Method for producing cell spheroids

Abstract

A novel method for producing undifferentiated cell spheroids is provided. [Solution] We provide a method for culturing undifferentiated cells on the cell-adhesive surface of a cell culture sheet, based on the fact that culturing on a culture surface having a specific surface not only makes it possible to produce undifferentiated cell spheroids but also makes it possible to maintain the undifferentiated state, and that culturing on a culture surface having a specific surface makes it possible to produce cell spheroids that express integrins more predominantly than when cultured in suspension on a non-cell-adhesive surface.

Description

[Technical Field]

[0001] The present invention relates to a method for producing cell spheroids. More specifically, the present invention relates to a method for producing undifferentiated cell spheroids, a method for maintaining the undifferentiated state of the spheroids, and cell spheroids obtained by the method. [Background technology]

[0002] In recent years, there has been growing momentum to utilize undifferentiated stem cells, such as iPS cells, in regenerative medicine, and active research is being conducted into technologies for culturing cells that maintain an undifferentiated state for clinical application.

[0003] For example, Patent Document 1 discloses that the cell culture carrier has specific pores, and therefore when seeding undifferentiated stem cells such as ES cells and iPS cells, suction can be applied from the back side of the carrier and / or pressure can be applied from the front side, and cells seeded outside the wells can be guided into the wells and efficiently aggregated, thereby allowing the cells to proliferate while maintaining their undifferentiated state and forming spheroids.

[0004] In Non-Patent Document 1, spheroids are formed by seeding embryonic stem cells into each well of a V-bottom low cell-adhesion plate.

[0005] Furthermore, Patent Document 2 discloses that reprogrammed somatic cells can be efficiently induced into iPS cells by seeding them on fibronectin-coated culture vessels and culturing them in a serum-free medium without the use of feeder cells, and that under these culture conditions the undifferentiated state and pluripotency of the iPS cells can be maintained for a long period of time. Furthermore, low-cell-adhesion plates are used to subsequently form embryoid bodies, as in Non-Patent Document 1.

[0006] On the other hand, Patent Document 3 discloses that by culturing iPS cells under a specific gravity environment, they can be proliferated while maintaining their undifferentiated state, even in the absence of feeder cells or coating agents, and can form and grow spheroids. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Publication No. 2017-212972 [Patent Document 2] Japanese Patent Application Laid-Open No. 2015-123079 [Patent Document 3] Patent No. 6421374 [Non-patent literature]

[0008] [Non-Patent Document 1] Cell Stem Cell 10, 771-785, June 14, 2012 Summary of the Invention [Problem to be solved by the invention]

[0009] However, while the cell culture carrier of Patent Document 1 allows for the formation of spheroids, it is a porous material produced by firing ceramics, making it opaque and difficult to directly observe the inside of the container. Furthermore, the containers of Non-Patent Document 1 and Patent Document 2 do not have adhesive surfaces, and since one spheroid is prepared per well, it is difficult to obtain large quantities of cells. Furthermore, the method of Patent Document 3 requires complex operations to achieve a gravitational environment by adjusting the rotation speed of the rotating bioreactor, and there are concerns that the rotation may cause shear stress on the cells, resulting in a decrease in cell function. Therefore, there is still room for improvement in both methods, and further improved technologies are needed.

[0010] One aspect of the present invention aims to provide a novel method for producing undifferentiated cell spheroids, a novel method for maintaining the undifferentiated state of the spheroids, and undifferentiated cell spheroids obtained by the method. Another aspect of the present invention aims to provide a novel method for producing cell spheroids that predominantly express integrin, and an integrin-expressing cell spheroid obtained by the method. [Means for solving the problem]

[0011] Generally, culturing undifferentiated cells such as iPS cells requires culturing them on feeder cells or on a substrate coated with a biological component such as collagen. However, the present inventors conducted extensive research to achieve the above-mentioned object and discovered that culturing on a culture surface having a specific surface not only enables the production of undifferentiated cell spheroids but also enables the maintenance of undifferentiation, thereby completing one aspect of the present invention. Furthermore, the present inventors discovered that culturing on a culture surface having a specific surface enables the production of cell spheroids that express integrins preferentially compared to suspension culture on a non-cell-adhesive surface, thereby completing another aspect of the present invention.

[0012] That is, the present invention relates to the following [1] to

[14] . [1] A method for producing undifferentiated cell spheroids, comprising a step of culturing undifferentiated cells on the cell-adhesive surface of a cell culture sheet. [2] A method for maintaining the undifferentiated state of cell spheroids, comprising the step of culturing undifferentiated cell spheroids on the cell adhesive surface of a cell culture sheet. [3] A method for producing integrin-expressing (integrin-containing) cell spheroids, comprising a step of culturing cells (cells that express or can express integrin, particularly undifferentiated cells) on the cell adhesive surface of a cell culture sheet. [4] The method according to any one of [1] to [3] above, wherein the cell adhesive surface is composed of a substance that exhibits cell adhesiveness. [5] The method according to any one of [1] to [4] above, wherein the cell adhesive surface comprises a polyimide resin. [6] The method according to any one of [1] to [5] above, wherein the cells are cultured on a flat cell adhesive surface. [7] The method according to any one of [1] to [6] above, wherein the cells are cultured on a cell culture sheet having a plurality of recesses with an opening diameter of 2000 μm or less, the inner side surfaces of the recesses having a non-cell-adhesive surface, and the bottom surfaces of the recesses having a cell-adhesive surface. [8] The method according to any one of [1] to [7] above, wherein the culture is carried out in the absence of feeder cells. [9] The method according to any one of [1] to [8] above, wherein the cells are undifferentiated stem cells or progenitor cells.

[10] The method according to [9] above, wherein the stem cells are hematopoietic stem cells, mesenchymal stem cells, neural stem cells, tissue stem cells, embryonic stem cells, or pluripotent stem cells.

[11] A cell spheroid obtained by the method according to any one of [1] to

[10] above.

[12] Compared to spheroids cultured in suspension on a non-cell-adhesive surface, 5 Undifferentiated cell spheroids in which the expression level of undifferentiated markers per cell is more than three times higher than the relative gene expression level at the mRNA level.

[13] A cell spheroid in which the expression level of integrin at the protein level is 1.2 times or more higher than that of a spheroid cultured in suspension on a non-cell-adhesive surface.

