iPS cells in a state ready for passage (cells cultured using Ajinomoto's StemFit cell culture medium and Nippi's iMatrix-511 ECM, 7 days after seeding) were prepared (Step A in Figure 5). The surface of the prepared iPS cells was washed once with PBS, and the cells were detached into single cells using 0.5x TrypLE Express (Step B in Figure 5). 4 cells / cm 2 The cells were suspended in StemFit containing 10 μM Y-27632 at a density of 10 μM and seeded onto various substrates including the previously described samples S1 to S4 (Step C in Figure 5), and cultured in an environment without ECM (Step D in Figure 5).

[0045] Figure 13 shows the results of culturing iPS cells on various substrates, including samples S1 to S4, and confirming their maintenance of an undifferentiated state. Here, a standard tissue culture plate was used as the plastic dish. "Substrate (I)" in Figure 13 is a control, and iPS cells were seeded on the plastic dish coated with iMatrix-511 ECM. "Substrates (II)" to "Substrates (XI)" in Figure 13 were not coated with ECM. "Substrate (II)" in Figure 13 is a plastic substrate, and "Substrate (III)" is a glass substrate. "Substrates (IV)" to "Substrates (VII)" in Figure 13 were prepared by sputtering titanium (Ti), zirconium (Zr), aluminum (Al), and platinum (Pt), respectively, onto a circular cover glass under the same conditions as samples S1 to S4. Three-dimensional nanostructures were formed on the smooth cover glass substrate. "Substrate (VIII)" to "Substrate (XI)" correspond to the above-mentioned samples S1 to S4, respectively. That is, the above-mentioned NST-Ti, NST-Zr, NST-Al, and NST-Pt were transferred onto circular cover glasses, respectively.

[0046] Figure 13 shows the results of staining cells seeded on each substrate ("Substrate (I) through (XI)") with rBC2LCN-FITC (Fujifilm Wako Pure Chemical Industries, Ltd.) on day 7 after seeding. This reagent stains only undifferentiated iPS cells, allowing observation under an inverted microscope by placing the cells facing downward, even on opaque substrates. Under culture conditions using the control "Substrate (I)" (normal culture conditions for undifferentiated iPS cells), iPS cells proliferated horizontally, and colonies of undifferentiated cells were uniformly stained across the entire field of view. In contrast, on "Substrate (II)" and "Substrate (III)," tissue culture plates without an ECM coating, iPS cells did not engraft. Cell death by anoikis or removal of viable cells during medium changes prevented cell engraftment. Even on "Substrate (IV) through "Substrate (VII)," in which a 3D nanostructure was formed on the surface by sputtering, no cell engraftment or proliferation was observed without an ECM coating. In contrast, when cultured using "Substrate (VIII)" to "Substrate (XI)" (samples S1 to S4), which are fibrous structures with a three-dimensional nanostructure on their surface, iPS cells were observed to take root and proliferate in a dome-like shape even without an ECM coating.

[0047] These results suggest that the effect of using "Substrates (VIII)" to "Substrates (XI)," which enables iPS cell engraftment and proliferation, is not simply due to the properties of the metals or metal compounds present on the substrate surface or the three-dimensional nanostructure with nano-level irregularities formed by sputtering. Specifically, the substrate must be fibrous, have a three-dimensional mesh structure formed by the intersection of these fibers, and have a three-dimensional nanostructure formed on the surface of each fiber. Furthermore, this three-dimensional nanostructure must be formed by metal-containing particles, including the metals and metal compounds described above. This provides a three-dimensional structure and electrostatic distribution that mimics ECM molecules with RGD sequences, providing a favorable growth surface for pluripotent stem cells.

