Spheroid formation medium

Abstract

To provide a highly versatile spheroid-forming medium that can produce spheroid-shaped cells without being limited by the type of cells to be cultured or the type of culture plate to be used, and to provide a spheroid-forming method that can easily form a spheroid without being limited by the type of cells to be cultured or the type of culture plate to be used.SOLUTION: Provided are a spheroid-forming medium containing a swelling layered silicate, and a spheroid-forming method comprising: mixing the spheroid-forming medium and a cell suspension to obtain a mixture; culturing the cells contained in the resulting mixture; and promoting the formation of a spheroid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a medium for spheroid formation and a method for forming spheroids. [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, development of media suitable for use in three-dimensional cell culture methods is underway. For example, Patent Document 1 describes a culture medium containing a polymer formed by polymerizing one or more compounds selected from the group consisting of D-glucosamine, D-galactosamine, D-glucuronic acid, L-iduronic acid, and D-galactose. Patent Document 2 describes a culture medium containing an exogenous cell aggregating agent, which includes any one of an antibody, a lectin, and a cell adhesion molecule.

[0005] As described above, 3D cell culture can be performed using special media. However, media used for 3D cell culture 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, depending on the type of cells being cultured and the type of culture plate used, cells may not form spheroids, and even if spheroids are formed, they may not have the desired shape or size, or the number of spheroids formed may be small. Therefore, there is a need for the development of a versatile medium for spheroid formation that can produce spheroid-shaped cells without being limited by the type of cells to be cultured or the type of culture plate to be used.

[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 3. Furthermore, Patent Document 4 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. However, there have been no reports of practical use of swellable layered silicates themselves in spheroid formation media. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 2017-147944 [Patent Document 2] Japanese Patent Application Laid-Open No. 2008-22743 [Patent Document 3] Japanese Patent Application Laid-Open No. 2011-121789 [Patent Document 4] International Publication No. 2012 / 133695 Summary of the Invention [Problem to be solved by the invention]

[0008] Although media for spheroid formation have been commercially available, the current situation is that spheroid formation may not occur depending on the type of cells being cultured and the type of culture plate used, and conventional media are not very versatile. Therefore, it has been difficult to perform general spheroid formation for a variety of cell types using conventional techniques.

[0009] Therefore, an object of the present invention is to provide a highly versatile medium for spheroid formation that can produce cells in the form of spheroids without being limited by the type of cells to be cultured or the type of culture plate to be used. Another object of the present invention is to provide a method for forming spheroids that can easily form spheroids without being limited by the type of cells to be cultured or the type of culture plate to be used. [Means for solving the problem]

[0010] In view of the above problems, the present inventors have conducted extensive research and found that by using a medium containing a swellable layered silicate, spheroids can be easily formed without being limited by the type of cells to be cultured or the type of culture plate to be used. The present invention has been completed based on these findings.

[0011] The above-mentioned problems of the present invention have been solved by the following means. (1) A medium for spheroid formation containing a swellable layered silicate. (2) The spheroid-forming medium according to (1) above, wherein the cation exchange capacity of the swelling layered silicate is 10 meq / 100 g or more. (3) The spheroid-forming medium according to (1) or (2), wherein the median diameter of the swellable layered silicate in the aqueous dispersion is 10 nm or more and 1000 nm or less. (4) The spheroid-forming medium according to any one of (1) to (3), wherein the amount of bovine serum albumin adsorbed per 1 mg of the swellable layered silicate is 10 μg or more and 500 μg or less. (5) The spheroid-forming medium according to any one of (1) to (4) above, wherein the swellable layered silicate is derived from a mineral belonging to the smectite group. (6) The spheroid-forming medium according to any one of (1) to (5), wherein the swellable layered silicate is at least one selected from the group consisting of montmorillonite, hectorite, stevensite, saponite, beidellite, nontrite, sauconite, and swellable mica.

[0012] (7) A method for forming spheroids, comprising mixing the spheroid-forming medium according to any one of (1) to (6) above with a cell suspension to obtain a mixture, culturing the cells contained in the obtained mixture, and forming spheroids. (8) The method for forming spheroids according to (7) above, 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. (9) The method for forming spheroids according to (7) or (8), wherein the cells are cultured in any one plate selected from the group consisting of a culture plate for adherent cells, a culture plate for suspension cells, and a plate for low-adhesion three-dimensional cell culture, to form spheroids. [Effects of the Invention]

[0013] The spheroid-forming medium of the present invention can be used to produce cells in the form of spheroids without being limited by the type of cells to be cultured or the type of culture plate to be used, and is therefore highly versatile. Furthermore, the method for forming spheroids of the present invention can easily form spheroids without being limited by the type of cells to be cultured or the type of culture plate to be used. [Brief explanation of the drawings]

