Spheroid formation promoter

Abstract

This agent for promoting spheroid formation includes adsorbent inorganic particles as an active ingredient. This method for promoting spheroid formation comprises: mixing a dispersion of the agent for promoting spheroid formation and a cell suspension to obtain a mixture in which the adsorbent inorganic particles contained in the agent for promoting spheroid formation are dispersed; and culturing the cells contained in the resulting mixture to promote spheroid formation.

Description

[Technical Field]

[0001] The present invention relates to a spheroid formation promoter, a spheroid formation kit, a method for producing spheroids, and a method for promoting spheroid formation. [Background technology]

[0002] In recent years, mammalian cells, such as those from humans, have been used as research materials in a wide variety of studies. In the traditional method of culturing animal cells on a flat surface (two-dimensional cell culture), cells spread and grow in two dimensions. Therefore, a certain amount of culture area is required to obtain a certain number of cultured cells. Furthermore, because the cultured cells are monolayer cells spread in two dimensions, there is the issue that the functions of the cells themselves cannot necessarily be reproduced as they are in vivo.

[0003] Therefore, there is a strong demand for three-dimensional cell culture utilizing the inherent aggregation reaction of adherent animal cells. Spheroids (aggregates) obtained by three-dimensional cell culture exhibit functions different from those of cells obtained by two-dimensional cell culture, realizing a cellular environment closer to that of the living body and possessing high cellular activity. Therefore, spheroids are useful for drug screening and toxicity testing in drug discovery, as well as for constructing safety assessment models as an alternative to animal testing, and are extremely important as biomaterials from which practical information can be obtained.

[0004] In general three-dimensional cell culture methods, it is important to control the adhesiveness of cells, and development of scaffolds, culture vessels, culture media, and the like that are suitable for three-dimensional cell culture methods is underway. For example, porous membranes and hydrogels used as scaffolds are commercially available. Patent Document 1 describes a method for culturing cells using chitin gel, which is prepared by gelling chitosan through treatment with an acetylating agent, as a culture substrate. Patent Document 2 describes a cell culture substrate in which a chimeric protein having a hydrophobic material binding site and a cell adhesion site is immobilized on the fiber surface of a fibrous structure formed from a polymer compound and having a specific average fiber diameter. Furthermore, Non-Patent Document 1 describes the control of cell adhesion by using a hydrogel in which an inorganic layered compound is present within a polymer matrix. Regarding culture vessels, microfabricated plates for non-adhesive treatment and U-bottom plates designed to collect cells can be used for three-dimensional cell culture methods and are commercially available. Furthermore, Patent Document 3 describes that spheroid formation is promoted by culturing a mixture of a cell sample and a polymer formed by polymerizing one or more selected from the group consisting of D-glucosamine, D-galactosamine, D-glucuronic acid, L-iduronic acid, and D-galactose in a spheroid-forming culture vessel. Patent Document 4 describes a method for producing spheroids in which cells are cultured in the presence of an exogenous cell aggregating agent containing any of an antibody, a lectin, and a cell adhesion molecule.

[0005] As described above, three-dimensional cell culture can be performed using special scaffolds, culture vessels, and media. However, these scaffolds, culture vessels, and media are generally expensive. Therefore, when a large number of spheroids are required, such as for drug screening in drug discovery, screening of drug efficacy in primary culture of clinical specimens, or production of useful proteins, the cost burden is high. Furthermore, there is a need for the development of a three-dimensional cell culture method that has a high spheroid formation rate and is highly versatile, without being limited to target biological tissues or cell types.

[0006] Swellable layered silicates such as smectite are a type of clay mineral that swells and becomes viscous when it absorbs liquids such as water. Smectite is composed of nanosheets that extend two-dimensionally. Furthermore, when smectite is swelled and dispersed in water, a colloidal dispersion with viscosity and thixotropy is obtained. In the dispersion, smectite exists in a state separated into individual layers. The smectite crystal layers in the dispersion themselves have a permanent negative charge, and cations such as sodium ions are incorporated into the crystal layers to compensate for this permanent negative charge. Taking advantage of these properties, swellable layered silicates such as smectite have long been widely used industrially. For example, a protein crystal formation control agent containing a layered silicate compound containing fluorine atoms is described in Patent Document 5. Furthermore, Patent Document 6 describes a protein crystallization condition exploration agent that contains a water-swellable layered silicate having fluorine atoms and hydroxyl groups, in which the fluorine atoms are covalently bonded to the silicate by isomorphous substitution with the hydroxyl groups.

[0007] Patent Document 7 describes the use of body fluids collected from the region from the epidermis to the muscle tissue of certain fish species with mucous skin, or body fluids collected from the trunk or liver of cephalopods, as a spheroid formation promoter. Patent Document 8 describes a method for concentrating or purifying the spheroid formation promoter by using aluminum silicate or natural allophane powder to remove odorous components and other impurities from the spheroid formation promoter.

[0008] However, there have been no practical reports on the promotion of spheroid formation by inorganic particulate materials such as swellable layered silicates. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Japanese Patent Application Laid-Open No. 2017-55727 [Patent Document 2] Japanese Patent Application Laid-Open No. 2010-63411 [Patent Document 3] Japanese Patent Application Publication No. 2017-147944 [Patent Document 4] Japanese Patent Application Laid-Open No. 2008-22743 [Patent Document 5] Japanese Patent Application Laid-Open No. 2011-121789 [Patent Document 6] International Publication No. 2012 / 133695 [Patent Document 7] Japanese Patent Application Laid-Open No. 2011-62129 [Patent Document 8] Japanese Patent Application Publication No. 2019-163212 [Non-patent literature]

[0010] [Non-Patent Document 1] Advanced Materials, 2013, Volume 25, Issue 30, p. 4069-4086 Summary of the Invention [Problem to be solved by the invention]

[0011] As mentioned above, the current method for producing spheroids requires the use of special scaffolds, culture vessels, and media. Furthermore, since spheroid production relies on the inherent adhesive force of cells, it can take a long time to form spheroids. Therefore, it has been difficult to form spheroids from a variety of cell types in a simple, rapid, and general-purpose manner using conventional methods.

[0012] Therefore, the present invention provides a spheroid formation promoter that enables simple and rapid production of cells in spheroid form. The present invention also provides a method for producing spheroids, which allows for simple and rapid formation of spheroids. Another objective of the present invention is to provide a method for promoting spheroid formation, which can promote cell aggregation or organization and rapidly form spheroids. A further object of the present invention is to provide a kit for spheroid formation that can be suitably used in the method for producing spheroids or the method for promoting spheroid formation. [Means for solving the problem]

[0013] In view of the above-mentioned problems, the present inventors have conducted extensive research. As a result, they have found that particles made of specific inorganic substances, such as swellable layered silicates, silicates such as kaolinite, mica, and zeolite, colloidal silica, alumina hydrate, magnesium oxide, and hydroxyapatite, have the effect of promoting cell aggregation or organization. Furthermore, they have found that when three-dimensional cell culture is performed in a state in which a predetermined amount of specific inorganic particles is dispersed in a cell suspension for forming spheroids, cells in a spheroid form can be obtained easily and in a short period of time. The present invention has been completed based on these findings.

[0014] The above-mentioned problems of the present invention have been solved by the following means. (1) A spheroid formation promoter containing inorganic particles with adsorptive properties as the active ingredient. (2) The spheroid formation promoter according to (1), wherein the inorganic particles are particles made of at least one inorganic substance selected from the group consisting of silicate, colloidal silica, alumina hydrate, carbonate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, aluminum silicate, aluminum hydroxide, magnesium oxide-aluminum oxide solid solution, hydrotalcite, and hydroxyapatite. (3) The spheroid formation promoter according to (1) or (2) above, wherein the amount of bovine serum albumin adsorbed per 1 mg of the adsorptive inorganic particles is 10 μg or more and 500 μg or less. (4) The spheroid formation promoter according to any one of (1) to (3), wherein the inorganic particles are particles made of at least one type of swellable layered silicate selected from the group consisting of montmorillonite, hectorite, stevensite, saponite, beidellite, nontrite, sauconite, and swellable mica. (5) The spheroid formation promoter according to any one of (1) to (3), wherein the inorganic particles are particles made of at least one type of phyllosilicate mineral selected from the group consisting of kaolinite and mica, or particles made of at least one type of tectosilicate mineral selected from the group consisting of zeolite. (6) The spheroid formation promoter according to any one of (1) to (3) above, wherein the inorganic particles are particles made of colloidal silica. (7) The spheroid formation promoter according to any one of (1) to (3) above, wherein the inorganic particles are particles made of alumina hydrate. (8) A kit for spheroid formation, comprising the spheroid formation promoter according to any one of (1) to (7) above.