[14] The spheroid according to any one of

[11] to

[13] above, wherein the diameter of the spheroid is 10 to 1500 μm. [Effects of the Invention]

[0013] According to the present invention, spheroids that maintain an undifferentiated state can be produced simply and efficiently. [Brief explanation of the drawings]

[0014] [Figure 1]FIG. 1 is a diagram showing a schematic cross-sectional structure of one embodiment of a cell culture sheet that can be used in the present invention. [Figure 2] FIG. 2 is a diagram showing a schematic cross-sectional structure of one embodiment of the cell culture sheet that can be used in the present invention. [Figure 3] FIG. 3 is a diagram showing a schematic cross-sectional structure of one embodiment of the cell culture sheet that can be used in the present invention. [Figure 4] FIG. 4 is a diagram schematically illustrating one embodiment of cells cultured on a cell culture sheet that can be used in the present invention. [Figure 5] FIG. 5 is a diagram schematically illustrating one embodiment of cells cultured on a cell culture sheet that can be used in the present invention. [Figure 6] FIG. 6 is a diagram showing a schematic cross-sectional structure of one embodiment of the cell culture sheet that can be used in the present invention. [Figure 7] 7 is a photograph of the embryoid bodies formed in Example 1. The scale bar indicates 200 μm. [Figure 8] 8 is a photograph of the embryoid bodies formed in Comparative Example 1. The scale bar indicates 200 μm. [Figure 9] FIG. 9 shows the relative gene expression levels of undifferentiation markers. [Figure 10] 10A and 10B are photographs of one embodiment of a cell culture sheet or cell culture vessel that can be used in the present invention, taken from the bottom side of the well. The scale bar in Fig. 10A represents 1 mm, and the scale bar in Fig. 10B represents 5 cm. [Figure 11] FIG. 11 shows the protein expression levels of undifferentiation markers. [Figure 12] FIG. 12 shows the expression level of integrin αv. [Figure 13] 13 is a fluorescence micrograph of a cell spheroid cultured using a cell culture sheet that can be used in the present invention. The scale bar indicates 100 μm. DETAILED DESCRIPTION OF THE INVENTION

[0015] One aspect of the present invention provides a method for producing undifferentiated cell spheroids, which includes a step of culturing undifferentiated cells on the cell adhesive surface of a cell culture sheet. Note that "undifferentiated cell spheroids" refers to cell masses that can be induced to differentiate into target cells by adding cell growth factors, differentiation inducers, etc. Another aspect of the present invention provides a method for producing integrin-expressing cell spheroids, which includes a step of culturing cells on the cell adhesive surface of a cell culture sheet.

[0016] The term "cell-adhesive surface" as used herein refers to a surface on which, when cells settle in a culture solution, they adhere with a certain number of adhesion points. Alternatively, it refers to a surface on which cells adhere so as to be immobilized to the extent that they can be detached by liquid flow, such as pipetting. Furthermore, it refers to a surface on which cells adhere to form a three-dimensional or three-dimensional tissue, such as a layer or spheroid, rather than a surface on which cells adhere and maintain or grow two-dimensionally. Such a surface is presumed to allow cells or undifferentiated cells to adhere appropriately to the sheet, and furthermore, to allow appropriate cell-cell interactions to occur, enabling close intercellular communication, leading to proliferation while maintaining an undifferentiated state, as well as the formation of spheroids (cell masses) (and thus the formation of adherent spheroids that can be detached without the use of chemicals such as enzymes). Additionally, cells cultured on such a cell-adhesive surface are subjected to stimuli different from those in a normal floating state, resulting in high expression of integrins, which affect cell migration, proliferation, differentiation, and survival. In spheroids with high integrin expression, cell death is suppressed even within the spheroid due to the action of integrins. Such spheroids are presumed to contribute to tissue regeneration because they can efficiently adhere, spread, proliferate, and differentiate at the transplant site. Furthermore, high integrin expression is believed to strongly contribute to the suppression of stem cell differentiation, and when undifferentiated cells are cultured, high integrin expression makes it easier for the cells to maintain their undifferentiated state. However, this presumption does not limit the present invention.

[0017] The cell adhesive surface may be made of a substance that exhibits at least cell adhesiveness.

[0018] The cell adhesive substance can be any substance to which the cells used for culture adhere or which can bind to cell surface molecules, such as proteins or sugar chains, present in the cell membrane of the cells used. While either hydrophilic or hydrophobic substances may be used, from the viewpoints of cell adhesiveness, spheroid formability, etc., hydrophilic substances (particularly, hydrophilic but not superhydrophilic) or hydrophobic substances (particularly, hydrophobic but not superhydrophobic) are preferred, with hydrophobic substances being more preferred. Furthermore, the degree of adhesiveness of the cell adhesive substance may be such that the cells do not protrude from the wells. Examples of such substances include substances obtained from living organisms or synthesized, such as proteins (collagen, fibronectin, laminin, etc.) and synthetic resins (fluororesins, polyimide resins, polysulfones, polyethersulfones, polydimethylsiloxanes, mixtures thereof, etc.). When a synthetic resin is selected, the strength and heat resistance of the synthetic resin itself allow for a cell culture sheet with excellent handleability. Furthermore, it is preferable to select a synthetic resin such as a polyimide resin from the viewpoints of biocompatibility, improved spheroid uniformity, ease of medium replacement due to moderate adhesion to various cells, the production of adhesive spheroids, and the improved uniformity of the resulting spheroids. By selecting a non-biologically derived component such as a polyimide resin, spheroids obtained using a cell culture sheet containing a polyimide resin can be easily applied to fields such as regenerative medicine and drug discovery.

[0019] Examples of polyimide resins include polyimide resins containing a structural unit represented by the following formula (I). Furthermore, from the viewpoint of favorable spheroid formation, resins containing fluorine atoms in the molecule are preferred, and fluorine-containing polyimides (fluorine-containing polyimide resins) are more preferred. The polyimide resin used in the present invention is typically obtained by imidizing polyamic acid obtained by polymerizing at least one acid dianhydride and at least one diamine. The polyimide resin may contain polyamic acid as part of its chemical structure. A known method for producing the polyimide resin may be used. For example, a two-stage synthesis method can be used. The two-stage synthesis method for polyimide resins involves synthesizing a polyamic acid as a precursor and converting the polyamic acid into a polyimide. The polyamic acid as a precursor may be a polyamic acid derivative. Examples of polyamic acid derivatives include polyamic acid salts, polyamic acid alkyl esters, polyamic acid amides, polyamic acid derivatives from bismethylidene pyromelitides, polyamic acid silyl esters, and polyamic acid isoimides. Examples of polyimides include polyimides made from acid anhydrides such as pyromellitic dianhydride, biphenyltetracarboxylic dianhydride, and benzophenonetetracarboxylic dianhydride, and diamines such as oxydiamine, paraphenylenediamine, metaphenylenediamine, and benzophenonediamine. Examples of resins having fluorine atoms include fluorine-containing polyimide resins containing a structural unit represented by the following formula (I), such as 4,4'-hexafluoroisopropylidenediphthalic anhydride (6FDA) / 1,4-bis(aminophenoxy)benzene (TPEQ) copolymer, 6FDA / 4,4'-oxydiphthalic anhydride (ODPA) / TPEQ copolymer, 4,4'-(4,4'-isopropylidenediphenoxy)diphthalic acid (BPADA) / 2,2-bis[4-(4-aminophenoxy)phenyl]hexafluoropropane (HFBAPP), and 6FDA / 2,2-bis(4-(4-aminophenoxy)phenyl)propane (BAPP) copolymer; and ethylene-tetrafluoroethylene copolymer.