[0014] [Figure 1] These are micrographs of suspension cell culture plates after culturing human glioblastoma cells U-251MG for 3 days. Figure 1(A) is a micrograph of a plate containing medium containing sodium fluoride hectorite, Figure 1(B) is a micrograph of a plate containing medium containing sodium stevensite, Figures 1(C) and 1(D) are micrographs of plates containing commercially available spheroid formation medium, and Figure 1(E) is a micrograph of a plate containing medium not containing swellable layered silicate. [Figure 2] Figure 2(A) is an enlarged micrograph of Figure 1(A), Figure 2(B) is an enlarged micrograph of Figure 1(B), Figure 2(C) is an enlarged micrograph of Figure 1(C), Figure 2(D) is an enlarged micrograph of Figure 1(D), and Figure 2(E) is an enlarged micrograph of Figure 1(E). The size of the scale bars in Figures 2(A) to (E) is 100 μm. [Figure 3] Figure 3(A) is an enlarged micrograph of Figure 1(A), Figure 3(B) is an enlarged micrograph of Figure 1(B), Figure 3(C) is an enlarged micrograph of Figure 1(C), and Figure 3(D) is an enlarged micrograph of Figure 1(D). The size of the scale bars in Figures 3(A) to (D) is 100 μm. [Figure 4]These are micrographs of suspension cell culture plates after culturing human glioblastoma cells U-251MG for 5 days. Figure 4(A) is a micrograph of a plate containing medium containing sodium fluoride hectorite, Figure 4(B) is a micrograph of a plate containing medium containing sodium stevensite, and Figures 4(C) and (D) are micrographs of plates containing commercially available spheroid formation medium. The scale bars in Figures 4(A) to (D) are 100 μm. [Figure 5] Figure 5 shows micrographs of low-adhesion 3D culture plates after culturing human glioblastoma cells U-251MG for 3 days. Figure 5(A) shows a plate containing medium containing sodium fluoride hectorite, Figure 5(B) shows a plate containing medium containing sodium stevensite, Figures 5(C) and 5(D) show plates containing commercially available spheroid formation medium, and Figure 5(E) shows a plate containing medium without swellable layered silicate. [Figure 6] Figure 6(A) is a magnified micrograph of Figure 5(A), Figure 6(B) is a magnified micrograph of Figure 5(B), Figure 6(C) is a magnified micrograph of Figure 5(C), Figure 6(D) is a magnified micrograph of Figure 5(D), and Figure 6(E) is a magnified micrograph of Figure 5(E). The size of the scale bars in Figures 6(A) to (E) is 100 μm. [Figure 7] Figure 7(A) is a magnified micrograph of Figure 5(A), Figure 7(B) is a magnified micrograph of Figure 5(B), Figure 7(C) is a magnified micrograph of Figure 5(C), Figure 7(D) is a magnified micrograph of Figure 5(D), and Figure 7(E) is a magnified micrograph of Figure 5(E). The size of the scale bars in Figures 7(A) to (E) is 100 μm. [Figure 8]These are micrographs of low-adhesion 3D culture plates after culturing human glioblastoma cells U-251MG for 5 days. Figure 8(A) is a micrograph of a plate containing medium containing sodium fluoride hectorite. Figure 8(B) is a micrograph of a plate containing medium containing sodium stevensite. Figures 8(C) and 8(D) are micrographs of plates containing commercially available spheroid formation medium. Figure 8(E) is a micrograph of a plate containing medium without swellable layered silicates. The scale bars in Figures 8(A) to 8(E) are 100 μm. [Figure 9] These are micrographs of adherent cell culture plates after culturing human glioblastoma colon adenocarcinoma cells (HT29) for 6 days. Figure 9(A) is a micrograph of a plate containing medium containing sodium hectorite, Figure 9(B) is a micrograph of a plate containing medium containing sodium stevensite, Figures 9(C) and (D) are micrographs of plates containing commercially available spheroid formation medium, and Figure 9(E) is a micrograph of a plate containing medium without swellable layered silicates. The scale bars in Figures 9(A) to 9(E) are 100 μm. [Figure 10] These are magnified micrographs of adherent cell culture plates after culturing human glioblastoma colon adenocarcinoma cells (HT29) for 7 days. Figure 10(A) is a magnified micrograph of a plate containing sodium hectorite, Figure 10(B) is a magnified micrograph of a plate containing sodium stevensite, and Figures 10(C) and 10(D) are magnified micrographs of plates containing commercially available spheroid formation media. The scale bars in Figures 10(A) to 10(D) are 100 μm. [Figure 11] 1 is a graph showing the time course of proliferation of human glioblastoma colon adenocarcinoma cells HT29 cultured in Test Example 3. [Figure 12]These are micrographs of suspension cell culture plates after culturing human glioblastoma colon adenocarcinoma cells HT29 for 6 days. Figure 12(A) is a micrograph of a plate containing medium containing sodium hectorite, Figure 12(B) is a micrograph of a plate containing medium containing sodium stevensite, Figures 12(C) and (D) are micrographs of plates containing commercially available spheroid formation medium, and Figure 12(E) is a micrograph of a plate containing medium without swellable layered silicates. The scale bars in Figures 12(A) to (E) are 100 μm. [Figure 13] These are magnified micrographs of suspension cell culture plates after culturing human glioblastoma colon adenocarcinoma cells HT29 for 7 days. Figure 13(A) is a magnified micrograph of a plate containing sodium hectorite, Figure 13(B) is a magnified micrograph of a plate containing sodium stevensite, Figures 13(C) and 13(D) are micrographs of plates containing commercially available spheroid formation media, and Figure 13(E) is a micrograph of a plate containing media without swellable layered silicates. The scale bars in Figures 13(A) to 13(E) are 100 μm. [Figure 14] 1 is a graph showing the time course of proliferation of human glioblastoma colon adenocarcinoma cells HT29 cultured in Test Example 4. [Figure 15] These are micrographs of low-attachment 3D culture plates for human glioblastoma colon adenocarcinoma HT29 cells after 6 days of culture. Figure 15(A) is a micrograph of a plate containing sodium hectorite, Figure 15(B) is a micrograph of a plate containing sodium stevensite, Figures 15(C) and 15(D) are micrographs of plates containing commercially available spheroid formation media, and Figure 15(E) is a micrograph of a plate containing media without swellable layered silicates. The scale bars in Figures 15(A) to 15(E) are 100 μm. [Figure 16]These are magnified micrographs of low-attachment 3D culture plates for human glioblastoma colon adenocarcinoma HT29 cells after 7 days of culture. Figure 16(A) is a magnified micrograph of a plate containing sodium hectorite, Figure 16(B) is a magnified micrograph of a plate containing sodium stevensite, Figures 16(C) and (D) are micrographs of plates containing a commercially available spheroid formation medium, and Figure 16(E) is a micrograph of a plate containing a medium without swellable layered silicates. The scale bars in Figures 16(A) to (E) are 100 μm. [Figure 17] 1 is a graph showing the time course of proliferation of human glioblastoma colon adenocarcinoma cells HT29 cultured in Test Example 5. [Figure 18] These are micrographs of adherent cell culture plates after culturing human alveolar basal epithelial adenocarcinoma cells A549 for 6 days. Figure 18(A) is a micrograph of a plate containing a medium containing sodium fluoride hectorite, Figures 18(B) and (C) are micrographs of plates containing a commercially available spheroid formation medium, and Figure 18(D) is a micrograph of a plate containing a medium not containing swellable layered silicate. The scale bars in Figures 18(A) to (D) are 100 μm. [Figure 19] Figure 19(A) is a magnified micrograph of Figure 18(A), Figure 19(B) is a magnified micrograph of Figure 18(B), Figure 19(C) is a magnified micrograph of Figure 18(C), and Figure 19(D) is a magnified micrograph of Figure 18(D). The size of the scale bars in Figures 19(A) to (D) is 100 μm. [Figure 20]These are micrographs of suspension cell culture plates after culturing human alveolar basal epithelial adenocarcinoma cells A549 for 6 days. Figure 20(A) is a micrograph of a plate containing medium containing sodium hectorite, Figure 20(B) is a micrograph of a plate containing medium containing fluorinated sodium hectorite, Figure 20(C) is a micrograph of a plate containing medium containing sodium stevensite, Figures 20(D) and (E) are micrographs of plates containing commercially available spheroid formation media, and Figure 20(F) is a micrograph of a plate containing medium without swellable layered silicates. The scale bars in Figures 20(A) to (F) are 100 μm. [Figure 21] Figure 21(A) is a magnified micrograph of Figure 20(A), Figure 21(B) is a magnified micrograph of Figure 20(B), Figure 21(C) is a magnified micrograph of Figure 20(C), Figure 21(D) is a magnified micrograph of Figure 20(D), and Figure 21(E) is a magnified micrograph of Figure 20(E). The size of the scale bars in Figures 21(A) to (E) is 100 μm. [Figure 22] These are micrographs of low-adhesion 3D culture plates for human alveolar basal epithelial adenocarcinoma cells A549 after 6 days of culture. Figure 22(A) is a micrograph of a plate containing medium containing sodium hectorite, Figure 22(B) is a micrograph of a plate containing medium containing fluorinated sodium hectorite, Figure 22(C) is a micrograph of a plate containing medium containing sodium stevensite, Figures 22(D) and (E) are micrographs of plates containing commercially available spheroid formation medium, and Figure 22(F) is a micrograph of a plate containing medium without swellable layered silicates. The scale bars in Figures 22(A) to (F) are 100 μm. [Figure 23]Figure 23(A) is a magnified micrograph of Figure 22(A), Figure 23(B) is a magnified micrograph of Figure 22(B), Figure 23(C) is a magnified micrograph of Figure 22(C), Figure 23(D) is a magnified micrograph of Figure 22(D), and Figure 23(E) is a magnified micrograph of Figure 22(E). The size of the scale bars in Figures 23(A) to (E) is 100 μm. [Figure 24] These are micrographs of suspension cell culture plates after culturing human neuroblastoma SH-SY5Y cells for 9 days. Figure 24(A) is a micrograph of a plate containing sodium hectorite, Figures 24(B) and (C) are micrographs of plates containing commercially available spheroid formation media, and Figure 24(D) is a micrograph of a plate containing a medium that does not contain swellable layered silicates. The scale bars in Figures 24(A) to (D) are 100 μm. [Figure 25] These are micrographs of suspension cell culture plates after culturing human neuroblastoma SH-SY5Y cells for 6 days. Figure 25(A) is a micrograph of a plate containing sodium hectorite, and Figures 25(B) and (C) are micrographs of plates containing commercially available spheroid formation media. The size of the scale bars in Figures 25(A) to (C) is 100 μm. [Figure 26] These are micrographs of low-attachment 3D culture plates for human neuroblastoma SH-SY5Y cells after 9 days of culture. Figure 26(A) is a micrograph of a plate supplemented with medium containing sodium hectorite. Figure 26(B) is a micrograph of a plate supplemented with medium containing sodium stevensite. Figures 26(C) and (D) are micrographs of plates supplemented with commercially available spheroid formation medium. Figure 26(E) is a micrograph of a plate supplemented with medium not containing swellable layered silicates. The scale bars in Figures 26(A) to (E) are 100 μm. [Figure 27]These are micrographs of low-attachment 3D culture plates for human neuroblastoma SH-SY5Y cells after 6 days of culture. Figure 27(A) is a micrograph of a plate containing sodium hectorite, Figure 27(B) is a micrograph of a plate containing sodium stevensite, Figures 27(C) and (D) are micrographs of plates containing commercially available spheroid formation media, and Figure 27(E) is a micrograph of a plate containing media without swellable layered silicates. The scale bars in Figures 27(A) to (E) are 100 μm. [Figure 28] Figure 28 shows micrographs of low-attachment 3D culture plates after culturing human alveolar basal epithelial adenocarcinoma cells A549 for 6 days. Figure 28(A) shows a plate containing a medium prepared by mixing a commercially available spheroid formation medium with sodium hectorite. Figure 28(B) shows a plate containing a medium prepared by mixing a commercially available spheroid formation medium with sodium fluoride hectorite. Figure 28(C) shows a plate containing a medium prepared by mixing a commercially available spheroid formation medium with sodium Stevensite. Figure 28(D) shows a plate containing a commercially available spheroid formation medium. [Figure 29] Figure 29 shows micrographs of low-adhesion 3D culture plates after culturing human alveolar basal epithelial adenocarcinoma cells A549 for 6 days. Figure 29(A) shows a micrograph of a plate containing a medium prepared by mixing a commercially available spheroid formation medium with sodium hectorite. Figure 29(B) shows a micrograph of a plate containing a medium prepared by mixing a commercially available spheroid formation medium with sodium fluoride hectorite. Figure 29(C) shows a micrograph of a plate containing a medium prepared by mixing a commercially available spheroid formation medium with sodium Stevensite. Figure 29(D) shows a micrograph of a plate containing a commercially available spheroid formation medium. The scale bars in Figures 29(A) to 29(D) are 100 μm. DETAILED DESCRIPTION OF THE INVENTION