[0015] (9) Mixing a dispersion of the spheroid formation promoter according to any one of (1) to (7) above with a cell suspension to obtain a mixture in which the adsorptive inorganic particles contained in the spheroid formation promoter are dispersed, The method for producing spheroids comprises culturing the cells contained in the obtained mixture to form spheroids. (10) A method for promoting spheroid formation, comprising mixing a dispersion of the spheroid formation promoter according to any one of (1) to (7) above with a cell suspension to obtain a mixture in which the adsorptive inorganic particles contained in the spheroid formation promoter are dispersed, and culturing the cells contained in the obtained mixture to promote spheroid formation. (11) The inorganic particles are particles made of at least one swellable layered silicate selected from the group consisting of montmorillonite, hectorite, stevensite, saponite, beidellite, nontrite, sauconite, and swellable mica, The method according to (9) or (10) above, wherein the solids concentration of the swellable layered silicate in the mixture is 0.0003 w / v % or more and less than 0.333 w / v %. (12) The inorganic particles are particles made of at least one phyllosilicate mineral selected from the group consisting of kaolinite and mica, The method according to (9) or (10), wherein the solids concentration of the phyllosilicate mineral in the mixture is 0.001 w / v% or more and less than 0.333 w / v%. (13) The inorganic particles are particles made of at least one tectosilicate mineral selected from the group consisting of zeolite groups, Item (9) The method according to item (10), wherein the solids concentration of the tectosilicate mineral in the mixture is 0.0005 w / v% or more and less than 0.333 w / v%. (14) The inorganic particles are particles made of colloidal silica, The method according to (9) or (10), wherein the solid concentration of colloidal silica in the mixture is 0.0005 w / v% or more and less than 0.04 w / v%. (15) The inorganic particles are particles made of alumina hydrate, The method according to (9) or (10) above, wherein the solids concentration of the alumina hydrate in the mixture is 0.001 w / v % or more and less than 0.5 w / v %. (16) The method according to any one of (9) to (15), wherein the cells are cells collected from an animal, cells cultured from cells collected from an animal, cells collected from an animal that have been subjected to various treatments, or a cultured cell line. (17) The method according to any one of (9) to (16) above, wherein the cells are mammalian cells. (18) The method according to any one of (9) to (17) above, wherein the cells are cultured in a culture plate for suspension cells to form spheroids. [Effects of the Invention]

[0016] The spheroid formation promoter of the present invention promotes cell aggregation and organization, enabling the production of spheroids to be achieved simply and quickly. Furthermore, according to the method for producing spheroids of the present invention, spheroids can be formed simply and quickly. Furthermore, the method for promoting spheroid formation of the present invention can promote cell aggregation or organization, and can rapidly form spheroids. Furthermore, the spheroid formation kit of the present invention can be suitably used in the above-mentioned method for producing spheroids or method for promoting spheroid formation. The above and other features and advantages of the present invention will become more apparent from the following description, taken in conjunction with the accompanying drawings where appropriate. [Brief explanation of the drawings]

[0017] [Figure 1] This is a micrograph of spheroids formed after culturing human pancreatic cancer cells, strain PANC-1, for three days in a suspension cell culture plate in a mixture of swellable layered silicate (sodium fluoride hectorite) dispersed at a solids concentration of 0.0052 w / v%. [Figure 2] This is a micrograph of spheroids formed after culturing human pancreatic cancer cells, strain PANC-1, for three days in a suspension cell culture plate in a mixture of swellable layered silicate (sodium hectorite powder) dispersed at a solids concentration of 0.0052 w / v%. [Figure 3] This is a micrograph of spheroids formed after culturing ACHN human kidney cancer cells for three days in a suspension cell culture plate in a mixture of swellable layered silicate (sodium fluoride hectorite) dispersed at a solids concentration of 0.0052 w / v%. [Figure 4] This is a micrograph of spheroids formed after culturing ACHN human kidney cancer cells for three days in a suspension cell culture plate in a mixture of swellable layered silicate (sodium hectorite powder) dispersed at a solids concentration of 0.0052 w / v%. [Figure 5]1 is a micrograph showing the state of human pancreatic cancer cells PANC-1 strain after culturing the cells for three days in a cell suspension containing no swellable layered silicate. [Figure 6] 1 is a micrograph showing the state of human kidney cancer cells of the ACHN strain after culturing them for three days in a cell suspension containing no swellable layered silicate. [Figure 7] These are micrographs of spheroids formed after culturing human pancreatic cancer cells of the PANC-1 strain in a mixture in which swellable layered silicate (sodium hectorite) was dispersed so that the solids concentration of the swellable layered silicate was 0.0052 w / v%, where Figure 7(a) is a photograph showing the condition on the first day after the start of culture, Figure 7(b) is a photograph showing the condition on the third day after the start of culture, and Figure 7(c) is a photograph showing the condition on the seventh day after the start of culture. [Figure 8] These are micrographs of spheroids formed after culturing human renal cancer cells of the ACHN strain in a mixture in which swellable layered silicate (sodium hectorite) was dispersed so that the solids concentration of the swellable layered silicate was 0.0052 w / v%, where Figure 8(a) is a photograph showing the condition on the first day after the start of culture, Figure 8(b) is a photograph showing the condition on the third day after the start of culture, and Figure 8(c) is a photograph showing the condition on the seventh day after the start of culture. [Figure 9]These are micrographs of spheroids of human colon cancer cells HT29 after culturing them for four days in the presence of various inorganic particles. Figure 9(a) is a micrograph of spheroids in a mixture containing 0.005 w / v% colloidal silica (trade name: Snowtex ST-C), Figure 9(b) is a micrograph of spheroids in a mixture containing 0.0025 w / v% colloidal silica (trade name: Snowtex ST-CXS), and Figure 9(c) is a micrograph of spheroids in a mixture containing 0.02 w / v% alumina hydrate particles (trade name: Aluminasol AS-200). Figure 9(d) is a micrograph of spheroids in a mixture containing alumina hydrate particles (product name: Alumina Sol AS-520-A) dispersed at a concentration of 0.08 w / v%, Figure 9(e) is a micrograph of spheroids in a mixture containing phyllosilicate mineral particles (product name: Kaolin JP-100) dispersed at a concentration of 0.02 w / v%, and Figure 9(f) is a micrograph of spheroids in a mixture containing phyllosilicate mineral particles (product name: MK-100) dispersed at a concentration of 0.04 w / v%. [Figure 10] Figure 10(a) shows micrographs of spheroids formed in a mixture containing 0.02 w / v% tectosilicate mineral particles (trade name: Zeoal-ZSM-5), 0.04 w / v% tectosilicate mineral particles (trade name: HSZ-940NHA), 0.04 w / v% magnesium oxide particles (trade name: Kyowamag MF-150), and 0.04 w / v% hydroxyapatite particles (trade name: SHAp). [Figure 11]Figure 11 shows double-stained observation images of spheroids formed by adding a swellable layered silicate (sodium hectorite) carrying a fluorescent marker to the SW620 human colon cancer cell line and culturing them for five days. Figure 11(a) is an image observed in bright field, Figure 11(b) is an image observed in fluorescent field, and Figure 11(c) is a composite image obtained by superimposing the bright field and fluorescent field images. DETAILED DESCRIPTION OF THE INVENTION

[0018] In the present invention, inorganic particles having adsorptive properties (hereinafter also simply referred to as "adsorptive inorganic particles") are used as an active ingredient of the spheroid formation promoter. As demonstrated in the Examples below, adsorptive inorganic particles promote cell aggregation and organization. Furthermore, spheroids can be formed easily and quickly by performing three-dimensional cell culture in a state where a predetermined amount of adsorptive inorganic particles is dispersed. The spheroid formation promoter, spheroid formation method, spheroid formation promoting method, and spheroid formation kit of the present invention are described below based on preferred embodiments, but the present invention is not limited thereto.

[0019] As used herein, "three-dimensional cell culture" refers to a method of culturing cells in an artificially created environment while allowing them to interact three-dimensionally with their surrounding environment. Three-dimensional cell culture differs from two-dimensional cell culture in that in vivo As with cells in in In vitro This allows cells to grow in all directions. Then, through three-dimensional cell culture, specific cells gather together using their adhesive properties to form tiny clusters of cells called "spheroids."

[0020] The inorganic particles used in the present invention have adsorptive properties. As used herein, "adsorbent properties" refers to the ability to adsorb proteins such as serum components and growth factors. Furthermore, the inorganic particles used in the present invention, or composite particles in which proteins are adsorbed onto inorganic particles, also adsorb to the surface of individual cells that form spheroids, thereby promoting cell aggregation and forming spheroids. The adsorptive properties of inorganic particles can be determined by standard methods. For example, the adsorptive capacity of inorganic particles can be measured using a bovine serum albumin (hereinafter simply referred to as "BSA") reagent. For example, a predetermined amount of colloidal particle dispersion is added to a solution containing dissolved BSA, followed by solid-liquid separation by centrifugation, and the amount of BSA in the supernatant is quantified. Since the BSA adsorbed by the inorganic particles migrates to the solid component after centrifugation, the amount of BSA adsorbed by the inorganic particles can be calculated by subtracting the amount of BSA in the supernatant from the actual amount of BSA. In this way, the protein adsorptive capacity of inorganic particles can be determined. Inorganic particles with protein adsorptive capacity are effective for spheroid formation and can be used in the present invention.

[0021] The inorganic particles used in the present invention are not particularly limited as long as they have adsorptive properties and do not impair the effects of the present invention, but are preferably particles made of at least one inorganic substance selected from the group consisting of silicates, colloidal silica, alumina hydrate, carbonates, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, aluminum silicate, aluminum hydroxide, magnesium oxide-aluminum oxide solid solution, hydrotalcite, and hydroxyapatite. Among these, particles made of at least one inorganic substance selected from the group consisting of silicates, colloidal silica, and alumina hydrate are preferred, and particles made of silicates are more preferred. Among particles made of silicates, particles made of swellable layered silicates are particularly preferred.