[0048] (Reseeding of iPS cells grown on NST and confirmation of maintenance of undifferentiated state) iPS cells were cultured and replated using "Substrate (VIII)" to "Substrate (XI)" (samples S1 to S4) to confirm the maintenance of the undifferentiated state. iPS cells grown on "Substrate (VIII)" to "Substrate (XI)" for one week (one passage) (step D in Figure 5) were detached from the substrate in the same manner as at the start of the first passage of culture and then replated onto both an iMatrix-511-coated tissue culture plate (the same substrate as "Substrate (I)") and the same substrate as the first passage (either of "Substrate (VIII)" to "Substrate (XI)") (steps F and E in Figure 5). After replated, the medium was changed daily and the cells were cultured for one week, after which the undifferentiated state was confirmed using rBC2LCN-FITC.

[0049] FIG. 14 is an explanatory diagram showing the results of confirming the maintenance of the undifferentiated state after culturing for one week (1st passage / 1p) using any of "substrates (VIII)" to "substrates (XI)" (step D in FIG. 5), reseeding on "substrate (I)", and culturing for another week (2nd passage / p2) (step F in FIG. 5). In FIG. 14, the results of culturing for the first passage (p1) using "substrate (VIII)" are shown as "NST-Ti p1 →TC p2 The results of the first passage (p1) culture using "Substrate (IX)" are shown as "NST-Zr p1 →TC p2 The results of the first passage (p1) culture using "Substrate (X)" are shown as "NST-Al p1 →TC p2 The results of the first passage (p1) culture using "Substrate (XI)" are shown as "NST-Pt p1 →TC p2 In Figure 14, the results of using "Substrate (I)" for both the first and second passages are shown as "TC p1 →TC p2As shown in Figure 14, when reseeded onto the ECM-coated "substrate (I)," it was confirmed that normal iPS cells proliferated while maintaining an undifferentiated state, regardless of whether the substrate used in the first generation was any of "substrate (VIII)" to "substrate (XI)," i.e., regardless of whether the metal constituting the metal-containing particles of the first generation substrate was Ti, Zr, Al, or Pt.

[0050] FIG. 15 is an explanatory diagram showing the results of confirming the maintenance of the undifferentiated state after culturing for one week (1st passage / 1p) using any of "substrates (VIII)" to "substrates (XI)" (step D in FIG. 5), reseeding on the same substrate as in the 1st passage (step E in FIG. 5), and culturing for another week (2nd passage / p2). In FIG. 15, the results using "substrate (VIII)" are compared with those using "NST-Ti p1 →NST-Ti p2 " and the results using "Substrate (IX)" are shown as "NST-Zr p1 →NST-Zr p2 " and the results using "Substrate (X)" are shown as "NST-Al p1 →NST-Al p2 " and the results using "Substrate (XI)" are shown as "NST-Pt p1 →NST-Pt p2 As shown in Figure 15, when reseeded on the same substrate as the first passage, "Substrate (VIII)" (NST-Ti) showed good growth and undifferentiated state. Furthermore, on "Substrate (X)" (NST-Al), the growth of undifferentiated iPS cell colonies was observed after the second passage of culture, although the number was smaller than on "Substrate (VIII)." On "Substrate (IX)" and "Substrate (XI)" (NST-Zr and NST-Pt), no proliferation was observed at the second passage. Based on the above, NST-Ti and NST-Al are preferable substrates for growth at the first passage, and NST-Ti is more preferable for continuous culture at two or more passages.

[0051] (Confirmation of maintenance of undifferentiated state after repeated passage on NST-Ti) Substrate (XIII) (NST-Ti), which is a substrate for culturing iPS cells without a protein-containing coating material and is considered to be particularly suitable for continuous culture for two or more passages, was used to perform longer-term subculture and confirm the repeatability and undifferentiation of the cells. That is, after one week of culture on NST-Ti (step D in Figure 5), the iPS cells were dispersed into single cells using the dispersion method described above and replated on NST-Ti (step E in Figure 5), followed by one week of culture. After one week of culture, the iPS cells were confirmed to have grown sufficiently and remained undifferentiated by staining with rBC2LCN-FITC, and the same sample was dispersed again and replated on NST-Ti (step E in Figure 5), and this process was repeated up to the 10th passage. Below, n-passage culture on NST-Ti is referred to as "P (n) " is expressed as ".