[0015] The spheroid-forming medium of the present invention (hereinafter also referred to simply as "the medium of the present invention") contains a swellable layered silicate. As demonstrated in the Examples below, the swellable layered silicate promotes cell aggregation and organization, leading to the formation of spheroids, regardless of the type of cells to be cultured or the type of culture plate used. The spheroid-forming medium and spheroid-forming method of the present invention are described below based on preferred embodiments, but the present invention is not limited thereto.

[0016] 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."

[0017] The mechanism by which spheroids are formed by three-dimensional cell culture using the medium of the present invention containing swellable layered silicates is unclear. However, it is speculated that the presence of swellable layered silicates in the medium as nanoscale flat particles allows the swellable layered silicates to function as small scaffolds for spheroid formation. Furthermore, because the layer surfaces of the swellable layered silicates are negatively charged, proteins (serum components) in the medium may adsorb to the swellable layered silicates, leading to their delivery to the cell surface via the swellable layered silicates. For example, proteins such as serum or growth factors contained in the culture medium of spheroid-forming cells may adsorb to the swellable layered silicates, resulting in the formation of complexes of serum or proteins with the swellable layered silicates, which then adsorb to the cell surface and act as scaffolds, leading to spheroid formation.

[0018] The "swellable layered silicate" contained in the culture medium of 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 mixture of these sheets. 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, thixotropy, and cation exchange. Furthermore, because they are inorganic, 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. Therefore, it is possible to provide a spheroid-forming medium that has excellent uniform dispersibility and can stably maintain the uniform dispersion state for a long period of time.

[0019] The particle size of the swellable layered silicate in the aqueous dispersion used in the present invention can be determined by measuring the particle size distribution using water as a 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 swellable layered silicate in the aqueous dispersion, the median diameter of the swellable layered silicate in the aqueous dispersion is preferably 10 nm or more, more preferably 20 nm or more, and is preferably 1000 nm or less, more preferably 500 nm or less. When measuring the median diameter of swellable layered silicates, photon correlation spectroscopy is preferred because the dispersed particle size is nanoscale. 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 swellable layered silicate in water and then diluting it to approximately 0.1 w / v%. Any commercially available photon correlation spectroscopy device can be used as the measurement device. 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.

[0020] The swellable layered silicate used in the present invention may have any cation exchange capacity as long as the effects of the present invention are not impaired. 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 a suitable range imparts a negative charge to the swellable layered silicate that is suitable for adsorbing proteins (serum components). From the viewpoint of achieving excellent swelling properties and dispersion stability, the swellable layered silicate used in the present invention is preferably a silicate of a monovalent cation type such as sodium. 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 the Japan Bentonite Industry Association's Standard Test Method JBAS-106-77. For example, the amount of leached cations in montmorillonite can be calculated by leaching interlayer cations in montmorillonite with 100 mL of 1 M ammonium acetate aqueous solution per 0.5 g of montmorillonite for 4 hours or more, 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 used in the present invention, but a practical upper limit is 120 meq / 100 g or less.

[0021] The swellable layered silicate used in the present invention preferably has adsorptive properties. As used herein, "adsorbability" refers to the ability to adsorb proteins such as serum components and growth factors. Furthermore, the swellable layered silicate used in the present invention or composite particles in which proteins are adsorbed onto the swellable layered silicate also adsorb to the surface of individual cells forming spheroids, thereby promoting cell aggregation and forming spheroids. The adsorptivity of swellable layered silicates can be determined by standard methods. For example, the adsorptivity of swellable layered silicates can be measured using a bovine serum albumin (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 swellable layered silicate migrates to the solid component after centrifugation, the amount of BSA adsorbed by the swellable layered silicate can be calculated by subtracting the amount of BSA in the supernatant from the initial amount of BSA. In this way, the protein adsorptivity of the swellable layered silicate can be determined. Swellable layered silicates with protein adsorptivity are effective for spheroid formation and are preferably used in the present invention. Protein quantification can be performed using the Bradford method, the BCA method, the Lowly method, or the like. The amount of BSA adsorbed per mg of the swellable layered silicate 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 mg of swellable layered silicate, with 500 μg or less being practical and 490 μg or less being preferred. It is expected that the swellable layered silicate will function as a scaffold capable of efficiently providing growth factors to cells through protein adsorption.