[0020] [ka]

[0021] In the above formula (I), X 0 represents an oxygen atom, a sulfur atom, a carbonyl group, a sulfonyl group, or a divalent organic group; Y represents a divalent organic group; Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , and Z 6 each independently represents a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, p is 0 or 1. In the polyimide resin, the chemical structure represented by formula (I) may be different for each structural unit of the resin, or may be the same. 0 , Y, Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , and Z 6 At least one of them preferably contains one or more fluorine atoms.

[0022] In the above formula (I), when p=0, X 0 may not exist (in other words, the left and right benzene rings may be directly bonded), but when p=1, the left and right benzene rings are 0 Connect via.

[0023] X 0 Specific examples of the divalent organic group represented by the formula (I) include an alkylene group, an arylene group, an aryleneoxy group, and an arylenethio group. Also included are a fused ring type divalent hydrocarbon group, a heterocyclic fused ring type divalent hydrocarbon group, and oxy and thio groups thereof. Among these, alkylene groups, aryleneoxy groups, and arylenethio groups are preferred, and alkylene groups and aryleneoxy groups are more preferred, which may be substituted with a fluorine atom. The alkylene group has, for example, 1 to 12 carbon atoms, and preferably 1 to 6 carbon atoms.

[0024] X 0 Examples of alkylene groups substituted with fluorine atoms include -C(CF3)2-, -C(CF3)2-C(CF3)2-, etc. 0 Among the alkylene groups mentioned above, which are examples of -C(CF3)2-, is preferred.

[0025] X 0 Examples of the arylene group include the following:

[0026] [ka]

[0027] X 0 Examples of the aryleneoxy group include the following:

[0028] [ka]

[0029] X 0 Examples of the arylene thio group include the following:

[0030] [ka]

[0031] From the viewpoint of being able to form spheroids well on the substrate, X 0 The divalent organic group represented by may be one selected from the group consisting of the above b-2 to b-10 and c-2 to c-10, or may be one selected from the group consisting of the above b-7 to b-9 and c-7 to c-9, or may be a structure represented by b-8.

[0032] X 0The above-mentioned arylene group, aryleneoxy group, and arylenethio group, which are examples of the aryl group, may each independently be substituted with a group selected from the group consisting of a halogen atom (e.g., a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, preferably a fluorine atom or a chlorine atom, more preferably a fluorine atom), a methyl group, and a trifluoromethyl group. There may be a plurality of these substituents, and in such cases, the types of the substituents may be the same or different. Preferred substituents on the arylene group, aryleneoxy group, and arylenethio group are a fluorine atom and / or a trifluoromethyl group, and preferably a fluorine atom. When Y does not contain a fluorine atom, the arylene group, aryleneoxy group, and arylenethio group are preferably substituted with at least one or more fluorine atoms.

[0033] In the above formula (I), the divalent organic group represented by Y is not particularly limited, but examples thereof include divalent organic groups having an aromatic ring. More specifically, examples thereof include a group consisting of one benzene ring, or a group having a structure in which two or more benzene rings are bonded via a carbon atom (i.e., a single bond or an alkylene group), an oxygen atom, a sulfur atom, or directly. Specific examples thereof include the following groups.

[0034] [ka]

[0035] [ka]

[0036] [ka]

[0037] [ka]

[0038] The divalent organic group having an aromatic ring, which is an example of Y, may be substituted with a group selected from the group consisting of a halogen atom (e.g., a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, preferably a fluorine atom or a chlorine atom, more preferably a fluorine atom), a methyl group, and a trifluoromethyl group, if possible. There may be a plurality of these substituents, and in this case, the types of the substituents may be the same or different. Suitable substituents substituted on the divalent organic group having an aromatic ring are particularly X 0 When does not contain a fluorine atom, it is preferably a fluorine atom and / or a trifluoromethyl group, and more preferably a fluorine atom.

[0039] From the viewpoint of spheroid formation, in the above formula (I), Y is preferably a structure selected from the group consisting of d-3, d-9, e-1 to e-4, f-6, and f-7, and more preferably a structure of e-1, e-3, or e-4.

[0040] In the above formula (I), Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , and Z 6 may be the same or different and are each independently selected from a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom; X 0 and Y do not contain a fluorine atom, Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , and Z 6 At least one of these is preferably a fluorine atom.

[0041] In a preferred embodiment of the present invention, from the viewpoint of spheroid formation, X 0is selected from the group consisting of -C(CF3)2-, the above b-2 to b-10, and c-2 to c-10; and Y is selected from the group consisting of d-3, d-9, e-1 to e-4, f-6, and f-7. In a more preferred embodiment of the present invention, in the above formula (I), X 0 is selected from the group consisting of -C(CF3)2-, b-7 to b-9, and c-7 to c-9; and Y is selected from the group consisting of e-1, e-3, and e-4.

[0042] A polyimide resin comprising the structural unit represented by formula (I) above can be obtained by baking a polyamic acid obtained by polymerizing an acid dianhydride and a diamine. The imidization rate of the "polyimide resin comprising the structural unit represented by formula (I)" does not have to be 100%. That is, a polyimide resin comprising the structural unit represented by formula (I) may be composed solely of the structural unit represented by formula (I), but may also contain a structural unit in which the cyclic imide structure does not undergo dehydration ring closure and remains as an amic acid, as long as the intended effect of the present invention is not impaired.

[0043] The polyamic acid synthesis reaction is preferably carried out in an organic solvent. The organic solvent used in the polyamic acid synthesis reaction is not particularly limited, as long as it allows the reaction between the raw materials, acid dianhydride and diamine, to proceed efficiently and is inert to these raw materials. Examples of suitable organic solvents include polar solvents such as N-methylpyrrolidone (NMP), N,N-dimethylacetamide, N,N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide, sulfolane, methyl isobutyl ketone, acetonitrile, benzonitrile, nitrobenzene, nitromethane, acetone, methyl ethyl ketone, isobutyl ketone, and methanol; and nonpolar solvents such as toluene and xylene. Among these, polar solvents are preferred. These organic solvents may be used alone or in combination. The reaction mixture after the amidation reaction may be subjected directly to thermal imidization. The concentration of the polyamic acid in the polyamic acid solution is not particularly limited, but is preferably 5% by weight or more, more preferably 10% by weight or more, and preferably 50% by weight or less, more preferably 40% by weight or less, from the viewpoints of the polymerization reactivity of the resulting resin composition, the viscosity after polymerization, and ease of handling during subsequent film formation and baking. The viscosity of the resin composition is not particularly limited, but can be measured according to a known measurement method, and is, for example, within the range of 1 to 20 Pa·s, preferably 3 to 15 Pa·s at 23°C.