[0022] In the present invention, the mechanism by which 3D cell culture promotes spheroid formation is unclear. However, it is thought that proteins such as serum or growth factors contained in the culture medium of spheroid-forming cells adsorb to the adsorptive inorganic particles, and the resulting complex of proteins and adsorptive inorganic particles adsorbs to the surface of the cells, acting as a scaffold to promote spheroid formation. In particular, particles made of swellable layered silicate that can be preferably used in the present invention are present in the medium as nanoscale flat particles, and it is thought that the swellable layered silicate serves as a small scaffold for spheroid formation. Furthermore, because the layer surfaces of the swellable layered silicate are negatively charged, proteins (serum components) in the medium are adsorbed onto the swellable layered silicate, and the proteins are thought to be delivered to the cell surface via the swellable layered silicate.

[0023] "Silicate," which can be used as an active ingredient in the spheroid formation promoter of the present invention, refers to a compound containing an anion with one or more silicon atoms at the center surrounded by electronegative ligands. In the majority of silicates, the silicon atom forms a tetrahedron structure surrounded by four oxygen atoms. The degree to which these tetrahedra are connected varies depending on the type of silicate, and the structure of the tetrahedra can vary widely, including pairs, clusters, rings, chains, double-chains, layers, and three-dimensional networks.

[0024] Among the silicates, the "swellable layered silicate" that can be particularly preferably used in the present invention refers to a compound that exhibits water swelling and has a layer structure in which many two-dimensional unit layers are stacked, and the layer structure is composed of at least silicon atoms and electronegative ligands. The layered silicate may have a single layer of tetrahedral sheets and / or octahedral sheets, or a mixed sheet of these. The tetrahedral sheets have silicon ions (Si 4+ ) into four oxygen ions (O 2-) and share three vertices with the neighboring tetrahedrons, forming a hexagonal network that is connected in a sheet shape. The octahedral sheet is connected by magnesium ions (Mg 2+ ), or aluminum ions (Al 3+ ) to six oxygen ions (O 2- ), or hydroxide ions (OH - ) are octahedra surrounded by tetrahedrons that share their edges and spread out two-dimensionally. When a tetrahedron sheet and an octahedron sheet are combined, the oxygen ions at the vertices of the tetrahedron sheets are shared. Furthermore, some of the tetrahedron sheets are aluminum ions, and some of the octahedron sheets are aluminum, magnesium, and iron ions (Fe 2+ , Fe 3+ ), or lithium ion (Li + Negative charges are generated by isomorphous substitution of ions such as tetrahedrons and octahedrons. Negative charges are also generated by the presence of voids in some of the tetrahedron and octahedron sheets. Cations exist between the layers to neutralize these negative charges. Swellable layered silicates have various properties, such as swelling, viscosity-increasing, thixotropy, and cation exchange. Furthermore, because they are inorganic substances, they are hardly decomposed or altered by microorganisms, making them safe for the human body. Furthermore, because the layered silicates used in the present invention are water-swellable, they are less likely to spontaneously settle even in cell suspensions. This makes it possible to provide a spheroid formation promoter that has excellent uniform dispersibility and can stably maintain a uniformly dispersed state for a long period of time. The swellable layered silicate that can be used in the present invention is preferably derived from a mineral belonging to the smectite group. Minerals belonging to the smectite group have a 2:1 layer structure. Primary particles of smectite are plate-like crystals with a thickness of 1 nm and an extent of 20 nm to 2 μm. As mentioned above, in aqueous dispersions, cations such as sodium ions are incorporated into the smectite crystal layers to compensate for the permanent negative charge of the crystalline layers themselves (see "Clay Handbook," Third Edition, Edited by the Clay Science Society of Japan, May 2009, p. 65). Specific examples of swellable layered silicates that can be used in the present invention include montmorillonite, hectorite, stevensite, saponite, beidellite, nontrite, sauconite, and swellable mica. The swellable layered silicate usable in the present invention may have any cation exchange capacity as long as it does not impair the effects of the present invention. The cation exchange capacity of the swellable layered silicate is preferably 10 meq / 100 g or more, more preferably 30 meq / 100 g or more. A cation exchange capacity within the preferred range imparts a negative charge to the swellable layered silicate that is suitable for adsorption of proteins (serum components). From the viewpoint of achieving excellent swelling properties and dispersion stability, the swellable layered silicate usable in the present invention is preferably a silicate of monovalent cation type such as sodium, and from the viewpoint of achieving excellent swelling properties and dispersion stability, a swellable layered silicate of monovalent cation type such as sodium is more preferred. The cation exchange capacity of the swellable layered silicate can be measured by a method based on the Schollenberger method (Clay Handbook, Third Edition, Edited by the Clay Science Society of Japan, May 2009, pp. 453-454). More specifically, it can be measured by the method described in JBAS-106-77, the standard test method of the Japan Bentonite Industry Association. For example, the amount of leached cations in montmorillonite can be calculated by leaching interlayer cations in montmorillonite for 4 hours or more using 100 mL of 1 M ammonium acetate aqueous solution per 0.5 g of montmorillonite, and measuring the concentrations of various cations in the resulting solution using ICP emission spectrometry, atomic absorption spectrometry, or the like. There is no particular upper limit to the cation exchange capacity of the swellable layered silicate that can be used in the present invention, but a practical upper limit is 120 meq / 100 g or less.

[0025] As silicate, in addition to the swelling layered silicate, silicate minerals made of silicate can be used.There is no particular limitation on the silicate minerals that can be used, and they include olivine, garnet, zircon, sillimanite, andalusite, kyanite, titanite, topaz, staurolite, datolite, braunite, chloritoid, alleghanyite, spurrite, dumortierite, malayite, dioptase, willemite, phenakite, knebelite, glaucochroite, Nesosilicate minerals such as humite, homilite, euclase, and gadolinite; sorosilicate minerals such as melilite, gehlenite, lawsonite, zoisite, clinozoisite, epidote, piemontite, allanite (Ce), vesuvianite (idocrase), hemimorphite, ilvaite (lievrite), zunyite, and bertrandite; and axinite group (axinite). Cyclosilicate minerals such as quartz group, beryl, cordierite, osumilite, sugilite, sogdianite, and tourmaline;Inosilicate minerals such as the pyroxene group, pyroxenoid group, pectolite, babingtonite, amphibole group, and plancheite; phyllosilicate minerals such as kaolinite, halloysite, serpentine, garnierite, pyrophyllite, talc, mica group, chlorite group, forsterite, gyrolite, okenite, prehnite, apophyllite group, and chrysocolla; feldspar group and feldspathoid group. These include tectosilicate minerals such as scapolite, zeolite group, leucite, ammonioleucite, inesite, danburite, helvite, and danalite, among which phyllosilicate minerals such as kaolinite, halloysite, serpentine, garnierite, pyrophyllite, talc, mica group, chlorite group, vermiculite, gyrolite, okenite, prehnite, apophyllite group, and chrysocolla;Preferred are tectosilicate minerals such as feldspar group, feldspathoid group, scapolite, zeolite group, leucite, ammonium leucite, inesite, danburite, helvite, and danalite, and more preferred are kaolinite, mica group, and zeolite group;

[0026] The "colloidal silica" that can be preferably used in the present invention is a colloid of silicon dioxide (SiO2) or its hydrate. Additionally, "alumina hydrate" is a general term for crystalline substances and gels that contain aluminum hydroxide and can be expressed by the chemical formula Al2O3·nH2O.

[0027] The particle size of the adsorptive inorganic particles in the aqueous dispersion used in the present invention varies depending on the measurement conditions, but is generally determined by measuring the particle size distribution using water as the dispersion medium, and can be set arbitrarily within a range that does not impair the effects of the present invention. From the viewpoint of efficiently and uniformly dispersing the adsorptive inorganic particles in the cell suspension, the median diameter of the adsorptive inorganic particles is preferably 10 nm or more, more preferably 20 nm or more, and is preferably 10,000 nm or less, more preferably 5,000 nm or less. When measuring the median diameter of inorganic adsorptive particles, if the dispersed particle size is nanoscale, photon correlation spectroscopy is preferred, and the median diameter can be expressed as the particle size distribution obtained as the diffusion coefficient equivalent diameter. Measurements can be performed by dispersing the inorganic adsorptive particles in water and then diluting the dispersion to approximately 0.1% by mass. Commercially available photon correlation spectroscopy instruments can be used. Examples include the SZ-100 series manufactured by Horiba, Ltd. Other examples include the Zetasizer Nano series manufactured by Malvern Instruments and the DLS-6500 series manufactured by Otsuka Electronics Co., Ltd. When the adsorptive inorganic particles are microscale, the median diameter can be measured using a laser diffraction / scattering method using a measuring device such as the LA-960 series manufactured by Horiba, the Mastersizer series manufactured by Malvern Instruments, or the ELSZ series manufactured by Otsuka Electronics.

[0028] The viscosity of the adsorptive inorganic particles used in the present invention can be set arbitrarily as long as the effects of the present invention are not impaired. For example, for the swellable layered silicate that can be preferably used in the present invention, the viscosity of a 1% by mass aqueous dispersion at 25°C, measured at 60 rpm using a Brookfield viscometer, is preferably 100 mPa·s or less, and more preferably 50 mPa·s or less. Setting the viscosity within this range facilitates accurate measurement of the dispersion when diluting it to adjust the concentration for cell culture.