[0022] The swellable layered silicate 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 smectite crystal layers themselves (see "Clay Handbook," Third Edition, Edited by the Clay Science Society of Japan, May 2009, p. 65). Specific examples of the swellable layered silicate used in the present invention include montmorillonite, hectorite, stevensite, saponite, beidellite, nontrite, sauconite and swellable mica, of which hectorite and stevensite are preferred.

[0023] The viscosity of the swellable layered silicate used in the present invention can be set arbitrarily as long as it does not impair the effects of the present invention. For example, the viscosity of a 1 w / v% 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.

[0024] The swellable layered silicate used in the present invention may be a natural product or may be synthesized according to a conventional method. When a natural product is used as the swellable layered silicate, the natural product may contain foreign substances and impurities, but from the viewpoint of spheroid formation, it is preferable that the foreign substances and impurities have been removed. Examples of methods for synthesizing swellable layered silicates include hydrothermal synthesis, melt synthesis, high-pressure synthesis, solid reaction, flame fusion, and alteration. For the synthesis method of swellable layered silicates, reference can be made to the method described in JP 2008-13401 A. In the present invention, commercially available swellable layered silicates can also be used. Examples of commercially available products include Kunipia-F (median diameter: 347.4 nm, BSA adsorption amount: 222 μg / mg, cation exchange capacity: 108 meq / 100 g, viscosity of 1 w / v% 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 w / v% 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 w / v% aqueous dispersion at 25° C.: 6 mPa·s). s), Sumecton-SWF (median diameter: 69.8 nm, BSA adsorption amount: 388 μg / mg, cation exchange capacity: 73 meq / 100 g, viscosity of 1 w / v% 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 w / v% 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.

[0025] Furthermore, the commercially available swellable layered silicates 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 the commercially available swellable layered silicates 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.

[0026] The basal medium of the medium of the present invention is not particularly limited and can be appropriately selected from commonly used basal media containing amino acids, vitamins, inorganic salts, and carbon sources such as glucose, depending on the cells to be cultured, etc. Examples include at least one basal medium selected from the group consisting of D-MEM, E-MEM, MEMα, RPMI, and Ham's F-12. In addition to the swellable layered silicate, the medium of the present invention may contain various additives, such as serum components necessary for spheroid formation and inhibitors of kinases, etc., within limits that do not impair the effects of the present invention. Furthermore, the medium of the present invention may already contain the swellable layered silicate and various additives. Alternatively, the medium of the present invention may be prepared by mixing the swellable layered silicate and various additives with a basal medium immediately before cell culture.

[0027] The solids concentration of the swellable layered silicate in the medium of the present invention is practically 0.001 w / v% or more, preferably 0.003 w / v% or more, more preferably 0.005 w / v% or more, and even more preferably 0.01 w / v% or more. The upper limit of the solids concentration is practically less than 1.000 w / v%, preferably 0.600 w / v% or less, more preferably 0.500 w / v% or less, more preferably 0.250 w / v% or less, and even more preferably 0.125 w / v% or less. If the solids concentration of the swellable layered silicate is too low, the amount of swellable layered silicate will be insufficient to form spheroids. Furthermore, when preparing a mixture of the desired concentration, the components in the medium will be overly diluted. On the other hand, if the solids concentration of the swellable layered silicate is too high, the amount of swellable layered silicate will increase, resulting in a large number of aggregates of the swellable layered silicate, making the medium unsuitable for three-dimensional cell culture. The swellable layered silicate used in the present invention may be of one type only, or may be a mixture of two or more types.

[0028] In the spheroid formation method of the present invention, the medium of the present invention is mixed with a cell suspension to obtain a mixture, and the cells contained in the obtained mixture are cultured. In order to form spheroids, it is necessary to ensure that the swellable layered silicate is uniformly present in the mixture, and it is preferable to appropriately adjust the concentration of the swellable layered silicate in the mixture. The dispersion medium used to prepare the mixture is not particularly limited as long as it is non-toxic to the cells forming the 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.

[0029] 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.

[0030] The mixture of the medium and cell suspension of the present invention contains serum components commonly used in spheroid formation. Serum components that can be used in the present invention include fetal calf serum (hereinafter also referred to as "FCS"), bovine serum, horse serum, and porcine serum. Serum contains growth factors, primarily proteins, and growth factors such as cytokines may also be used in so-called serum-free media. The mixture of the medium and cell suspension of the present invention may also contain human platelet lysate (hPL) or inhibitors of kinases, etc. By mixing the cell suspension with the medium of the present invention, the serum components adsorbed to the swellable layered silicate are supplied to the surface of the cells.

[0031] The amount of swellable layered silicate in the mixture may be sufficient to form spheroids, and can be set arbitrarily depending on the type of cells in the mixture, the number of cells, the culture environment, etc. For example, the solids concentration of the swellable layered silicate in the mixture 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, and even more preferably 0.0027 w / v% or more. The upper limit of the solids concentration 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. If the solids concentration of the swellable layered silicate is too low, the amount of swellable layered silicate will be insufficient to form spheroids. On the other hand, if the solids concentration of the swellable layered silicate is too high, the swellable layered silicate will form a gel in the mixture, inhibiting the formation of spheroids.

[0032] In the present invention, the solids concentration of the swellable layered silicate contained in the mixture can be determined by a conventional method. For example, the solids concentration of the swellable layered silicate can be determined by drying the mixture containing the swellable layered silicate by freeze-drying or the like, and then measuring the chemical composition, methylene blue adsorption amount, and cation exchange capacity.

[0033] 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, and it can be set appropriately taking into consideration the cell type, culture conditions, the purpose of use of the spheroids, etc. For example, 1 cell / μL to 10,000 cells / μL is preferred, and 10 cells / μL to 1,000 cells / μL is more preferred.

[0034] 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.

[0035] The cells contained in the mixture obtained by mixing the cell suspension with the swellable layered silicate 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 are not particularly limited and can be in the form of vessels used for cell culture in general spheroid culture methods, such as culture plates for adherent cells, culture plates for suspension cells, and plates for low-adhesion three-dimensional cell culture, and are not particularly limited in terms of material, shape, or size. Materials for vessels 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, since the swellable layered silicate itself also functions as a scaffold in the mixture, it is preferable to culture without using a commonly used scaffold from the viewpoint of cost reduction.

[0036] 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 swellable layered silicate and a cell suspension can be maintained in an environment suitable for cell culture, such as a 5% CO atmosphere at 37°C, for a given period of time, thereby achieving rapid spheroid formation. [Example]

[0037] 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, the median diameter of various swellable layered silicates was measured using a 0.1 w / v% aqueous dispersion of the swellable layered silicate with a nanoparticle analyzer (nano Partica SZ-100V2, manufactured by Horiba, Ltd.). The amount of BSA adsorption onto the swellable layered silicate was determined by adding 0.2 mL of a 1 w / v% dispersion of the swellable layered silicate or distilled water (blank) to 1 mL of a 1 mg / mL BSA solution. The mixture was then vortexed and incubated 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 method and compared with the blank. The 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 amount of BSA in the supernatant was quantified using a TaKaRa Bradford Protein Assay Kit (Takara Bio Inc.) and measuring the absorbance at 595 nm with a spectrophotometer (ASV11D-H, AS ONE Corp.). The amount of adsorption was determined by subtracting the amount of BSA in the reaction supernatant of the swellable layered silicate dispersion from the amount of BSA in the blank, and this was taken as the amount of BSA adsorbed to the swellable layered silicate.