[0044] The polyamic acid is imidized by either thermal imidization or chemical imidization to obtain a resin composition containing a fluorine-containing polyimide. In a specific embodiment, the polyamic acid is imidized by heat treatment (thermal imidization) to obtain a resin composition containing a fluorine-containing polyimide. Polyimides obtained by thermal imidization are less likely to contain residual catalyst and are therefore more preferable for cell culture applications.

[0045] When imidization is performed by thermal imidization, for example, the polyamic acid is baked in air, or more preferably in an inert gas atmosphere such as nitrogen, helium, or argon, or in vacuum, preferably at a temperature of 50 to 400°C, more preferably 100 to 380°C, for preferably 0.1 to 10 hours, more preferably 0.2 to 5 hours, to carry out the imidization reaction, thereby obtaining a polyimide-containing resin composition.

[0046] The polyamic acid to be subjected to the thermal imidization reaction is preferably in a form dissolved in a suitable solvent. The solvent may be any solvent that dissolves the polyamic acid, and the solvents described above for the polyamic acid synthesis reaction may also be used.

[0047] In the case of imidization by chemical imidization, polyamic acid can be directly imidized in an appropriate solvent using a cyclodehydration reagent described below.

[0048] The cyclodehydration reagent can be any reagent capable of chemically cyclodehydrating a polyamic acid to form a polyimide, and is not particularly limited. As the cyclodehydration reagent, a tertiary amine compound is preferably used alone, or a tertiary amine compound and a carboxylic acid anhydride are preferably used in combination, in order to efficiently promote imidization.

[0049] Examples of tertiary amine compounds include trimethylamine, triethylamine, tripropylamine, tributylamine, pyridine, 1,4-diazabicyclo[2.2.2]octane (DABCO), 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,5-diazabicyclo[4.3.0]non-5-ene, N,N,N',N'-tetramethyldiaminomethane, N,N,N',N'-tetramethylethylenediamine, N,N,N',N'-tetramethyl-1,3-propanediamine, N,N,N',N'-tetramethyl-1,4-phenylenediamine, N,N,N',N'-tetramethyl-1,6-hexanediamine, N,N,N',N'-tetraethylmethylenediamine, and N,N,N',N'-tetraethylethylenediamine. Among these, pyridine, DABCO, and N,N,N',N'-tetramethyldiaminomethane are particularly preferred, with DABCO being more preferred. Only one type of tertiary amine may be used, or two or more types may be used.

[0050] Examples of carboxylic acid anhydrides include acetic anhydride, trifluoroacetic anhydride, propionic anhydride, butyric anhydride, isobutyric anhydride, succinic anhydride, and maleic anhydride. Among these, acetic anhydride and trifluoroacetic anhydride are particularly preferred, and acetic anhydride is more preferred. Only one type of carboxylic acid anhydride may be used, or two or more types may be used.

[0051] In chemical imidization, a polar solvent with excellent solubility is suitable as a solvent for dissolving polyamic acid. Examples include tetrahydrofuran, N,N-dimethylacetamide, N,N-dimethylformamide, N-methylpyrrolidone, and dimethyl sulfoxide. Among these, one or more solvents selected from the group consisting of N,N-dimethylacetamide, N,N-dimethylformamide, and N-methylpyrrolidone are particularly preferred from the viewpoint of achieving a uniform reaction. When such a solvent is used as a solvent for the amidation reaction, the polyamic acid can be used for chemical imidization directly without being separated from the reaction mixture after the amidation reaction.

[0052] The weight-average molecular weight of the polyimide resin is, for example, 5,000 to 2,000,000, preferably 8,000 to 1,000,000, and more preferably 20,000 to 500,000. In this specification, the weight-average molecular weight of the resin can be measured according to a known measurement method, and when the weight-average molecular weight is in the above range, the synthesis and handling of the polyimide resin, film formation, and spheroid formability are improved.

[0053] The cell adhesive surface may further contain additive components such as a plasticizer and an antioxidant in addition to the above-mentioned substance that exhibits cell adhesiveness.

[0054] A cell-adhesive substance or cell-adhesive surface [a hydrophobic (particularly, non-superhydrophobic) cell-adhesive substance or cell-adhesive surface] may have a static water contact angle of 70° or more, a sliding angle of 15° or more, or a static water contact angle of 70° or more and a sliding angle of 15° or more. When a cell-adhesive surface satisfies these conditions, spheroid formation is further promoted. From the viewpoint of spheroid formability, the static water contact angle is more preferably greater than 75°, even more preferably greater than 77°, even more preferably greater than 79°, and particularly preferably greater than 80° (e.g., greater than 80°). The upper limit of the static water contact angle is, for example, less than 150°, preferably less than 120° (e.g., less than 120°), more preferably less than 110°, even more preferably less than 100° (e.g., less than 99°, 98° or less, 97° or less, 95° or less, etc.), and particularly preferably less than 90°. On the other hand, the static water contact angle of a substance or cell adhesive surface exhibiting cell adhesiveness [a substance or cell adhesive surface exhibiting hydrophilic (particularly, hydrophilic but not superhydrophilic) cell adhesiveness] may be preferably 65° or less, more preferably 55° or less, and even more preferably 50° or less. The lower limit may be 0° or more, preferably 5° or more, and more preferably 10° or more. From the viewpoint of spheroid formation, the sliding angle of a cell-adhesive substance or cell-adhesive surface is preferably higher in the following order: 18° or more, 19° or more, 20° or more, 22° or more, 24° or more, 26° or more, 28° or more, and 30° or more. The upper limit of the sliding angle is, for example, less than 80°, preferably 70° or less (e.g., less than 70°), more preferably 60° or less (e.g., less than 60°), and even more preferably 50° or less (e.g., less than 50°). The surface properties can be measured according to known methods.

[0055] The cell adhesive surface of the cell culture sheet may be the entire or a part of the sheet surface, may be compartmentalized, or may be flat, as long as the surface with which the cells come into contact is the cell adhesive surface. For example, the entire surface of the cell culture sheet may be a flat cell adhesive surface. Furthermore, when the cell culture sheet has an uneven surface and the cell culture surface is compartmentalized, it is sufficient that the portion to which the cells adhere is composed of the cell adhesive surface.