[0029] The adsorptive inorganic particles used in the present invention are capable of adsorbing proteins. The amount of protein adsorption is measured using BSA as a representative reagent. For example, 0.2 mL of a 1% by mass dispersion of adsorptive inorganic particles or distilled water for comparison is added to 1 mL of a 1 mg / mL BSA solution, mixed using a vortex mixer, and allowed to react for 1 hour on a rotator. The particles are then allowed to settle using a centrifuge, the supernatant is collected, and the BSA contained in the supernatant is quantified. The amount of BSA adsorbed to the adsorptive inorganic particles can then be quantified by comparing it with the supernatant obtained without the addition of adsorptive inorganic particles. Protein quantification can be performed using the Bradford method, the BCA method, the Lowly method, or the like. The amount of BSA adsorbed per 1 mg of adsorptive inorganic particles used in the present invention is preferably 10 μg or more, more preferably 15 μg or more, more preferably 20 μg or more, more preferably 30 μg or more, more preferably 40 μg or more, more preferably 100 μg or more, more preferably 200 μg or more, and even more preferably 300 μg or more. There is no particular upper limit to the amount of BSA adsorbed per 1 mg of adsorptive inorganic particles, with 500 μg or less being practical and 490 μg or less being preferred. It is expected that protein adsorption will allow the inorganic particles to function as a scaffold capable of efficiently providing growth factors to cells.

[0030] The adsorptive inorganic particles used in the present invention may be natural products or may be synthesized according to conventional methods. When natural products are used as the adsorptive inorganic particles, the natural products may contain contaminants and impurities, but from the viewpoint of promoting spheroid formation, it is preferable that the contaminants and impurities have been removed.

[0031] Examples of synthesis methods for swellable layered silicates include hydrothermal synthesis, melt synthesis, high-pressure synthesis, solid-state reaction, flame fusion, and alteration. For the synthesis method of swellable layered silicates, the method described in JP 2008-13401 A can be referenced. Typical representative methods for synthesizing colloidal silica include gas-phase synthesis, such as Aerosil synthesis by thermal decomposition of silicon tetrachloride, methods using water glass as a raw material, and liquid-phase synthesis, such as hydrolysis of alkoxides. Alumina hydrate is generally produced by the Bayer process, in which bauxite is dissolved in sodium hydroxide at high temperature.

[0032] In addition, commercially available adsorptive inorganic particles can also be used in the present invention. Commercially available swellable layered silicates include Kunipia-F (median diameter: 347.4 nm, BSA adsorption amount: 222 μg / mg, cation exchange capacity: 108 meq / 100 g, viscosity of 1 mass% aqueous dispersion at 25°C: 4 mPa·s), Sumecton-SA (median diameter: 85.7 nm, BSA adsorption amount: 437 μg / mg, cation exchange capacity: 70 meq / 100 g, viscosity of 1 mass% aqueous dispersion at 25°C: 5 mPa·s), and Sumecton-SWN (median diameter: 64.4 nm, BSA adsorption amount: 485 μg / mg, cation exchange capacity: 49 meq / 100 g, viscosity of 1 mass% aqueous dispersion at 25°C: 6 mPa·s), Sumecton-SWF (median diameter: 69.8 nm, BSA adsorption amount: 388 μg / mg, cation exchange capacity: 73 meq / 100 g, viscosity of 1 mass% aqueous dispersion at 25°C: 16 mPa·s), Sumecton-ST (median diameter: 42.4 nm, BSA adsorption amount: 474 μg / mg, cation exchange capacity: 30 meq / 100 g, viscosity of 1 mass% aqueous dispersion at 25°C: 2 mPa·s) (trade name, manufactured by Kunimine Industries Co., Ltd.), Somasif ME, Somasif MEB-3 (all trade names, manufactured by Katakura Co-op Agri Co., Ltd.), PDM-5B, PDM-800 (all trade names, manufactured by Topy Industries Co., Ltd.), and the like.

[0033] Commercially available layered silicates other than swellable layered silicates include Kaolin JP-100 (median diameter: 5.4 μm, BSA adsorption amount: 23 μg / mg, manufactured by Takehara Chemical Industry Co., Ltd.), MK-100 (median diameter: 5.1 μm, BSA adsorption amount: 24 μg / mg, manufactured by Katakura Co-op Agri Co., Ltd.), 1000F, FG-15F (all trade names, manufactured by Nippon Talc Co., Ltd.), Hydrite TS90, Hydrite UF90 (all trade names, manufactured by Hayashi Kasei Co., Ltd.), Zeoal 4A (median diameter: 51.6 nm, BSA adsorption amount: 15 μg / mg, manufactured by Nakamura Choukou Co., Ltd.), Zeoal Examples include ZSM-5 (median diameter: 168.0 nm, BSA adsorption capacity: 17 μg / mg, manufactured by Nakamura Choukou Co., Ltd.), HSZ-940NHA (median diameter: 3.2 μm, BSA adsorption capacity: 34 μg / mg, manufactured by Tosoh Corporation), HSZ-820NHA (median diameter: 6.2 μm, BSA adsorption capacity: 30 μg / mg, manufactured by Tosoh Corporation), and HSZ-320NAA (median diameter: 8.2 μm, BSA adsorption capacity: 12 μg / mg, manufactured by Tosoh Corporation).

[0034] Commercially available colloidal silica products include Snowtex-C (median diameter: 45.9 nm, BSA adsorption amount: 47 μg / mg), Snowtex-CXS (median diameter: 46.0 nm, BSA adsorption amount: 89 μg / mg), Snowtex-30 (median diameter: 16.0 nm, BSA adsorption amount: 79 μg / mg), Snowtex Na type (ST-XS, ST-S, ST-50-T, ST-30L, ST-YL, ST-ZL, MP-1040, MP-2040, MP-4540M, Examples include Snowtex O-type (ST-OXS, ST-OS, ST-O, ST-O-40, ST-OL, ST-OYL, ST-OUP, ST-PS-SO, ST-PS-MO), Snowtex N-type (ST-NXS, ST-NS, ST-N, ST-N-40), Snowtex C-type (ST-CM), and Snowtex AK-type (ST-AK, ST-AK-L, ST-AK-YL) (all trade names, manufactured by Nissan Chemical Industries, Ltd.).

[0035] Commercially available alumina hydrates include Aluminasol AS-200 (median diameter: 204.0 nm, BSA adsorption amount: 453 μg / mg, manufactured by Nissan Chemical Industries, Ltd.), Aluminasol AS-520-A (median diameter: 76.8 nm, BSA adsorption amount: 267 μg / mg, manufactured by Nissan Chemical Industries, Ltd.), Alumisol-10A, Alumisol-A2, Alumisol-10C, and Alumisol-F1000 (all trade names, manufactured by Kawaken Fine Chemicals Co., Ltd.).

[0036] Specific examples of other adsorptive inorganic particles include carbonates such as Alcamizer, Magcellar, DHT-4A, DHT-4A-2, DHT-4C, and Kyoward 500 (all trade names, manufactured by Kyowa Chemical Industry Co., Ltd.); magnesium oxides such as Kyowamag, Kyowamag MF, Pyrokisma 5301, Pyrokisma 3320, Magsarat, Magmic, and High Purity Magnesium Oxide 500-04R (all trade names, manufactured by Kyowa Chemical Industry Co., Ltd.); and Kisma (trade name, manufactured by Kyowa Chemical Industry Co., Ltd.). magnesium hydroxide; magnesium carbonate such as Magnesium Carbonate TT (trade name, manufactured by Naikai Salt Co., Ltd.); magnesium silicate such as Kyoward 600 (trade name, manufactured by Kyowa Chemical Co., Ltd.); aluminum silicate such as Kyoward 700 (trade name, manufactured by Kyowa Chemical Co., Ltd.); aluminum hydroxide such as Kyoward 200 (trade name, manufactured by Kyowa Chemical Co., Ltd.); magnesium oxide-aluminum oxide solid solution such as KW-2000 (trade name, manufactured by Kyowa Chemical Co., Ltd.); hydrotalcite such as STABIACE HT (trade name, manufactured by Sakai Chemical Industry Co., Ltd.); and hydroxyapatites such as SHAp, nano-SHAp (both trade names, manufactured by Sofsera), and HAP (trade name, manufactured by MP Biomedicals).

[0037] Furthermore, the commercially available inorganic adsorptive particles that can be used in the present invention may be pulverized to adjust the particle size in order to improve dispersibility. For example, the particle size of commercially available inorganic adsorptive particles can be further reduced by appropriately using a jet mill for dry pulverization, a bead mill for wet pulverization, or a pressurized wet pulverization device.

[0038] In the spheroid formation method of the present invention, a dispersion liquid containing adsorbent inorganic particles dispersed at a predetermined concentration is first prepared. To promote spheroid formation, it is necessary to ensure that the adsorbent inorganic particles are uniformly present in the dispersion liquid. It is therefore preferable to mix a dispersion medium with the adsorbent inorganic particles previously dispersed in an aqueous solution such as water or a buffer solution. This prevents aggregation and precipitation of the adsorbent inorganic particles in the dispersion liquid. The adsorptive inorganic particles used in the present invention may be of one type only, or may be a mixture of two or more types.

[0039] In the present invention, the concentration of the adsorptive inorganic particles in the dispersion can be appropriately set. For example, the solid content concentration of the adsorptive inorganic particles in the dispersion is practically 0.001% by mass or more, preferably 0.003% by mass or more, and more preferably 0.01% by mass or more. The upper limit of the solid content concentration is practically less than 1.000% by mass, preferably 0.600% by mass or less, more preferably 0.500% by mass or less, more preferably 0.250% by mass or less, and even more preferably 0.125% by mass or less. If the solids concentration of the adsorptive inorganic particles is too low, the amount of adsorptive inorganic particles is insufficient to promote spheroid formation. Furthermore, when a mixture of the particles is prepared to a predetermined concentration, the components in the medium are excessively diluted. On the other hand, if the solids concentration of the adsorptive inorganic particles is too high, the particles will aggregate when mixed with the medium components, making the mixture unsuitable for three-dimensional cell culture. The dispersion medium for preparing the adsorptive inorganic particle dispersion is not particularly limited as long as it is non-toxic to the cells that form spheroids and does not impair the proliferation or function of the cells. Specific examples include water, buffer, and cell culture medium. Examples of buffers include phosphate-buffered saline (PBS), HEPES buffer, and Hanks' buffer. Examples of culture medium include D-MEM, E-MEM, MEMα, RPMI, and Ham's F-12. The adsorptive inorganic particle dispersion that can be used in the present invention may further contain various additives within limits that do not impair the effects of the present invention.