[0052] Each passage number (P (n) ) and then stained with rBC2LCN-FITC to confirm the undifferentiated state, the cells were reseeded on the NST-Ti and also seeded on an ECM-coated tissue culture plate (substrate (I)) to confirm the undifferentiated state (step F in Figure 5). Then, after culturing on the plate to confirm the undifferentiated state (P (n+1) ), and phase-contrast microscopy images after staining with rBC2LCN-FITC were used to perform detailed morphological observations to confirm whether the cells retained the morphology characteristic of undifferentiated iPS cells.

[0053] Figure 16 shows the results for each passage number (P (n) ), the undifferentiated state was confirmed by phase contrast microscopy after staining with rBC2LCN-FITC, and then the cells were cultured on a plate for confirming the undifferentiated state, and the adhesion and growth status of the cells was examined by phase contrast observation. The undifferentiated state was also verified by staining with rBC2LCN-FITC (Figure 16 shows P (n+1)(Figure 16) As shown in Figure 16, iPS cells maintained their undifferentiated state while growing on NST-Ti at all passage numbers. Furthermore, iPS cells grown on NST-Ti at each passage number were detached and plated on plates for confirming their undifferentiated state. Detailed microscopic observation of iPS cells stained with rBC2LCN-FITC confirmed the expression of undifferentiated markers on the cell surface in phase-contrast images. Furthermore, high-magnification (20x objective) phase-contrast images confirmed that the cells maintained their morphologically undifferentiated state (large nuclei with almost no cytoplasm, clear nucleolus contrast, and the formation of compact, tightly packed colonies with clear boundaries between adjacent cells). Thus, when NST-Ti was used as a substrate, it was confirmed that iPS cells could grow in an undifferentiated state even after 10 passages.

[0054] The present disclosure is not limited to the above-described embodiments, and can be realized in various configurations without departing from the spirit thereof. For example, the technical features in the embodiments corresponding to the technical features in each aspect described in the Summary of the Invention section can be appropriately replaced or combined to solve some or all of the above-described problems or achieve some or all of the above-described effects. Furthermore, if a technical feature is not described as essential in this specification, it can be appropriately deleted.

[0055] The present disclosure can also be realized in the following forms. [Application example 1] A scaffold substrate for pluripotent stem cells, comprising: It is formed as a fibrous structure in which fibers are arranged three-dimensionally, The fibers have a three-dimensional nanostructure on the surface of the fibers in which metal-containing particles composed of at least one of a metal and a metal compound and having a particle size of 1 nm to 60 nm are three-dimensionally arranged. Scaffold substrate for pluripotent stem cells. [Application example 2] A scaffold for pluripotent stem cells according to Application Example 1, The three-dimensional nanostructure is a primary structure constituted by the metal-containing particles having a particle size of 5 nm or more and 10 nm or less; a secondary structure formed by clusters of the metal-containing particles, the clusters having a diameter of 30 nm or more and 100 nm or less; Equipped with Scaffold substrate for pluripotent stem cells. [Application example 3] A scaffold for pluripotent stem cells according to Application Example 1 or 2, The fiber has a cross-sectional diameter of 100 nm or more and 700 nm or less. Scaffold substrate for pluripotent stem cells. [Application example 4] A scaffold for pluripotent stem cells according to any one of Application Examples 1 to 3, The fiber structure is formed by folding two or more layers of the fibers. Scaffold substrate for pluripotent stem cells. [Application example 5] A scaffold for pluripotent stem cells according to any one of Application Examples 1 to 4, The spacing between the fibers constituting the fiber structure is 0.5 μm or more and 10 μm or less. Scaffold substrate for pluripotent stem cells. [Application Example 6] A scaffold for pluripotent stem cells according to any one of Application Examples 1 to 5, The fibers are formed into a tubular or semi-tubular shape. Scaffold substrate for pluripotent stem cells. [Application Example 7] A scaffold for pluripotent stem cells according to any one of Application Examples 1 to 6, The metal-containing particles are composed of at least one of a metal and a metal oxide. Scaffold substrate for pluripotent stem cells. [Application Example 8] A scaffold for pluripotent stem cells according to any one of Application Examples 1 to 7, The metal element constituting the metal-containing particles includes at least one of titanium (Ti), zirconium (Zr), aluminum (Al), and platinum (Pt). Scaffold substrate for pluripotent stem cells. [Application Example 9] A scaffold for pluripotent stem cells according to any one of Application Examples 1 to 8, The metal element constituting the metal-containing particles includes at least one of titanium (Ti) and aluminum (Al). Scaffold substrate for pluripotent stem cells. [Application Example 10] A scaffold for pluripotent stem cells according to any one of Application Examples 1 to 9, The metal element constituting the metal-containing particles includes titanium (Ti). Scaffold substrate for pluripotent stem cells. [Application Example 11] A cell culture vessel comprising the scaffold substrate for pluripotent stem cells according to any one of Application Examples 1 to 10. [Application Example 12] A method for culturing pluripotent stem cells, comprising a culturing step of culturing pluripotent stem cells using the scaffold for pluripotent stem cells according to any one of Application Examples 1 to 10. [Explanation of symbols]