[0038] [Preparation Example 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 (Thinky Corporation) to obtain Dispersion 1 with a hectorite solids concentration of 1 w / v %. 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.

[0039] [Preparation Example 2] Dispersion 2 with a hectorite solids concentration of 1 w / v% 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.

[0040] [Preparation Example 3] Instead of the sodium fluoride hectorite powder, 150 g of sodium stevensite powder (trade name: Sumecton-ST, manufactured by Kunimine Industries Co., Ltd.; hereinafter, simply referred to as "ST") was added to 4.5 kg of a 50% by mass aqueous isopropyl alcohol solution (water / isopropyl alcohol = 50 / 50 (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 4.5 kg of a 50% by mass aqueous isopropyl alcohol solution (water / isopropyl alcohol = 50 / 50 (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 34.1 meq / 100 g. The median diameter of sodium stevensite in the dispersion was 42.4 nm. The adsorption capacity of BSA per mg of sodium stevensite was 474 μg / mg.

[0041] (Test Example 1) Cell culture of human glioblastoma cells (1) A basal medium (hereinafter simply referred to as "EMEM-10%FCS") was prepared by adding 10 parts by mass of FCS (Gibco) to 100 parts by mass of E-MEM (Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter simply referred to as "EMEM"), a cell culture medium. The dispersions obtained in Preparation Examples 1 and 3 were added to the prepared EMEM-10%FCS so that the final concentration of the swellable layered silicate was 0.01 w / v%, to prepare swellable layered silicate-containing media (EMEM-10%FCS+SWF and EMEM-10%FCS+ST).

[0042] The swellable layered silicate-containing medium was added at 50 μL / well to a suspension cell culture plate (product name: Non-treated 96-well microplate, Iwaki, hereinafter simply referred to as "Iwaki"). Human glioblastoma (astrocytoma) cells, U-251MG strain (hereinafter simply referred to as "U251MG"; obtained from ATCC), were seeded at 2,000 cells / 50 μL / well. The concentration of the swellable layered silicate after cell seeding was 0.005 w / v%. After cell seeding, the plate was cultured in an incubator at 37°C in a 5% CO2 atmosphere. For comparison, human glioblastoma cells were cultured in the same manner, except that commercially available spheroid culture media (Cancer Stem Cell Media Premium (trade name, manufactured by ProMab, hereinafter also referred to as "CC-Premium") and 3D Tumorsphere Medium XF (trade name, manufactured by Takara Bio, hereinafter also referred to as "3D-TMXF")), which do not contain swellable layered silicate, and EMEM-10% FCS were used instead of the swellable layered silicate-containing medium. The plates were observed under a microscope (trade name: Biozero, manufactured by Keyence Corporation) on days 3 and 5 after the start of culture to confirm the presence or absence of spheroid formation. The results are shown in Figures 1 to 4.

[0043] Furthermore, an ATP assay was performed according to the following procedure. 100 μL of ATP measurement reagent (product name: CellTiter-Glo 2.0 Cell Viability Assay, manufactured by Promega) was added per well to the plate where the cells were cultured, and after pipetting, the entire volume was transferred to the wells of a 96-well white plate (manufactured by Thermo Fisher Scientific). The white plate was agitated for 2 minutes using a plate shaker (manufactured by Scientific Industries), then left at room temperature for 5 minutes. Luminescence intensity was measured using a multilabel plate reader (product name: EnVisionXcite, manufactured by PerkinElmer). The results are shown in Table 1.

[0044] [Table 1]

[0045] When cells were cultured in EMEM-10% FCS medium without swellable layered silicates, the cells observed under a microscope were amorphous, two-dimensional cells (see Figure 2(E)). In contrast, when cells were cultured in medium containing swellable layered silicates, cells were formed throughout the plate (see Figures 1(A) and (B) and Figures 2(A) and (B)). The formed cells were confirmed to have a spheroid morphology (see Figures 3(A) and (B)). Furthermore, the formed cells (spheroids) were confirmed to have high proliferation potential (see Table 1), and the size of the spheroids increased with the passage of culture time (see Figures 4(A) and (B)). When commercially available CC-Premium medium, which does not contain swellable layered silicates, was used, spheroids were formed, but the cell size was small (see Figure 3(C)). When commercially available 3D-TMXF medium, which does not contain swellable layered silicates, was used, spheroid formation was confirmed (see Figure 3(D)), but the cell number was low and the cells were unevenly distributed (see Figures 1(D), 2(D), and 4(D)). Furthermore, when commercially available spheroid medium, which does not contain swellable layered silicates, was used, cell proliferation was also lower than when medium containing swellable layered silicates was used (see Table 1).

[0046] (Test Example 2) Cell culture of human glioblastoma cells (2) Human glioblastoma cells were cultured in the same manner as in Test Example 1, except that the suspension cell culture plate used in Test Example 1 was replaced with a low-adhesion three-dimensional cell culture plate (product name: Nunclon Sphera, manufactured by Thermo Fisher Scientific). The results are shown in Figures 5 to 8 and Table 2.

[0047] [Table 2]

[0048] When cells were cultured in EMEM-10% FCS medium without swellable layered silicate, spheroids were formed (see Figure 7(E)), but the cell numbers were low (see Figures 5(E) and 8(E)), and cell proliferation was also low (see Table 2). In contrast, when cells were cultured in medium containing swellable layered silicate, cells were formed throughout the plate (see Figures 5(A) and (B) and Figures 6(A) and (B)), and the formed cells were confirmed to have a spheroid morphology (see Figures 7(A) and (B)). Furthermore, the proliferation of the formed cells (spheroids) was confirmed to be high (see Table 2), and the size of the spheroids increased with the passage of culture time (see Figures 8(A) and (B)). When a commercially available spheroid medium not containing swellable layered silicate was used, spheroids were formed (see Figures 7(C) and (D)), but the number of cells formed was small (see Figures 5(C) and (D), Figures 6(C) and (D), and Figures 8(C) and (D)), and the cell proliferation rate was also low (see Table 2).

[0049] (Test Example 3) Cell culture of human glioblastoma colon adenocarcinoma cells (1) A basal medium (hereinafter simply referred to as "RPMI-10%FCS") was prepared by adding 10 parts by weight of FCS (Gibco) to 100 parts by weight of RPMI-1640 cell culture medium (Fujifilm Wako Pure Chemical Industries, Ltd.; hereinafter simply referred to as "RPMI") The dispersions obtained in Preparation Examples 2 and 3 were added to the prepared RPMI-10%FCS so that the final concentration of the swellable layered silicate was 0.01 w / v%, and the resulting mixture was filtered through a 5 μm pore filter (trade name: Acrodisc 32 mm Syringe Filter with 5 μm Supor Membrane Non-Pyrogenic, Pall Corporation) to prepare swellable layered silicate-containing media (RPMI-10%FCS+SWN and RPMI-10%FCS+ST).