[0040] Next, the prepared dispersion of adsorptive inorganic particles is mixed with a cell suspension in which a cell sample from which spheroids are to be formed is suspended in a solvent. The solvent for preparing the cell suspension is not particularly limited as long as it is non-toxic to the cells that form spheroids and does not impair the proliferation or function of the cells. Specific examples include water, buffer, and cell culture medium. Examples of buffers include phosphate-buffered saline (PBS), HEPES buffer, and Hanks' buffer. Examples of culture media include D-MEM, E-MEM, MEMα, RPMI, and Ham's F-12. The cell suspension used in the present invention may further contain various additives within a range that does not impair the effects of the present invention. Examples include phenol red indicator, buffers such as HEPES, EDTA, L-glutamine, antibiotics such as penicillin, and antifungal agents such as funginol.

[0041] At least one of the dispersion of adsorptive inorganic particles and the cell suspension contains serum components commonly used in spheroid formation. Serum components that can be used in the present invention include fetal bovine serum (FBS), bovine serum, horse serum, and porcine serum. Serum contains growth factors, primarily proteins, and growth factors such as cytokines may be used in so-called serum-free media. At least one of the dispersion of adsorptive inorganic particles and the cell suspension may also contain human platelet lysate (hPL). In the present invention, by mixing a cell suspension with a dispersion of adsorptive inorganic particles, serum components adsorbed to the adsorptive inorganic particles are supplied to the surface of the cells.

[0042] The amount of adsorptive inorganic particles to be mixed with the cell suspension need only be sufficient to achieve the effect of promoting spheroid formation, and can be set arbitrarily depending on the type of cells in the mixture of the cell suspension and the dispersion of the adsorptive inorganic particles, the number of cells constituting the mixture, the culture environment, etc. For example, when using swellable layered silicate as adsorbent inorganic particles, the solid content concentration of the swellable layered silicate in the mixture of cell suspension and dispersion of swellable layered silicate is preferably 0.0003 w / v% or more, more preferably 0.0005 w / v% or more, more preferably 0.0006 w / v% or more, more preferably 0.0010 w / v% or more, more preferably 0.0027 w / v% or more.The upper limit of the solid content concentration of swellable layered silicate is preferably less than 0.333 w / v%, preferably 0.1667 w / v% or less, more preferably 0.0833 w / v% or less, and even more preferably 0.0417 w / v% or less. When a phyllosilicate mineral is used as the adsorbent inorganic particle among the silicates mentioned above other than swellable layered silica, the solids concentration of the phyllosilicate mineral in the mixture of the cell suspension and the phyllosilicate mineral dispersion is preferably 0.001 w / v% or more, more preferably 0.005 w / v% or more, more preferably 0.01 w / v% or more, and even more preferably 0.02 w / v% or more. The upper limit of the solids concentration of the phyllosilicate mineral is preferably less than 0.333 w / v%, preferably 0.1667 w / v% or less, more preferably 0.0833 w / v% or less, and even more preferably 0.0417 w / v% or less. Furthermore, when a tectosilicate mineral is used, the solids concentration of the tectosilicate mineral in the mixture of the cell suspension and the tectosilicate mineral dispersion is preferably 0.0005 w / v% or more, more preferably 0.001 w / v% or more, more preferably 0.002 w / v% or more, and even more preferably 0.0025 w / v% or more. The upper limit of the solids concentration of the tectosilicate mineral is preferably less than 0.333 w / v%, preferably 0.1667 w / v% or less, more preferably 0.08 w / v% or less, and even more preferably 0.04 w / v% or less. When colloidal silica is used as the adsorptive inorganic particles, the solids concentration of the colloidal silica in the mixture of the cell suspension and the colloidal silica dispersion is preferably 0.0005 w / v% or more, more preferably 0.001 w / v% or more, more preferably 0.002 w / v% or more, and even more preferably 0.0025 w / v% or more. The upper limit of the solids concentration of the colloidal silica is preferably less than 0.04 w / v%, preferably 0.02 w / v% or less, more preferably 0.01 w / v% or less, and even more preferably 0.005 w / v% or less. When alumina hydrate is used as the adsorptive inorganic particles, the solids concentration of the alumina hydrate in the mixture of the cell suspension and the alumina hydrate dispersion is preferably 0.001 w / v% or more, more preferably 0.005 w / v% or more, more preferably 0.01 w / v% or more, more preferably 0.02 w / v% or more, and more preferably 0.04 w / v% or more. The upper limit of the solids concentration of the alumina hydrate is preferably less than 0.5 w / v%, preferably 0.333 w / v% or less, more preferably 0.1667 w / v% or less, and even more preferably 0.08 w / v% or less. If the solids concentration of the adsorptive inorganic particles is too low, the amount of adsorptive inorganic particles is insufficient to promote spheroid formation, whereas if the solids concentration of the adsorptive inorganic particles is too high, the adsorptive inorganic particles form a gel in the mixture, inhibiting spheroid formation.

[0043] In the present invention, the solids concentration of the adsorptive inorganic particles contained in a dispersion of adsorptive inorganic particles or a mixture of a dispersion and a cell suspension can be determined by a conventional method. For example, the solids concentration of the composite adsorptive inorganic particles can be determined by drying a dispersion or mixture containing adsorptive inorganic particles such as swellable layered silicate by freeze-drying or the like, and then measuring the chemical composition, methylene blue adsorption amount, and cation exchange capacity.

[0044] The cells contained in the cell sample used in the present invention may be any cells capable of forming spheroids, preferably adhesive cells. The cell suspension may contain only one type of cell, or two or more types of cells. There are no particular limitations on the number of cells in the mixture of the adsorptive inorganic particle dispersion and cell suspension, and it can be set appropriately taking into consideration the cell type, culture conditions, the purpose of use of the spheroids, etc. For example, the number is preferably 1 cell / μL or more and 10,000 cells / μL or less, and more preferably 10 cells / μL or more and 1,000 cells / μL or less.

[0045] There are no particular limitations on the cells used to form spheroids, and any of these may be used: cells collected from animals, cells cultured from animals, cells collected from animals that have been subjected to various treatments, cultured cell lines, etc. The type of animal from which the cells are derived is not particularly limited, and it is preferable to use mammalian cells such as human, monkey, dog, cat, rabbit, pig, cow, mouse, and rat. When the cells used are cells collected from an animal, the cells that can be used in the present invention may be any of epithelial tissue cells, muscle tissue cells, connective tissue cells, and nervous tissue cells. The site of collection is not particularly limited, and any of the cells may be somatic cells derived from bone, muscle, internal organs, nerves, brain, bone, skin, blood, etc., germ cells, embryonic stem cells (ES cells), etc. Furthermore, cells that have undergone various treatments include induced pluripotent stem cells (iPS cells) and cells after differentiation induction. Cultured cell lines may be obtained from ATCC (American Type Culture Collection) or ECACC (European Collection of Cell Cultures), or primary cultured cells of tissue cells collected from clinical tissues can be used. The cells that can be used in the present invention may be cells at any stage of the cell cycle, may be undifferentiated cells, may be differentiated cells, may be normal cells, or may be cells collected from pathological tissues such as cancer tissues.

[0046] The cells contained in the mixture obtained by mixing the cell suspension with the dispersion of adsorptive inorganic particles are cultured in a vessel for spheroid formation culture according to a conventional method. The culture vessels that can be used in the present invention may be any vessel used in cell culture using a general spheroid culture method, such as a culture plate for adherent cells or a culture plate for suspension cells, and are not particularly limited in terms of material, shape, or size. Furthermore, containers that suppress adhesion other than between cells, such as a culture plate for suspension cells, are preferred from the viewpoint of reducing the amount of adsorptive inorganic particles used. Materials for the vessel for spheroid formation and culture include, but are not limited to, glass, stainless steel, and plastic. Vessels for spheroid formation and culture include, but are not limited to, dishes, tubes, flasks, bottles, and plates. The treatment and operation for forming the scaffold may be performed in a vessel for spheroid formation and culture. However, as described above, adsorptive inorganic particles such as swellable layered silicates, which also function as a scaffold in a dispersion, are preferred from the viewpoint of cost reduction because they allow culture without the use of commonly used scaffolds.

[0047] The culture conditions in the vessel for spheroid formation and culture may be any environment suitable for the cell type used, and may be, for example, a commercially available culture medium recommended for culturing the cell type used, temperature conditions recommended for culturing the cell type used, etc. The culture time may also be set arbitrarily depending on the cell type used, the number of cells, and the desired size of spheroids. For example, a mixture of a dispersion of adsorptive inorganic particles such as swellable layered silicates and a cell suspension can be maintained for a desired period of time in an environment suitable for cell culture, such as a 5% CO atmosphere at 37°C, thereby rapidly achieving spheroid formation.