[0056] When the surface of the cell culture sheet is a flat cell-adhesive surface, it may have some irregularities as long as the seeded cells can be fixed and adhered thereto, or the cells may be adhered over the entire surface regardless of the irregularities.

[0057] A cell culture sheet, which is a flat cell adhesive surface, may be formed by physically or chemically fixing or placing a substance that exhibits cell adhesiveness on the surface of a substrate, or the sheet itself may be made of a substance that exhibits cell adhesiveness.

[0058] Immobilization of a substance on a substrate surface can be achieved by drying a solution containing the substance on the substrate surface, melting the substance and pressing it together, curing the substance applied to the substrate with UV or other energy rays, forming a covalent bond by chemically reacting functional groups on the substance with functional groups on the substrate (e.g., condensation reaction between functional groups such as carboxyl groups or amino groups), or bonding thiol groups on the substance with a thin metal (e.g., platinum or gold) film preformed on the substrate. The thickness of the substance to be immobilized is not particularly limited, and examples range from 0.01 to 1000 μm. Furthermore, sheeting can be achieved by applying the substance to a release sheet (e.g., an organic polymer film such as a polyethylene substrate, ceramics, metal, or glass) with a release-treated surface by casting, spray coating, dip coating, spin coating, roll coating, or other methods to a suitable thickness, followed by heating to form a layer of the substance into a sheet.

[0059] When the cell culture sheet has an uneven surface, it is preferable that cells adhere to the bottom of the recesses, and an embodiment in which the bottom is made of a material that exhibits cell adhesiveness can be mentioned. The inner surface of the recesses is not particularly limited, and may be made of, for example, a material that exhibits non-cell adhesiveness. Alternatively, the cell culture sheet may have a micropattern formed of a cell-adhesive surface and a non-cell-adhesive surface. Examples of such cell culture sheets include a sheet having a layer with a cell-adhesive material and a micropattern of a non-cell-adhesive material provided on the surface of the layer (i.e., a sheet having a layer with a cell-adhesive material and a mask with a micropattern of a non-cell-adhesive material). Cell culture sheets with shallow recesses, formed by forming recesses using a micropattern, are thought to contribute more to the expression of cell function than conventional containers that culture cells individually in each well or cavity, such as microtiter plates or microplates separated by high side walls. This is because when cells are seeded on such a cell culture sheet, the cells are compartmentalized on the cell adhesive surface of the shallow recesses, but the medium above the recesses is shared by the entire sheet, and humoral factors are shared by all the seeded cells, which is thought to result in interactions between cells and improved expression of cell function. The formation of fine patterns may be carried out by, for example, microcontact printing, spin coating, casting, roll coating, die coating, gravure coating, spray coating, bar coating, flexographic printing, dip coating, inkjet printing, and a patterning method in which a desired substance is injected into the gaps of an uneven structure formed on the surface of a base material by capillary force generated in the gaps. The cell non-adhesive substance can be any substance that does not bind to cell surface molecules, such as proteins or sugar chains, present in the cell membrane of the cells used, and may be biocompatible or non-biocompatible. Furthermore, the substance may be either hydrophobic or hydrophilic, for example, ultrahydrophobic (superhydrophobic) or superhydrophilic. From the viewpoints of cell non-adhesiveness, spheroid uniformity, and formability, hydrophobic (particularly superhydrophobic) substances [e.g., more hydrophobic (particularly superhydrophobic) relative to a hydrophobic or hydrophilic (e.g., hydrophobic) cell adhesive surface or the resin constituting the surface (and further its contact angle)] are particularly preferred, but hydrophilic (particularly superhydrophilic) substances [e.g., more hydrophilic (particularly superhydrophilic) relative to a hydrophilic or hydrophobic (e.g., hydrophobic) cell adhesive surface or the resin constituting the surface (and further its contact angle)] are also preferred. Examples of such substances include polyethylene glycol and its derivatives, MPC (2-methacryloyloxyethyl phosphorylcholine) and its derivatives, compounds containing HEMA (hydroxyethyl methacrylate) and its derivatives, or polymers of these compounds, compounds containing SPC (segmented polyurethane) and its derivatives, proteins obtained from living organisms (such as albumin), and sugar chains to which cells do not adhere (such as agarose and cellulose). Of these, MPC and its derivatives, or polymers thereof, are preferred from the standpoint of adhesiveness to synthetic resins, simplification of the cell culture sheet production process, and improvement in the uniformity of the resulting spheroids. The non-cell-adhesive substance may be appropriately modified depending on the ease of handling and the desired degree of hydrophobicity (e.g., superhydrophobicity) or hydrophilicity (e.g., superhydrophilicity). For example, a hydrophilic substance may be crosslinked to achieve both hydrophilicity and low solubility in water. Furthermore, a raw material (e.g., hydrophobic or hydrophilic) may be appropriately hydrophobized or hydrophilized (e.g., by introducing hydrophobic or hydrophilic groups) to obtain a material with the desired hydrophobic or hydrophilic properties.

[0060] A non-cell-adhesive surface is a surface on which, when cells settle in a culture solution, the cells hardly change shape and do not adhere at all, or, even if they temporarily adhere weakly, they naturally detach. Such a surface may be formed, for example, by physically or chemically fixing a substance exhibiting cell non-adhesive properties to the surface of a sheet that forms recesses, or the sheet itself may be made of a substance exhibiting cell non-adhesive properties.

[0061] From the viewpoint of making the size of the formed spheroids uniform and improving the circularity, a cell non-adhesive surface can be determined using as an index the surface characteristics, for example, the static water contact angle described below. For example, when the non-cell-adhesive surface is a hydrophobic surface formed from the above-mentioned substances, the static water contact angle is preferably 90° or more, more preferably 93° or more, and even more preferably 95° or more. The static water contact angle may be 150° or less, preferably 130° or less, and more preferably 120° or less. On the other hand, when the non-cell-adhesive surface is a hydrophilic surface, the static water contact angle is preferably 65° or less, more preferably 55° or less, and even more preferably 50° or less. The static water contact angle may be 0° or more, and is preferably 5° or more, and more preferably 10° or more. For example, a highly hydrophobic MPC (or a surface formed from the MPC) can achieve a static water contact angle of, for example, 90° or more, or 100° or more. Such a static water contact angle may be a value on a non-cell-adhesive surface, or may be a value on a substance that exhibits non-cell adhesiveness (or a substance that constitutes a non-cell-adhesive surface). The static water contact angle may be measured, for example, by the method described below.