[0050] The swellable layered silicate-containing medium was added at 50 μL / well to a culture plate for adherent cells (trade name: Tissue culture plate VTC-P96, manufactured by Violamo; hereinafter simply referred to as "Violamo"). Human glioblastoma colon adenocarcinoma cells, HT29 strain (hereinafter simply referred to as "HT29"; obtained from ATCC), were seeded at 1,000 cells / 50 μL / well. The concentration of the swellable layered silicate after cell seeding was 0.005 w / v%. After cell seeding, the plate was cultured in an incubator at 37°C in a 5% CO2 atmosphere. For comparison, human glioblastoma colon adenocarcinoma cells were cultured in the same manner, except that CC-Premium, 3D-TMXF, and RPMI-10% FCS, which do not contain swellable layered silicates, were used instead of the swellable layered silicate-containing medium.

[0051] The plates were observed under a microscope (trade name: Biozero, manufactured by Keyence Corporation) on days 6 and 7 after the start of culture to confirm the presence or absence of spheroid formation. The results are shown in Figures 9 and 10. Furthermore, an ATP assay was performed on days 3 and 6 after the start of culture in the same manner as in Test Example 1 to evaluate cell proliferation. The results are shown in Figure 11.

[0052] When cells were cultured in RPMI-10% FCS medium without swellable layered silicate, the cells observed under a microscope were amorphous, two-dimensional cells (see Figure 9(E)). In contrast, when cells were cultured in a medium containing swellable layered silicate, cells were formed throughout the plate (see Figures 9(A) and (B)), and the formed cells were confirmed to have a spheroid morphology (see Figures 10(A) and (B)). Furthermore, the formed cells (spheroids) had high proliferation potential, and it was confirmed that the proliferation rate of the cells increased with the passage of culture time (see Figure 11). Spheroid formation was confirmed when commercially available 3D-TMXF medium, which does not contain swellable layered silicates, was used (see Figure 10(D)). However, when CC-Premium medium, which does not contain swellable layered silicates, was used, spheroids were formed, but the cell size was small (see Figure 10(C)). Furthermore, when commercially available spheroid medium, which does not contain swellable layered silicates, was used, cell proliferation was lower than when medium containing swellable layered silicates was used (see Figure 11).

[0053] (Test Example 4) Cell culture of human glioblastoma colon adenocarcinoma cells (2) Human glioblastoma colon adenocarcinoma cells were cultured in the same manner as in Test Example 3, except that the culture plate for adherent cells used in Test Example 3 was replaced with Iwaki culture plates for suspension cells. The results are shown in Figures 12 to 14.

[0054] When cells were cultured using RPMI-10% FCS medium that did not contain swellable layered silicate, spheroids were formed (see Figure 13(E)), but the cell count was low (see Figure 12(E)), and cell proliferation was also low (see Figure 14). In contrast, when cells were cultured using a medium that contained swellable layered silicate, cells were formed throughout the plate (see Figures 12(A) and (B)), and it was confirmed that the formed cells were in spheroid form (see Figures 13(A) and (B)). Furthermore, it was confirmed that the proliferation of the formed cells (spheroids) was high, and that the proliferation of the cells increased with the passage of culture time (see Figure 14). When a commercially available spheroid medium without swellable layered silicates was used, the number of cells formed was smaller (see Figures 12(C) and (D)) and cell proliferation was also lower (see Figure 14) compared to when a medium containing swellable layered silicates was used. Furthermore, when CC-Premium medium without swellable layered silicates was used, the size of the cells formed was smaller (see Figure 13(C)). When a commercially available 3D-TMXF medium without swellable layered silicates was used, irregularly shaped spheroids in which the cells fused and gathered in almost one place were observed (see Figure 13(D)).

[0055] (Test Example 5) Cell culture of human glioblastoma colon adenocarcinoma cells (3) Human glioblastoma colon adenocarcinoma cells were cultured in the same manner as in Test Example 3, except that the adherent cell culture plate used in Test Example 3 was replaced with the Nunclon Sphera plate for low-adhesion three-dimensional cell culture. The results are shown in Figures 15 to 17.

[0056] When cells were cultured using RPMI-10% FCS medium that did not contain swellable layered silicate, spheroids were formed (see Figure 16(E)), but the number of cells was small and unevenly distributed (see Figure 15(E)). Furthermore, cell proliferation was also low (see Figure 17). In contrast, when cells were cultured using a medium that contained swellable layered silicate, cells were formed throughout the plate (see Figures 15(A) and (B)), and it was confirmed that the formed cells were in spheroid form (see Figures 16(A) and (B)). Furthermore, it was confirmed that the proliferation of the formed cells (spheroids) was high, and that the proliferation of the cells increased with the passage of culture time (see Figure 17). When commercially available CC-Premium medium, which does not contain swellable layered silicates, was used, spheroid formation was confirmed, but the cells were irregular in shape and some cells were adhered (see Figure 16(D)). When commercially available 3D-TMXF medium, which does not contain swellable layered silicates, was used, spheroid formation was also confirmed (see Figure 16(D)), but they were unevenly distributed (see Figure 15(D)). Furthermore, when commercially available spheroid medium, which does not contain swellable layered silicates, was used, fewer cells were formed (see Figures 15(C) and (D)) and cell proliferation was also low (see Figure 17) compared to when medium containing swellable layered silicates was used.

[0057] (Test Example 6) Cell culture of human alveolar basal epithelial adenocarcinoma cells (1) A basal medium (hereinafter simply referred to as "DMEM-10%FCS") was prepared by adding 10 parts by mass of FCS (Gibco) to 100 parts by mass of D-MEM (Fujifilm Wako Pure Chemical Industries, Ltd., hereinafter simply referred to as "DMEM"), a cell culture medium. The dispersion obtained in Preparation Example 1 was added to the prepared DMEM-10%FCS so that the final concentration of the swellable layered silicate was 0.01 w / v%, and the resulting mixture was filtered through a 5 μm pore size filter (trade name: Acrodisc 32 mm Syringe Filter with 5 μm Supor Membrane Non-Pyrogenic, Pall Corporation) to prepare a swellable layered silicate-containing medium (DMEM-10%FCS+SWF).

[0058] The medium containing the swellable layered silicate was added to a Violamo culture plate for adherent cells at 50 μL / well. Human alveolar basal epithelial adenocarcinoma cells A549 (hereinafter simply referred to as "A549"; obtained from ATCC) were seeded at 1,000 cells / 50 μL / well. The concentration of the swellable layered silicate after cell seeding was 0.005 w / v%. After cell seeding, the plate was cultured in an incubator at 37°C in a 5% CO2 atmosphere. For comparison, human alveolar basal epithelial adenocarcinoma cells were cultured in the same manner, except that CC-Premium, 3D-TMXF, and DMEM-10% FCS, which do not contain swellable layered silicates, were used instead of the swellable layered silicate-containing medium.

[0059] On day 6 after the start of culture, the plates were observed under a microscope (product name: Biozero, manufactured by Keyence Corporation) to confirm the presence or absence of spheroid formation. The results are shown in Figures 18 and 19. Furthermore, on day 6 after the start of culture, an ATP assay was performed in the same manner as in Test Example 1 to evaluate cell proliferation. The results are shown in Table 3.