[0048] The spheroid formation kit of the present invention contains a spheroid formation promoter containing adsorptive inorganic particles as an active ingredient. The spheroid formation kit of the present invention may also contain a spheroid formation culture vessel, a dispersion medium for preparing a dispersion of adsorptive inorganic particles such as swellable layered silicate, a solvent for preparing a cell suspension, serum components necessary for spheroid formation, a cell culture medium, etc. In this way, by providing a kit containing the reagents, tools, etc. necessary for promoting spheroid formation, spheroid formation can be performed more easily and in a shorter time. [Example]

[0049] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples. In the following examples, when the median diameter of various adsorptive inorganic particles was nanoscale, the median diameter was measured using a 0.1% by mass aqueous dispersion of the inorganic particles with a nanoparticle analyzer (nano Partica SZ-100V2, manufactured by Horiba, Ltd.) For microscale particles, the median diameter was measured with a laser diffraction / scattering particle size distribution analyzer (Partica LA-950V2, manufactured by Horiba, Ltd.). The amount of BSA adsorption to inorganic particles was determined by adding 0.2 mL of a 1% by weight dispersion of adsorptive inorganic particles or distilled water (blank) to 1 mL of a 1 mg / mL BSA solution, stirring with a vortex mixer, and then incubating for 1 hour using a rotator (MTR-103, AS ONE Corporation). The particles were then centrifuged to settle the supernatant, which was then quantified by the Bradford assay and compared with the blank. A 1 mg / mL BSA solution was prepared by dissolving BSA powder (Fujifilm Wako Pure Chemical Industries, Ltd.) in distilled water. Centrifugation was performed at 15,000 G for 15 minutes using a high-speed centrifuge (CT15E, Hitachi Koki Holdings Co., Ltd.). The BSA content in the supernatant was quantified using a TaKaRa Bradford Protein Assay Kit (Takara Bio Inc.) and measuring the absorbance at 595 nm using a spectrophotometer (ASV11D-H, AS ONE Corporation). The amount of adsorption was determined by subtracting the amount of BSA in the inorganic particle dispersion reaction supernatant from the amount of BSA in the blank, and this value was taken as the amount of BSA adsorbed onto the adsorptive inorganic particles.

[0050] (Test Example 1) Three-dimensional cell culture using swellable layered silicate [Preparation Example 1-1] 100 g of sodium fluoride hectorite (trade name: Sumecton-SWF, manufactured by Kunimine Industries Co., Ltd.; hereinafter simply referred to as "SWF") was added to 2 kg of an 80% by mass aqueous solution of isopropyl alcohol (water / isopropyl alcohol = 20 / 80 (mass ratio)). The mixture was then stirred at 100 rpm for 20 minutes at 25°C using a propeller mixer (manufactured by AS ONE). The resulting suspension was suction filtered using a Nutsche filter to separate the solid and liquid, yielding a dehydrated cake. The resulting cake was washed by passing 2 kg of an 80% by mass aqueous solution of isopropyl alcohol (water / isopropyl alcohol = 20 / 80 (mass ratio)) through the filter, and the same washing process by passing the liquid through and filtering was repeated twice. The washed dehydrated cake was recovered and dried in a dryer at 105°C, and the dried powder was recovered. The cation exchange capacity of the obtained powder was measured and found to be 73.3 meq / 100 g. The powder and distilled water were then mixed using a planetary mixer (manufactured by Thinky Corporation) to obtain Dispersion 1 with a hectorite solids concentration of 1 mass %. The median diameter of the sodium fluoride hectorite in the dispersion was 42.1 nm. The BSA adsorption capacity per mg of sodium fluoride hectorite was 388 μg / mg.

[0051] [Preparation Example 1-2] Dispersion 2 having a hectorite solids concentration of 1% by mass was obtained in the same manner as in Preparation Example 1, except that sodium hectorite powder (product name: Sumecton-SWN, manufactured by Kunimine Industries Co., Ltd., hereinafter simply referred to as "SWN") was used instead of the fluorinated sodium hectorite powder. The cation exchange capacity of the obtained powder was 53.1 meq / 100 g. The median diameter of the sodium hectorite in the dispersion was 71.0 nm. The BSA adsorption capacity per mg of sodium hectorite was 485 μg / mg.

[0052] [Three-dimensional cell culture] (1) Preparation of medium-1 Ten parts by weight of FBS (Gibco) was added to 100 parts by weight of cell culture medium: RPMI-1640 (Fujifilm Wako Pure Chemical Industries, Ltd., hereafter referred to as "RPMI"), D-MEM (Fujifilm Wako Pure Chemical Industries, Ltd., hereafter referred to as "DMEM"), or E-MEM (Fujifilm Wako Pure Chemical Industries, Ltd., hereafter referred to as "EMEM"). Cell suspensions were prepared using these media, adjusting the cell numbers. 100 μL of the resulting suspensions were seeded into two types of 96-well plates (adherent cell culture plates) (trade name: Tissue culture plate VTC-P96, Violamo, hereafter referred to as "Violamo") and suspension cell culture plates (non-treated 96-well microplate, Iwaki, hereafter referred to as "Iwaki"). The number of cells seeded onto the plates is shown in Tables 1 to 4. The cells used were the human pancreatic cancer cell line PANC-1 (hereinafter simply referred to as "PANC-1"), the human renal cancer cell line ACHN (hereinafter simply referred to as "ACHN"), the human glioma (astrocytoma) cell line U-251MG (hereinafter simply referred to as "U251MG"), and the human bladder cancer cell line UMUC3 (hereinafter simply referred to as "UMUC3"), and these cells were obtained from the American Type Culture Collection (ATCC).

[0053] (2) Preparation of medium-2 100 parts by mass of D-MEM (Fujifilm Wako Pure Chemical Industries, Ltd.), a cell culture medium, was mixed with 5 parts by mass of FBS (Gibco). Cell suspensions were prepared using this medium, adjusting the cell numbers of various cells. 100 μL of the resulting suspension was seeded into a 96-well plate (suspension cell culture plate) (trade name: Non-treated 96-well microplate, Iwaki). The number of cells seeded onto the plate is shown in Table 4. The cells used were the human glioma cell line KNS-81 (hereinafter simply referred to as "KNS81") and the human glioblastoma cell line LN-299 (hereinafter simply referred to as "LN299"), which were obtained from the American Type Culture Collection (ATCC).

[0054] (3) Addition of swellable layered silicate Dispersions 1 and 2 prepared in Preparation Examples 1-1 and 1-2 were diluted with sterilized water to 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 128-fold, 256-fold, or 512-fold to prepare dispersions. 50 μL of Dispersions 1 and 2 and their diluted dispersions (corresponding to 1, 0.5, 0.25, 0.125, 0.0625, 0.031, 0.016, 0.008, 0.004, and 0.002 w / v%, respectively) was added to 100 μL of cell suspension seeded in a 96-well plate. The final concentrations of the swellable layered silicate in the medium at the time of culture were 0.33, 0.17, 0.083, 0.042, 0.021, 0.010, 0.0052, 0.0026, 0.0013, and 0.00065 w / v%, respectively. After adding the swellable layered silicate, the plates were cultured in an incubator at 37°C in a 5% CO2 atmosphere.

[0055] (4) Evaluation of spheroid formation After the culture, the plate was observed under a microscope to confirm whether or not spheroids had formed. The results of the presence or absence of spheroid formation are shown in Tables 1 to 4. The results of three-dimensional cell culture without using the dispersions prepared in Preparation Examples 1-1 and 1-2 are also shown. Regarding the "state of cells after culture" in Tables 1 to 4, "one-dimensional" means that the observed cells existed as single spheres. "two-dimensional" means that the observed cells existed in a single layer, amorphous and adhered to the plate, and no spheroid formation was confirmed. "three-dimensional" means that spheroid formation was confirmed. "three-dimensional-like" means that the cells were not completely spheroid-like, but some spheroid-like cells were present. 1 (Example 8 of the present invention), 2 (Example 20 of the present invention), 3 (Example 12 of the present invention), and 4 (Example 24 of the present invention) show micrographs of the state of human pancreatic cancer cells PANC-1 or human renal cancer cells ACHN after culturing for 3 days in a mixture prepared by dispersing swellable layered silicate in a suspension cell culture plate so that the solids concentration of the swellable layered silicate was 0.0052 w / v%. Furthermore, 5 (Reference Example 2) and 6 (Reference Example 4) show micrographs of the state of human pancreatic cancer cells PANC-1 or human renal cancer cells ACHN after culturing for 3 days in a cell suspension that does not contain swellable layered silicate. Furthermore, Figures 7 and 8 show micrographs of the changes in the state of the human pancreatic cancer cells PANC-1 and human renal cancer cells ACHN after culturing them in a mixture in which the swellable layered silicate was dispersed so that the solids concentration of the swellable layered silicate in dispersion liquid 2 was 0.0052 w / v% after 1 day, 3 days, and 7 days, respectively.

[0056] TIFF0007742055000001.tif243170

[0057] TIFF0007742055000002.tif248164

[0058] TIFF0007742055000003.tif119170

[0059] TIFF0007742055000004.tif106170

[0060] In the absence of swellable layered silicate (Reference Examples 1 to 4), no spheroids were formed even after cell culture for 3 days (see FIGS. 5 and 6). In contrast, when three-dimensional cell culture was performed in the presence of swellable layered silicate (Examples 1 to 36 of the present invention), spheroids were formed as early as one day after the start of culture, and generally within about three days (see Figures 1 to 4 and Figures 7 and 8). These results indicate that swellable layered silicates dispersed in cell suspensions under three-dimensional cell culture conditions promote cell aggregation and organization, regardless of the cell type, and promote spheroid formation. Furthermore, the use of swellable layered silicates enables the production of spheroids without the need for special scaffolds, culture vessels, or media.