[0062] From the viewpoint of improving the size uniformity and circularity of the resulting spheroids, it is preferable to balance the degree of adhesion between the cell-adhesive surface and the non-cell-adhesive surface. Therefore, even if the non-cell-adhesive surface exhibits cell adhesion, it is sufficient if it exhibits lower adhesion than the cell-adhesive surface. For example, when the static water contact angle is used as an indicator, the difference (or its absolute value) between the static water contact angle on the non-cell-adhesive surface (or the substance exhibiting cell non-adhesive properties) and the non-cell-adhesive surface (or the substance exhibiting cell non-adhesive properties) is preferably 3° or more (e.g., 5° or more), more preferably 10° or more (e.g., 12° or more), and even more preferably 15° or more. Furthermore, the upper limit of the difference (or its absolute value) can be appropriately selected depending on the hydrophobicity / hydrophilicity of the cell-adhesive surface (or the substance exhibiting cell non-adhesive properties), and is not particularly limited, and may be, for example, 100°, 90°, 80°, 70°, 60°, 50°, 40°, 30°, etc.

[0063] A specific example of a cell culture sheet having an uneven surface is a cell culture sheet having a plurality of recesses with an opening diameter of 1000 μm or less, the inner surface of each recess having a non-cell-adhesive surface, and the bottom surface of each recess having a cell-adhesive surface.

[0064] Here, the structure of a cell culture sheet with an uneven surface will be explained using an example of a cross-sectional view of the sheet cut perpendicular to the recesses. As shown in the schematic diagram of Figure 1, there are multiple recesses 11 on the sheet surface 12, and each recess 11 is composed of an inner surface 11a and a bottom surface 11b, and spheroids are formed in the recesses 11.

[0065] The number of recesses 11 on the sheet surface 12 cannot be determined in general, depending on the sheet area, the type of cells to be cultured, etc., and can be set appropriately according to the relevant technical common sense. For example, 2 The lower limit number per recess may be 1, 10, 30, 50, etc., and the upper limit number may be 1000, 500, 300, 200, 100, etc. The total number of recesses on the sheet surface may be set as appropriate, for example, to 10 or more, 100 or more, 1000 or more, 10000 or more, 50000 or more, etc.

[0066] The shape of the opening of the well is not limited to a circle, and may be, for example, a polygon or an ellipse. The diameter of the opening may be, for example, 2000 μm or less, and can be appropriately set according to common technical knowledge depending on the size of the cells to be cultured and the size of the desired spheroids. In the present invention, the pore diameter refers to the diameter (maximum length) of a circle formed so as to enclose the target site, regardless of the shape of the target site, and the pore diameter of the opening refers to, for example, the length indicated by D(11) in FIG. 1. Examples of the pore diameter of the opening are within the ranges of 10 to 2000 μm, 10 to 1000 μm, 10 to 800 μm, and 10 to 500 μm.

[0067] The shape of the bottom of the recess is not limited to a circle, but may be, for example, a polygon or an ellipse, and may be the same as or different from the shape of the opening. The bottom may be flat (flat-bottomed) or curved. The area of ​​the bottom per recess is, for example, 1.0 × 10 -1 cm 2 , 5.0×10 -2 cm 2 , 2.0×10 -2 cm 2 , 1.0×10 -2 cm 2 , 5.0×10 -3 cm 2 , 2.5×10 -3 cm 2 , 2.0×10 -3 cm 2 , 1.0×10 -3 cm 2 and the like are examples. The diameter (length) of the bottom hole may be the same as or different from the diameter of the opening hole. The diameter of the bottom hole may be smaller than or larger than the diameter of the opening hole. The diameter of the bottom hole is, for example, the length indicated by DB (11b) in FIG. 1, and examples of the diameter of the bottom hole are within the ranges of 10 to 2000 μm, 10 to 1000 μm, 10 to 800 μm, 10 to 500 μm, 10 to 400 μm, and 10 to 300 μm. For example, when the diameter of the bottom hole is the same as the diameter of the opening hole, the recess has a cylindrical shape. When the diameter of the bottom hole is smaller than the diameter of the opening hole, the recess has a tapered shape toward the bottom of the recess.

[0068] The ratio of the pore size of the bottom surface to the pore size of the opening (pore size of the bottom surface / pore size of the opening) is not particularly limited, but examples include 5 / 1 to 1 / 5, 3 / 1 to 1 / 3, and 1 / 1 to 1 / 2 from the viewpoint of ease of cell seeding and recovery.

[0069] The distance (gap) between an opening and an adjacent opening is, for example, the length indicated by D(12) in Figure 1, and is not particularly limited, but examples include ranges such as 800 μm or less, 700 μm or less, 600 μm or less, 500 μm or less, 300 μm or less, 200 μm or less, and 100 μm or less, depending on the desired large-scale culture, as long as it is a finite value.

[0070] The depth of the recesses can be appropriately set according to the size of the cells to be cultured and in accordance with common general technical knowledge. The depth of the recesses is, for example, the length indicated by D(11a) in FIG. 1, and is exemplified by ranges of 10 to 1500 μm, 10 to 1000 μm, 10 to 800 μm, 10 to 500 μm, and 10 to 300 μm. Furthermore, when recesses are formed in a fine pattern, the depth of the recesses can be less than 10 μm. In this case, the lower limit of the depth of the recesses need only be the minimum thickness at which cells can recognize non-adhesiveness. For example, the depth can be set to 100 nm to 500 nm.

[0071] The ratio of the pore size of the opening to the depth of the recess (pore size of the opening / depth of the recess) is not particularly limited, but from the viewpoint of ease of seeding and recovery of cells, examples include 5 / 1 to 1 / 5, 3 / 1 to 1 / 3, and 2 / 1 to 1 / 1. When the ratio is within the above ranges, cells are less likely to jump out of the recess, and operations such as cell recovery and degassing treatment are easier.

[0072] The thickness of the bottom surface of the recess is not particularly limited and can be appropriately set in accordance with common technical knowledge.