[0060] [Table 3]

[0061] When cell culture was performed using DMEM-10% FCS medium that did not contain swellable layered silicate, cell proliferation was confirmed from the results of the ATP assay (see Table 3), but the cells confirmed by microscopic observation were two-dimensional cells (see Figure 19(D)). In contrast, when cell culture was performed using a medium containing swellable layered silicate, cells were formed throughout the plate (see Figure 18(A)), and it was confirmed that the formed cells were in spheroid form (see Figure 19(A)). Furthermore, it was confirmed that the formed cells (spheroids) had high proliferation potential (see Table 3). When CC-Premium medium, which does not contain swellable layered silicates, was used, the cells confirmed by microscopic observation were two-dimensional cells (see Figure 19(B)). When commercially available 3D-TMXF medium, which does not contain swellable layered silicates, was used, spheroid formation was confirmed, but the cells were irregular in shape and some of them were adhered (see Figures 18(C) and 19(C)). Furthermore, when commercially available spheroid medium, which does not contain swellable layered silicates, was used, cell proliferation was lower than when medium containing swellable layered silicates was used (see Table 3).

[0062] (Test Example 7) Cell culture of human alveolar basal epithelial adenocarcinoma cells (2) To 100 parts by mass of DMEM, 10 parts by mass of FCS (Gibco) was added to prepare DMEM-10% FCS. The dispersions obtained in Preparation Examples 1 to 3 were added to the prepared DMEM-10% FCS so that the final concentration of the swellable layered silicate was 0.01 w / v%, and the resulting mixture was filtered through a 5 μm pore size filter (product name: Acrodisc 32 mm Syringe Filter with 5 μm Super Membrane Non-Pyrogenic, Pall Corporation) to prepare swellable layered silicate-containing media (DMEM-10% FCS + SWN, DMEM-10% FCS + SWF, and DMEM-10% FCS + ST).

[0063] The medium containing the swellable layered silicate was added to an Iwaki suspension cell culture plate at 50 μL / well. A549 cells were seeded at 1,000 cells / 50 μL / well. The concentration of the swellable layered silicate after cell seeding was 0.005 w / v%. After cell seeding, the plate was cultured in an incubator at 37°C in a 5% CO2 atmosphere. For comparison, human alveolar basal epithelial adenocarcinoma cells were cultured in the same manner, except that CC-Premium, 3D-TMXF, and DMEM-10% FCS, which do not contain swellable layered silicates, were used instead of the swellable layered silicate-containing medium.

[0064] On day 6 after the start of culture, the plates were observed under a microscope (product name: Biozero, manufactured by Keyence Corporation) to confirm the presence or absence of spheroid formation. The results are shown in Figures 20 and 21. Furthermore, on day 6 after the start of culture, an ATP assay was performed in the same manner as in Test Example 1 to evaluate cell proliferation. The results are shown in Table 4.

[0065] [Table 4]

[0066] When cell culture was performed using DMEM-10% FCS medium that did not contain swellable layered silicate, cell proliferation was confirmed from the results of the ATP assay (see Table 4), but the cells confirmed by microscopic observation were two-dimensional cells (see Figure 20(F)). In contrast, when cell culture was performed using a medium containing swellable layered silicate, cells were formed throughout the plate (see Figures 20(A) to (C)), and it was confirmed that the formed cells were in spheroid form (see Figures 21(A) to (C)). Furthermore, it was confirmed that the formed cells (spheroids) had high proliferation potential (see Table 4). When CC-Premium medium, which does not contain swellable layered silicates, was used, spheroids were formed, but the cell size was small, the cell number was low, and the cell distribution was uneven (see Figures 20(D) and 21(D)). When commercially available 3D-TMXF medium, which does not contain swellable layered silicates, was used, spheroid formation was confirmed, but the cell shape was irregular and some cells were adhered (see Figures 20(E) and 21(E)). Furthermore, when commercially available spheroid medium, which does not contain swellable layered silicates, was used, cell proliferation was lower than when medium containing swellable layered silicates was used (see Table 4).

[0067] (Test Example 8) Cell culture of human alveolar basal epithelial adenocarcinoma cells (3) Human alveolar basal epithelial adenocarcinoma cells were cultured in the same manner as in Test Example 7, except that the adherent cell culture plate used in Test Example 7 was replaced with the Nunclon Sphera plate for low-adhesion three-dimensional cell culture. The results are shown in Figures 22 and 23 and Table 5.

[0068] [Table 5]

[0069] When cells were cultured using DMEM-10% FCS medium that did not contain swellable layered silicate, spheroids were formed, but the number of cells was small and unevenly distributed (see Figure 22(F)). Furthermore, cell proliferation was also low (see Table 5). In contrast, when cells were cultured using a medium that contained swellable layered silicate, cells were formed throughout the plate (see Figures 22(A) to (C)), and it was confirmed that the formed cells were in spheroid form (see Figures 23(A) to (C)). Furthermore, it was confirmed that the formed cells (spheroids) had high proliferation potential (see Table 5). When commercially available CC-Premium medium, which does not contain swellable layered silicates, was used, spheroids were formed, but the cell size was small, the cell number was low, and the cell distribution was uneven (see Figures 22(D) and 23(D)). When commercially available 3D-TMXF medium, which does not contain swellable layered silicates, was used, spheroid formation was also confirmed (see Figure 22(E)), but the cell distribution was uneven (see Figure 23(E)). Furthermore, when commercially available spheroid medium, which does not contain swellable layered silicates, was used, cell proliferation was lower than when medium containing swellable layered silicates was used (see Table 5).

[0070] (Test Example 9) Human neuroblastoma cell culture (1) A basal medium (hereinafter simply referred to as "Ham's-F-12:E-MEM-10%FCS") was prepared by adding 10 mass parts of FCS (Gibco) to 100 mass parts of Ham's F-12:E-MEM (mixing volume ratio = 1:1, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.; hereinafter simply referred to as "Ham's-F12:EMEM"), a cell culture medium. The dispersion obtained in Preparation Example 2 was added to the prepared Ham's-F12:EMEM-10%FCS so that the final concentration of the swellable layered silicate was 0.01 w / v%, and the resulting mixture was filtered through a 5 μm pore size filter (product name: Acrodisc 32 mm Syringe Filter with 5 μm Supor Membrane Non-Pyrogenic, manufactured by Pall Corporation) to prepare a medium containing the swellable layered silicate (Ham's-F12:EMEM-10%FCS+SWN).

[0071] The medium containing the swellable layered silicate was added to an Iwaki suspension cell culture plate at 50 μL / well. Human neuroblastoma cell line SH-SY5Y (hereinafter simply referred to as "SH-SY5Y"; obtained from ATCC) was seeded at 1,000 cells / 50 μL / well. The concentration of the swellable layered silicate after cell seeding was 0.005 w / v%. After cell seeding, the plate was cultured in an incubator at 37°C in a 5% CO2 atmosphere. For comparison, human neuroblastoma cells were cultured in the same manner except that CC-Premium, 3D-TMXF, and Ham's-F12:EMEM-10%FCS, which do not contain swellable layered silicates, were used instead of the swellable layered silicate-containing medium.

[0072] The plates were observed under a microscope (trade name: Biozero, manufactured by Keyence Corporation) on days 6 and 9 after the start of culture to confirm the presence or absence of spheroid formation. The results are shown in Figures 24 and 25.