[0061] Furthermore, the results shown in Tables 1 to 4 also show that in order to produce spheroids, it is necessary to set the solids concentration of the swellable layered silicate in the mixture within a predetermined range. Specifically, when culturing was performed without adding swellable layered silicate to the medium, no spheroid formation was observed, and all cultured cells were monolayer cells (Reference Examples 1 and 3). On the other hand, when the solid concentration of swellable layered silicate in the mixture was too high, pure spheroids were not formed (Comparative Example 1), or they were in a non-uniform spheroid-like state (Comparative Example 2). In contrast, as shown in the results of Inventive Examples 1 to 36, in order to form spheroids, it is important to set the solids concentration of the swellable layered silicate dispersed in the cell suspension within a predetermined range. Furthermore, as shown in Figures 7 and 8, spheroid formation was observed the day after the start of culture (see Figures 7(a) and 8(a)), and spheroid growth was confirmed by the third and seventh days after the start of culture (see Figures 7(b) and (c), and Figures 8(b) and (c)).

[0062] (Test Example 2) Three-dimensional cell culture using various adsorptive inorganic particles [Preparation Example 2-1] Colloidal silica (trade name: Snowtex ST-C, manufactured by Nissan Chemical Industries, Ltd., solid content concentration 20.5% by mass) and distilled water were mixed to obtain a 1% by mass colloidal silica dispersion. The median diameter of the colloidal silica in the dispersion was 45.9 nm, and the amount of BSA adsorbed per 1 mg of colloidal silica was 47 μg / mg.

[0063] [Preparation Example 2-2] Colloidal silica (trade name: Snowtex ST-CXS, manufactured by Nissan Chemical Industries, Ltd., solid content concentration 15% by mass) and distilled water were mixed to obtain a 1% by mass colloidal silica dispersion. The median diameter of the colloidal silica in the dispersion was 45.5 nm, and the amount of BSA adsorbed per mg of colloidal silica was 89 μg / mg.

[0064] [Preparation Example 2-3] Alumina hydrate particles (trade name: Aluminasol AS-200, manufactured by Nissan Chemical Industries, Ltd., solid content concentration 10.6% by mass) and distilled water were mixed to obtain a 1% by mass alumina hydrate dispersion. The median diameter of the alumina hydrate particles in the dispersion was 204.0 nm, and the amount of BSA adsorbed per 1 mg of alumina hydrate particles was 453 μg / mg.

[0065] [Preparation Example 2-4] Alumina hydrate particles (trade name: Aluminasol AS-520-A, manufactured by Nissan Chemical Industries, Ltd., solid content concentration 21% by mass) and distilled water were mixed to obtain a 1% by mass alumina hydrate dispersion. The median diameter of the alumina hydrate particles in the dispersion was 77.0 nm, and the amount of BSA adsorbed per 1 mg of alumina hydrate particles was 267 μg / mg.

[0066] [Preparation Example 2-5] Phyllosilicate mineral particles (product name: Kaolin JP-100, manufactured by Takehara Chemical Industry Co., Ltd.) were mixed with distilled water to obtain a 1% by mass phyllosilicate mineral dispersion. The median diameter of the phyllosilicate mineral particles in the dispersion was 5.4 μm. Furthermore, the amount of BSA adsorbed per mg of phyllosilicate mineral particles was 23 μg / mg.

[0067] [Preparation Example 2-6] Phyllosilicate mineral particles (mica) (trade name: MK-100, manufactured by Katakura Co-op Agri Co., Ltd.) were mixed with distilled water to obtain a 1% by mass phyllosilicate mineral dispersion. The median diameter of the phyllosilicate mineral particles in the dispersion was 5.1 μm. Furthermore, the amount of BSA adsorbed per mg of phyllosilicate mineral particles was 24 μg / mg.

[0068] [Three-dimensional cell culture] A medium was prepared by adding 10 parts by mass of FBS (Gibco) to 100 parts by mass of RPMI (Fujifilm Wako Pure Chemical Industries, Ltd.). The dispersions obtained in Preparation Examples 2-1 to 2-6 were diluted with the medium and poured into a 96-well plate (Iwaki). Human colon cancer cells, HT29 strain (hereinafter simply referred to as "HT29"), were seeded at 1,000 cells / 50 μL. The HT29 strain was obtained from the American Type Culture Collection (ATCC). After the cells were seeded, the plate was cultured in an incubator at 37°C in a 5% CO2 atmosphere for 4 days.

[0069] After the culture, the plate was observed under a fluorescence microscope (product name: Biozero, manufactured by Keyence Corporation) to confirm the presence or absence of spheroid formation. The results are shown in Figure 9. As shown in FIG. 9, it can be seen that spheroids can also be formed using the dispersions obtained in Preparation Examples 2-1 to 2-6.

[0070] Furthermore, spheroids were formed by varying the concentrations of the various inorganic particles contained in the dispersions obtained in Preparation Examples 2-1 to 2-6, and the solid content concentrations of the inorganic particles at which spheroids were formed were determined. The results are shown in Table 5.

[0071] TIFF0007742055000005.tif48170

[0072] The results shown in Table 5 show that in order to produce spheroids, it is necessary to set the solid concentration of each type of inorganic particle in the mixture within a predetermined range.

[0073] (Test Example 3) Three-dimensional cell culture using various adsorptive inorganic particles [Preparation Example 3-1] Tectosilicate mineral particles (zeolite) (trade name: Zeoal-ZSM-5, manufactured by Nakamura Choukou Co., Ltd., solid content concentration 32 mass %) and distilled water were mixed to obtain a 1 mass % tectosilicate dispersion. The median diameter of the tectosilicate mineral particles in the dispersion was 248.5 nm, and the amount of BSA adsorbed per mg of tectosilicate mineral particles was 17 μg / mg.

[0074] [Preparation Example 3-2] Tectosilicate mineral particles (zeolite) (product name: HSZ-940NHA, manufactured by Tosoh Corporation) were mixed with distilled water to obtain a 1% by mass tectosilicate dispersion. The median diameter of the tectosilicate mineral particles in the dispersion was 3.2 μm, and the amount of BSA adsorbed per mg of tectosilicate mineral particles was 34 μg / mg.

[0075] [Preparation Example 3-3] Magnesium oxide particles (trade name: Kyowamag MF-150, manufactured by Kyowa Chemical Industry Co., Ltd.) and distilled water were mixed to obtain a 1% by mass magnesium oxide dispersion liquid. The median diameter of the magnesium oxide particles in the dispersion was 6.6 μm. Furthermore, the amount of BSA adsorbed per mg of magnesium oxide particles was 104 μg / mg.

[0076] [Preparation Example 3-4] Hydroxyapatite particles (trade name: SHAp, manufactured by Sofsera) and distilled water were mixed to obtain a 1% by mass hydroxyapatite dispersion. The median diameter of the hydroxyapatite particles in the dispersion was 7.0 μm. Furthermore, the amount of BSA adsorbed per mg of hydroxyapatite particles was 17 μg / mg.

[0077] [Three-dimensional cell culture] A medium was prepared by adding 10 parts by mass of FBS (Gibco) to 100 parts by mass of RPMI (Fujifilm Wako Pure Chemical Industries, Ltd.). The dispersions obtained in Preparation Examples 3-1 to 3-4 were diluted with the medium and poured into a 96-well plate (Iwaki). Human kidney cancer cells, the ACHN strain, were seeded into the wells at 1,000 cells / 50 μL. After seeding the cells, the plate was cultured in an incubator at 37°C in a 5% CO2 atmosphere for 5 days.

[0078] After the culture, the plate was observed under a fluorescence microscope (product name: Biozero, manufactured by Keyence Corporation) to confirm the presence or absence of spheroid formation. The results are shown in Figure 10. As shown in FIG. 10, it can be seen that spheroids can also be formed using the dispersions obtained in Preparation Examples 3-1 to 3-4.

[0079] (Test Example 4) Confirmation of the adsorption properties of inorganic particles In order to clarify the effect of the adsorptive inorganic particles in the spheroids formed by the present invention and their location, a fluorescent marker was attached to the SWF, and the state of the spheroids was observed.

[0080] Rhodamine 6G (Kanto Chemical Co., Ltd.) was dissolved in distilled water as a fluorescent marker for SWF to prepare a 2 mM aqueous solution. 500 μL of distilled water and 500 μL of 2 mM rhodamine 6G aqueous solution were added to 1000 μL of the dispersion of Preparation Example 1-1, and the mixture was mixed using a vortex mixer. The mixed dispersion was passed through a syringe filter (pore size 0.45 μm) to obtain a dispersion of composite particles (R6G-SWF) in which rhodamine 6G was held in SWF (0.5 wt% R6G-SWF dispersion). 100 parts by mass of DMEM (Fujifilm Wako Pure Chemical Industries, Ltd.) was mixed with 10 parts by mass of FBS (Gibco) to obtain a 10% FBS / D-MEM medium. A 0.5 wt% R6G-SWF dispersion was diluted with this medium to adjust the composite particle concentration to 0.02 w / v%. The medium was then passed through a syringe filter (pore size 5 μm) and added to a 96-well plate (Iwaki) at 50 μL / well. Human colon cancer cells SW620 (1000 cells / 50 μL) prepared in 10% FBS / D-MEM medium were seeded at 50 μL / well. The SW620 cell line was obtained from the American Type Culture Collection (ATCC). After seeding, the plate was cultured in an incubator at 37°C under a 5% CO2 atmosphere.