[0073] The recess has a cell-non-adhesive surface on the inner side and a cell-adhesive surface on the bottom side. Specifically, for example, as shown in FIG. 1, the inner side 11a has a cell-non-adhesive surface and the bottom side 11b has a cell-adhesive surface. In addition, in a cell culture sheet, the sheet surface, which is also the periphery of the recess, preferably has a cell-non-adhesive surface from the viewpoint of simplifying the production of the cell culture sheet. The cell-non-adhesive surface of the sheet surface may be the same as the cell-non-adhesive surface of the inner side of the recess, or may be a different cell-non-adhesive surface. In the case where the cell-non-adhesive surface is the same, the sheet surface and the inner side of the recess have a continuous surface. The percentage of the bottom surface of the well that is occupied by the surface exhibiting cell adhesive properties is not particularly limited, but it is preferable that the surface occupies 90% or more, 95% or more, 99% or more, or essentially the entire bottom surface of the well. The proportion of the inner surface of the well that is occupied by the non-cell-adhesive surface is not particularly limited, but is preferably to an extent that reduces the adhesion of cultured cells. Preferably, it occupies 90% or more of the area of ​​the inner surface, more preferably 95% or more, and even more preferably the entire inner surface. The proportion of the sheet surface that is also the periphery of the well that is occupied by the non-cell-adhesive surface is not particularly limited, but it preferably occupies 90% or more, 95% or more, or 99% or more of the periphery of the well, or substantially the entire periphery. Preferably, the non-cell-adhesive surface occupies 90% or more, 95% or more, or 99% or more of the combined periphery and inner surface of the well, or substantially the entire periphery and inner surface. For example, as shown in the schematic diagram of Figure 2, a substance 21 exhibiting non-cell adhesive properties may be immobilized on the sheet surface 12 and the inner surface 11a of the recess 11. The sheeting and immobilization of the substance can be performed with reference to the section on the cell culture sheet, which is a flat cell adhesive surface.

[0074] The cell culture sheet having an uneven surface has a plurality of the recess structures described above, but it may also be a layered structure including a layer including the bottom surfaces of the recesses and a layer including the inner surfaces of the recesses. Here, the layer including the inner surfaces of the recesses itself constitutes a layer having through-holes. Therefore, one embodiment of the sheet is a laminate of a layer having a non-cell-adhesive surface with through-holes and a layer having a cell-adhesive surface. For example, as shown in the schematic diagram of FIG. 3, the region including the bottom surfaces 11b of the recesses 11 may be formed with a layer made of a cell-adhesive substance 22, and a layer having a non-cell-adhesive surface with through-holes may be formed on top of that. For reference, FIGS. 4 and 5 show schematic diagrams of spheroid formation in the recesses of the cell culture sheet.

[0075] From the viewpoint of forming the through-holes or simplifying the production of the cell culture sheet, the layer having a cell-non-adhesive surface is preferably one in which a substance exhibiting cell-non-adhesive properties is immobilized on a layered substrate.

[0076] Any layered substrate known in the art can be used. Examples include plates made of synthetic resins such as polystyrene, polyethylene, polypropylene, polycarbonate, polyamide, polyacetal, polyester (e.g., polyethylene terephthalate), polyurethane, polysulfone, polyacrylate, polymethacrylate, polyvinyl, polycycloolefin, polyether ketone, polyether ether ketone, polyimide, and silicone; synthetic rubbers such as EPDM (Ethylene Propylene Diene Monomer); natural rubbers; glass; ceramics; and metal materials such as stainless steel. A transparent substrate is also a preferred embodiment.

[0077] From the viewpoint of workability, it is preferable to immobilize a substance that exhibits non-cell adhesiveness onto a layered substrate after forming through-holes, which will be described later.

[0078] The through-hole preferably has a wall corresponding to the inner surface of the recess, and the diameter and shape of the opening and the opposite end thereof can be set in the same manner as the recess. The depth of the through-hole corresponds to the thickness of the layer having a non-cell-adhesive surface, but also corresponds to the depth of the recess and can be set in the same manner as the recess. When a substance exhibiting non-cell adhesive properties is immobilized on a layered substrate, the layer of the substance exhibiting non-cell adhesive properties is preferably, for example, 1 nm or more, more preferably 10 nm or more, and can be set appropriately as long as the thickness of the entire layer, including the layer of the substance exhibiting non-cell adhesive properties and the substrate, is within the range of the thickness of the layer having a non-cell-adhesive surface.

[0079] The formation of the through holes is not particularly limited as long as the through holes of the above-mentioned sizes can be formed, and can be carried out by such methods. For example, the through holes can be formed by drilling (drilling, etc.), optical microfabrication (laser (e.g., CO laser, excimer laser, semiconductor laser, YAG laser), etc.), etching, embossing, etc. The processing may be such that the through holes have a tapered shape, and in this case, the periphery of the end portion may be deformed, and a structure may be formed in which the layer thickness differs between the periphery of the opening and the portion located in the middle region between the openings adjacent to the opening, as shown in FIG. 5, for example.

[0080] The thickness of the layer having a cell adhesive surface is, for example, 1 nm or more and 4 mm or less, preferably 1 μm or more and 1 mm or less, and may be the same as the thickness of the bottom surface of the recess.

[0081] The cell culture sheet may further include an adhesive layer (sticky layer) between the layer having the cell adhesive surface and the layer having the non-cell adhesive surface. For example, Figure 6 shows an example of this, which includes an adhesive layer 23 between a layer consisting of a cell adhesive substance 22 and a portion where a non-cell adhesive substance 21 is immobilized.

[0082] Any adhesive layer known in the art can be used. Examples include acrylic resins, silicone resins, synthetic rubbers, and natural rubbers. Preferably, a low-elution adhesive layer can be used. Commercially available double-sided tape can also be used.

[0083] The thickness of the adhesive layer is not particularly limited and can be set appropriately within a range that does not impair the effects of the present invention, for example, 0.5 to 100 μm.

[0084] The cell culture sheet can be produced by laminating, in this order, a layer having a non-cell-adhesive surface with through-holes and a layer having a cell-adhesive surface. Layers other than those mentioned above may be laminated, and a layer having cavities may also be laminated.

[0085] When laminating each layer, each layer may be laminated in order, each layer may be prepared in advance, or a separate layer may be formed on a previously prepared layer, or a combination of these may be used. Specifically, for example, a layer having a cell-adhesive surface can be formed into a sheet by coating a substance exhibiting cell adhesiveness to an appropriate thickness by a method such as casting, spray coating, dip coating, spin coating, or roll coating on a release sheet (e.g., an organic polymer film such as a polyethylene substrate, ceramics, metal, glass, etc.) whose surface has been subjected to a release treatment, and then heating the coated surface. On the other hand, a layer having a cell-nonadhesive surface can be prepared in advance by forming through-holes in the substrate for the layer having a cell-nonadhesive surface and coating the surface with a substance exhibiting cell-nonadhesive properties. Then, after peeling off the release sheet for the layer having a cell-adhesive surface formed as described above, a separately prepared layer having a cell-nonadhesive surface can be laminated on top of the layer. When laminating a layer having a non-cell-adhesive surface and a layer having a cell-adhesive surface, the lamination may be performed using the adhesive layer (sticky layer) described above, or by welding (high-frequency welding, ultrasonic welding, etc.) or compression bonding (thermocompression bonding, etc.).

[0086] The thickness of the cell culture sheet is not particularly limited, but from the viewpoint of ease of handling, it is preferably 10 to 5000 μm, more preferably 10 to 2000 μm. The sheet area is also not particularly limited, and may be, for example, 0.01 to 10000 cm. 2 , preferably 0.03 to 5000 cm 2 is exemplified.