[0056] 10...Scaffold substrate for pluripotent stem cells 20...Fiber structure 22...Fiber 24...metal-containing particles 25…Base material space 26...Cluster 30...Cell culture container 32...Container part 34...Bottom 37...Culture medium for undifferentiated cells 38…ECM-free medium 40…pluripotent stem cells 42...pseudopod

Claims

1. A scaffold substrate for pluripotent stem cells, comprising: The fibers are formed as a fiber structure arranged three-dimensionally, The fibers have a three-dimensional nanostructure on the surface of the fibers in which metal-containing particles composed of at least one of a metal and a metal compound and having a particle size of 1 nm to 60 nm are three-dimensionally arranged. Scaffold substrate for pluripotent stem cells.

2. The scaffold substrate for pluripotent stem cells according to claim 1, The three-dimensional nanostructure is a primary structure constituted by the metal-containing particles having a particle size of 5 nm or more and 10 nm or less; a secondary structure formed by clusters of the metal-containing particles, the clusters having a diameter of 30 nm or more and 100 nm or less; Equipped with Scaffold substrate for pluripotent stem cells.

3. The scaffold substrate for pluripotent stem cells according to claim 1, The cross-sectional diameter of the fiber is 100 nm or more and 700 nm or less. Scaffold substrate for pluripotent stem cells.

4. The scaffold substrate for pluripotent stem cells according to claim 1, The fiber structure is formed by folding two or more layers of the fibers. Scaffold substrate for pluripotent stem cells.

5. The scaffold substrate for pluripotent stem cells according to claim 4, The spacing between the fibers constituting the fiber structure is 0.5 μm or more and 10 μm or less. Scaffold substrate for pluripotent stem cells.

6. The scaffold substrate for pluripotent stem cells according to claim 1, The fibers are formed into a tubular or semi-tubular shape. Scaffold substrate for pluripotent stem cells.

7. The scaffold substrate for pluripotent stem cells according to claim 1, The metal-containing particles are composed of at least one of a metal and a metal oxide. Scaffold substrate for pluripotent stem cells.

8. The scaffold substrate for pluripotent stem cells according to claim 1, The metal element constituting the metal-containing particles includes at least one of titanium (Ti), zirconium (Zr), aluminum (Al), and platinum (Pt). Scaffold substrate for pluripotent stem cells.

9. The scaffold substrate for pluripotent stem cells according to claim 8, The metal element constituting the metal-containing particles includes at least one of titanium (Ti) and aluminum (Al). Scaffold substrate for pluripotent stem cells.

10. The scaffold substrate for pluripotent stem cells according to claim 9, The metal element constituting the metal-containing particles includes titanium (Ti). Scaffold substrate for pluripotent stem cells.

11. A cell culture vessel comprising the scaffold substrate for pluripotent stem cells according to any one of claims 1 to 10.

12. A method for culturing pluripotent stem cells, comprising a culturing step of culturing pluripotent stem cells using the scaffold for pluripotent stem cells according to any one of claims 1 to 10.

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