[0081] On day 5 of culture, nuclei were stained using NucBlue Live ReadyProbes Reagent (ThermoFisher Scientific), and the spheroids were transferred to a 96-well plate (Greiner Bio-One). Z-stack images were taken at 0.3 μm intervals using a confocal laser microscope (CellVoyager CV8000, Yokogawa Electric Corporation, excitation wavelength 405 nm - 561 nm, detection wavelength 445 / 45 nm - 600 / 37 nm, 60x magnification). Maximum Intensity Projection (MIP) images were created using analysis software (CellPathfinder, Yokogawa Electric Corporation). The results are shown in Figure 11.

[0082] In the obtained MIP image, the nuclei of live cells are stained blue. The MIP image clearly shows the location of the cells forming spheroids. The present invention causes live cells to aggregate and form spheroids, and red-stained R6G-SWF is distributed around the live cells. In this way, the adsorption of inorganic particles to the cell surface promotes spheroid formation. Furthermore, since R6G-SWF is present in large amounts in the center of the spheroids, it is clear that it is present on the cell surface in the early stages of spheroid formation, and the spheroids themselves proliferate and grow outward.

[0083] Based on the above results, adsorptive inorganic particles can be used as an active ingredient in spheroid formation promoters. Furthermore, in three-dimensional cell culture, by setting the solids concentration of adsorptive inorganic particles dispersed in a cell suspension within a predetermined range, cell aggregation or organization is promoted regardless of the cell type, allowing for simple and rapid culture of spheroid-shaped cells.

[0084] While the present invention has been described in connection with embodiments thereof, we do not intend to limit our invention to any of the details of the description unless otherwise specified, and believe that the claims should be construed broadly without departing from the spirit and scope of the invention as set forth in the appended claims.

[0085] This application claims priority based on Japanese Patent Application No. 2020-049062, filed on March 19, 2020, the contents of which are incorporated herein by reference as part of the present specification.

Claims

1. A spheroid formation promoter containing adsorptive inorganic particles as an active ingredient, the inorganic particles are made of at least one inorganic substance selected from the group consisting of swellable layered silicates, phyllosilicate minerals, tectosilicate minerals, colloidal silica, and alumina hydrate; When the inorganic particles are particles made of a swellable layered silicate, the particles made of the swellable layered silicate are used at a solids concentration of 0.0010 w / v% or more and 0.1667 w / v% or less in a mixture for spheroid formation of a cell suspension and a dispersion of the swellable layered silicate, When the inorganic particles are particles made of a phyllosilicate mineral, the particles made of the phyllosilicate mineral are used at a solids concentration of 0.005 w / v% or more and 0.1667 w / v% or less in a mixture for forming spheroids, which mixture is a cell suspension and a dispersion of a swellable layered silicate; When the inorganic particles are particles made of a tectosilicate mineral, the particles made of a tectosilicate mineral are used at a solids concentration of 0.0025 w / v% or more and 0.1667 w / v% or less in a mixture for forming spheroids, which mixture is a cell suspension and a dispersion of a swellable layered silicate; When the inorganic particles are particles made of colloidal silica, the particles made of colloidal silica are used at a solids concentration of 0.001 w / v% or more and less than 0.04 w / v% in a mixture for forming spheroids, which mixture is a cell suspension and a dispersion of a swellable layered silicate; When the inorganic particles are particles made of alumina hydrate, the particles made of alumina hydrate are used at a solids concentration of 0.005 w / v% or more and 0.1667 w / v% or less in a mixture for forming spheroids, which mixture is a cell suspension and a dispersion of a swellable layered silicate. Spheroid formation promoter.

2. The spheroid formation promoter according to claim 1, wherein the inorganic particles are particles made of at least one type of swellable layered silicate selected from the group consisting of montmorillonite, hectorite, stevensite, saponite, beidellite, nontrite, sauconite, and swellable mica.

3. The spheroid formation promoter according to claim 1, wherein the inorganic particles are particles made of at least one phyllosilicate mineral selected from the group consisting of kaolinite and mica group.

4. The spheroid formation promoter according to claim 1, wherein the inorganic particles are particles made of at least one type of tectosilicate mineral selected from the group consisting of zeolite group.

5. The spheroid formation promoter according to claim 1 , wherein the inorganic particles are particles made of colloidal silica.

6. The spheroid formation promoter according to claim 1 , wherein the inorganic particles are particles made of alumina hydrate.

7. The spheroid formation promoter according to any one of claims 1 to 6, wherein the amount of bovine serum albumin adsorbed per 1 mg of the adsorptive inorganic particles is 10 µg or more and 500 µg or less.

8. A kit for spheroid formation, comprising the spheroid formation promoter according to any one of claims 1 to 7.

9. A method for producing spheroids, comprising: mixing a dispersion of the spheroid formation promoter according to any one of claims 1 to 7 with a cell suspension to obtain a mixture in which adsorptive inorganic particles contained in the spheroid formation promoter are dispersed; culturing the cells contained in the obtained mixture to form spheroids; the inorganic particles are made of at least one inorganic substance selected from the group consisting of swellable layered silicates, phyllosilicate minerals, tectosilicate minerals, colloidal silica, and alumina hydrate; When the inorganic particles are particles made of a swellable layered silicate, the particles made of the swellable layered silicate are used at a solids concentration of 0.0010 w / v% or more and 0.1667 w / v% or less in a mixture for spheroid formation of a cell suspension and a dispersion of the swellable layered silicate; When the inorganic particles are particles made of a phyllosilicate mineral, the particles made of the phyllosilicate mineral are used at a solids concentration of 0.005 w / v% or more and 0.1667 w / v% or less in a mixture for forming spheroids, which mixture is a cell suspension and a dispersion of a swellable layered silicate; When the inorganic particles are particles made of a tectosilicate mineral, the particles made of the tectosilicate mineral are used at a solids concentration of 0.0025 w / v% or more and 0.1667 w / v% or less in a mixture for forming spheroids, which mixture is a cell suspension and a dispersion of a swellable layered silicate; When the inorganic particles are particles made of colloidal silica, the particles made of colloidal silica are used at a solids concentration of 0.001 w / v% or more and less than 0.04 w / v% in a mixture for forming spheroids, which mixture is a cell suspension and a dispersion of a swellable layered silicate; When the inorganic particles are particles made of alumina hydrate, the particles made of alumina hydrate are used at a solids concentration of 0.005 w / v% or more and 0.1667 w / v% or less in a mixture for forming spheroids, which mixture is a cell suspension and a dispersion of a swellable layered silicate. Method for producing spheroids.

10. A method for promoting spheroid formation, comprising: mixing a dispersion of the spheroid formation promoter according to any one of claims 1 to 7 with a cell suspension to obtain a mixture in which adsorptive inorganic particles contained in the spheroid formation promoter are dispersed; culturing cells contained in the obtained mixture; and promoting spheroid formation, the inorganic particles are made of at least one inorganic substance selected from the group consisting of swellable layered silicates, phyllosilicate minerals, tectosilicate minerals, colloidal silica, and alumina hydrate; When the inorganic particles are particles made of a swellable layered silicate, the particles made of the swellable layered silicate are used at a solids concentration of 0.0010 w / v% or more and 0.1667 w / v% or less in a mixture for spheroid formation of a cell suspension and a dispersion of the swellable layered silicate; When the inorganic particles are particles made of a phyllosilicate mineral, the particles made of the phyllosilicate mineral are used at a solids concentration of 0.005 w / v% or more and 0.1667 w / v% or less in a mixture for forming spheroids, which mixture is a cell suspension and a dispersion of a swellable layered silicate; When the inorganic particles are particles made of a tectosilicate mineral, the particles made of the tectosilicate mineral are used at a solids concentration of 0.0025 w / v% or more and 0.1667 w / v% or less in a mixture for forming spheroids, which mixture is a cell suspension and a dispersion of a swellable layered silicate; When the inorganic particles are particles made of colloidal silica, the particles made of colloidal silica are used at a solids concentration of 0.001 w / v% or more and less than 0.04 w / v% in a mixture for forming spheroids, which mixture is a cell suspension and a dispersion of a swellable layered silicate; When the inorganic particles are particles made of alumina hydrate, the particles made of alumina hydrate are used at a solids concentration of 0.005 w / v% or more and 0.1667 w / v% or less in a mixture for forming spheroids, which mixture is a cell suspension and a dispersion of a swellable layered silicate. A method for promoting spheroid formation.

11. 11. The method according to claim 9 or 10, wherein the inorganic particles are particles made of at least one swellable layered silicate selected from the group consisting of montmorillonite, hectorite, stevensite, saponite, beidellite, nontrite, sauconite, and swellable mica.

12. 11. The method according to claim 9 or 10, wherein the inorganic particles are particles made of at least one phyllosilicate mineral selected from the group consisting of kaolinite and mica.

13. The method according to claim 9 or 10, wherein the inorganic particles are particles made of at least one tectosilicate mineral selected from the group consisting of zeolite minerals.

14. The method according to claim 9 or 10, wherein the inorganic particles are particles made of colloidal silica.

15. The method according to claim 9 or 10, wherein the inorganic particles are particles made of alumina hydrate.

16. The method according to any one of claims 9 to 15, wherein the cells are cells collected from an animal, cultured cells collected from an animal, cells collected from an animal that have been subjected to various treatments, or a cultured cell line.

17. The method according to any one of claims 9 to 16, wherein the cells are mammalian cells.

18. The method according to any one of claims 9 to 17, wherein the cells are cultured in a culture plate for suspension cells to form spheroids.

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