[0073] When cells were cultured using Ham's-F12:EMEM-10%FCS medium that did not contain swellable layered silicate, spheroids were formed, but the cell size was small, the cell number was low, and they were unevenly distributed (see Figure 24(D)). In contrast, when cells were cultured using a medium that contained swellable layered silicate, cells were formed throughout the plate (see Figure 24(A)), and it was confirmed that the formed cells were in spheroid form (see Figure 25(A)). When a commercially available medium not containing swellable layered silicate was used, spheroids were formed (see Figures 25(B) and (C)), but the number of cells was small and unevenly distributed (see Figures 24(B) and (C)).

[0074] (Test Example 10) Human neuroblastoma cell culture (2) Ham's-F-12:E-MEM-10%FCS was prepared by adding 10 parts by weight of FCS (Gibco) to 100 parts by weight of Ham's-F12:EMEM. The dispersions obtained in Preparation Examples 2 and 3 were added to the prepared Ham's-F12:EMEM-10%FCS so that the final concentration of the swellable layered silicate was 0.01 w / v%, and the resulting mixture was filtered through a 5 μm pore size filter (trade name: Acrodisc 32 mm Syringe Filter with 5 μm Super Membrane Non-Pyrogenic, Pall Corporation) to prepare swellable layered silicate-containing media (Ham's-F12:EMEM-10%FCS+SWN and Ham's-F12:EMEM-10%FCS+ST).

[0075] The swellable layered silicate-containing medium was added to Nunclon Sphera plates for low-adhesion three-dimensional cell culture at 50 μL / well. Human neuroblastoma SH-SY5Y cells (hereinafter simply referred to as "SH-SY5Y"; obtained from ATCC) were seeded at 1,000 cells / 50 μL / well. The concentration of the swellable layered silicate after cell seeding was 0.005 w / v%. After cell seeding, the plate was cultured in an incubator at 37°C in a 5% CO2 atmosphere. For comparison, human neuroblastoma cells were cultured in the same manner except that CC-Premium, 3D-TMXF, and Ham's-F12:EMEM-10%FCS, which do not contain swellable layered silicates, were used instead of the swellable layered silicate-containing medium.

[0076] The plates were observed under a microscope (trade name: Biozero, manufactured by Keyence Corporation) on days 6 and 9 after the start of culture to confirm the presence or absence of spheroid formation. The results are shown in Figures 26 and 27.

[0077] When cells were cultured in Ham's-F12:EMEM-10%FCS medium that did not contain swellable layered silicate, spheroids were formed, but the cells were irregular in shape and some cells were adhered (see Figures 26(E) and 27(E)). In contrast, when cells were cultured in a medium containing swellable layered silicate, cells were formed over the entire plate (see Figures 26(A) and (B)), and it was confirmed that the formed cells were in spheroid form (see Figures 27(A) and (B)). When a commercially available medium not containing swellable layered silicate was used, spheroids were formed (see Figures 27(D) and (E)), but the cell size was small, the number of cells was low, and the cells were unevenly distributed (see Figures 26(D) and (E)).

[0078] (Test Example 11) Cell culture of human alveolar basal epithelial adenocarcinoma cells (4) The dispersions obtained in Preparation Examples 1 to 3 were added to 100 parts by mass of CC-Premium or 3D-TMXF so that the final concentration of the swellable layered silicate was 0.01 w / v%, and the resulting mixture was filtered through a 5 μm pore size filter (product name: Acrodisc 32 mm Syringe Filter with 5 μm Supor Membrane Non-Pyrogenic, manufactured by Pall Corporation) to prepare swellable layered silicate-containing media (CC-Premium+SWN, CC-Premium+SWF, CC-Premium+ST, 3D-TMXF+SWN, 3D-TMXF+SWF, and 3D-TMXF+ST).

[0079] The swellable layered silicate-containing medium was added to a Nunclon Sphera plate for low-adhesion three-dimensional cell culture at 50 μL / well. A549 cells were seeded at 1,000 cells / 50 μL / well. The swellable layered silicate concentration after cell seeding was 0.005 w / v%. After cell seeding, the plate was cultured in an incubator at 37°C in a 5% CO2 atmosphere. As a comparison, human alveolar basal epithelial adenocarcinoma cells were cultured in the same manner except that CC-Premium and 3D-TMXF, which do not contain swellable layered silicate, were used.

[0080] On day 6 after the start of culture, the plate was observed under a microscope (product name: Biozero, manufactured by Keyence Corporation) to confirm the presence or absence of spheroid formation. The results are shown in Figures 28 and 29.

[0081] As shown in Figures 28(D) and 29(D) and in the above-mentioned Test Examples 6 to 8, desired spheroids were not formed even when commercially available spheroid culture media were used. In contrast, when cells were cultured using a medium prepared by adding swellable layered silicate to a commercially available spheroid medium, spheroid-shaped cells were formed over the entire plate (see Figures 28(A)-(C) and 29(A)-(C)).

[0082] The results of spheroid formation under various culture conditions are summarized in Table 6.

[0083] [Table 6]

[0084] Notes for Table 6 ◎: Spheroids formed throughout. ○: Spheroids are formed, but they are small, few, or unevenly distributed. △: Spheroids are formed, but some are not adhered or the shape is distorted. ×: Cells present in a single layer with an irregular shape and adhering to the plate. Violamo: Plates for Adherent Cells Iwaki: Plates for suspension cells Nunclon sphera: Plates for 3D culture

[0085] As shown in Table 6, when cell culture is performed using a medium that does not contain swellable layered silicate, spheroids may not form depending on the cell type being cultured and the plate used. In contrast, when cell culture is performed using the medium of the present invention containing a swellable layered silicate, spheroids can be formed regardless of the type of cells to be cultured or the type of culture plate used.

[0086] The above results demonstrate that the use of the spheroid-forming medium of the present invention enables three-dimensional culture of many types of cells. Furthermore, the spheroid-forming medium of the present invention is not limited to the culture plates that can be used. Therefore, the spheroid-forming medium of the present invention is more versatile than conventional three-dimensional culture media available on the market.

Claims

1. A medium for spheroid formation comprising a dispersed solid component made of a swellable layered silicate, The spheroid formation medium contains the swellable layered silicate at a solids concentration of 0.001 to 0.250 w / v %, and the swellable layered silicate is an active ingredient that exhibits a spheroid formation-promoting effect.

2. The spheroid-forming medium according to claim 1, wherein the cation exchange capacity of the swellable layered silicate is 10 meq / 100 g or more.

3. The spheroid-forming medium according to claim 1 or 2, wherein the median diameter of the swellable layered silicate in the aqueous dispersion is 10 nm or more and 1000 nm or less.

4. The spheroid-forming medium according to any one of claims 1 to 3, wherein the amount of bovine serum albumin adsorbed per 1 mg of the swellable layered silicate is 10 μg or more and 500 μg or less.

5. The spheroid-forming medium according to any one of claims 1 to 4, wherein the swellable layered silicate is derived from a mineral belonging to the smectite group.

6. The spheroid-forming medium according to any one of claims 1 to 5, wherein the swellable layered silicate is at least one selected from the group consisting of montmorillonite, hectorite, stevensite, saponite, beidellite, nontrite, sauconite, and swellable mica.

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