[0087] The cell culture sheet thus obtained may be appropriately sized to fit the size of the target device, regardless of whether the surface is uneven, so that it can be placed directly in a known cell culture device for use. Cell culture can be performed by placing a cell-containing medium on one surface of the sheet, and the sheet can be placed and fixed in various cell culture vessels such as a culture plate, each well of a plate, a culture dish (culture dish), a flask, or a culture bag, and then adding a cell-containing medium to the vessel so as to cover part or all of the fixed sheet.

[0088] The undifferentiated cells to be cultured on the cell-adhesive surface of the cell culture sheet are not particularly limited as long as they are undifferentiated cells capable of differentiation. Specifically, primary cells derived from any organ or tissue (brain, liver, pancreas, spleen, heart, lung, intestine, cartilage, bone, adipose tissue, kidney, nerve, skin, bone marrow, dental pulp, embryo, periosteum, synovium, muscle, placenta tissue, umbilical cord tissue (umbilical cord blood), peripheral blood, etc.) of humans or non-human animals (monkeys, pigs, dogs, rabbits, rats, mice, etc.), established cell lines, or genetically engineered cells thereof can be used. Furthermore, the cells that express integrins are not particularly limited as long as they are capable of expressing integrins, and may be undifferentiated cells. Examples of such cells include the same cells as those described above.

[0089] More specifically, stem cells and progenitor cells can be used, for example, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), neural stem cells, mesenchymal stem cells, tissue stem cells (somatic stem cells), hematopoietic stem cells, cancer stem cells, etc. As such cells, one type of cell can be used alone, or two or more types of cells can be mixed in any ratio.

[0090] The medium and culture conditions used in culturing undifferentiated cells or cells expressing integrins can be appropriately determined depending on the cells used. In the present invention, because cells are cultured on the cell-adhesive surface described above, there is no need to culture them on feeder cells or on a substrate coated with biological components such as collagen. Culture can be performed in the absence of feeder cells or components that affect the adhesiveness of cells (e.g., collagen, hyaluronic acid, Matrigel, laminin, fibronectin, gelatin, etc.). The term "feeder cells" refers to cells that can provide a culture environment for the undifferentiated cells or cells expressing integrins to be cultured. For example, mouse fibroblasts can be used as feeder cells when the cells to be cultured are human ES cells. For example, 5×10 undifferentiated cells or cells are placed on the cell adhesive surface. 3 ~3×10 4 cells / cm 2The cells are seeded at a density of 1000 μg / cm2 and cultured in a serum-free medium (e.g., StemFit® medium) for approximately 1 to 7 days, thereby forming undifferentiated cell spheroids according to one embodiment of the present invention or integrin-expressing cell spheroids according to another embodiment of the present invention. When using the cell culture sheet or cell culture vessel, degassing may be performed beforehand, if necessary. The degassing treatment is not particularly limited, and general treatments such as spraying, pipetting, shaking, temperature changes such as heating and cooling, centrifugation, vacuum degassing, and ultrasonic treatment can be used, with spraying, pipetting, and temperature changes being preferred. Furthermore, when seeding the cells, a medium containing a Rock inhibitor may be used to suppress cell death due to dispersion during seeding. If necessary, cells may be expanded and then treated with a detachment agent. The detachment agent is not particularly limited, but examples thereof include trypsin and TrypLE. TM , Accutase TM etc. can be used.

[0091] The diameter of the undifferentiated cell spheroids and integrin-expressing cell spheroids thus obtained is not particularly limited, but may be, for example, 10 to 1500 μm, 10 to 1000 μm, 10 to 800 μm, 10 to 600 μm, or 10 to 500 μm. The diameter of the spheroids can be measured by standard methods (e.g., image analysis software, particle size distribution analyzer) and can be expressed, for example, as a fluid diameter or a circle-equivalent diameter. Furthermore, the shape of the spheroids may be spherical or dome-shaped (hemispherical). If spherical, the circularity is, for example, 0.5 to 1.0, preferably 0.7 to 1.0.

[0092] The number of cells forming the undifferentiated cell spheroid and the integrin-expressing cell spheroid is not particularly limited, and may be, for example, 1 × 10 1 pcs or more, 1×10 2 pcs or more, 1×10 3 pcs or more, 1×10 4 pcs or more, 1×10 5 pcs or more, 1×10 6 pcs or more, 1×10 7 pcs or more, 1×108 pcs or more, 1×10 9 There is no particular upper limit, and for example, 1 × 10 10 It is fine as long as it is less than one.

[0093] Although the composition of undifferentiated cell spheroids and integrin-expressing cell spheroids is not necessarily determined by the constituent cells, in the present invention, spheroids are formed on the cell-adhesive surface described above, thereby reducing the risk of contamination and making it possible to form spheroids that do not contain serum-derived components, for example. While typical cells adhere to each other and to the extracellular matrix in vivo, they are always in a suspended state in suspension culture vessels. Cells that cannot adhere may undergo apoptosis, but liquid factors contained in serum are required to suppress this. On the other hand, the method of the present invention allows cells to adhere in the same way as in vivo, eliminating the need for liquid factors to suppress apoptosis. Furthermore, in the present invention, in addition to forming spheroids on the above-mentioned cell-adhesive surface, the spheroids can be detached and recovered without enzymatic treatment, so that the spheroids themselves have adhesive properties and can be recovered while still containing the extracellular matrix. In another aspect of the present invention, the tissue expresses integrin, and therefore has excellent cell adhesiveness and can adhere well to the wound site.

[0094] The undifferentiated nature of undifferentiated cell spheroids can be confirmed by detecting the expression of undifferentiated markers. Examples of undifferentiation markers include, but are not limited to, Oct3 / 4, Nanog, Sox2, POU5F1, c-Myc, and SSEA4. Detection of undifferentiation markers can be performed by standard methods (e.g., real-time PCR, protein arrays, etc.). For example, the cell spheroids obtained by the method of the present invention have undifferentiation marker expression levels at the mRNA level that are preferably 3-fold higher, more preferably 5-fold higher, as relative gene expression levels, compared to cell spheroids obtained by suspension culture on a non-cell-adhesive surface. Furthermore, the protein level is preferably 1.5-fold higher, more preferably 2-fold higher, and even more preferably 3-fold higher. The upper limit of these levels is not particularly limited, but may be, for example, about 20-fold higher.

[0095] Integrins are cell adhesion molecules present on the cell membrane surface, and are heterodimers consisting of two subunits, an α chain and a β chain. Examples of integrin α chains include α1, α2, α3, α4, α5, α6, α7, α8, α9, α10, α11, αv, and αIIb, and examples of β chains include, but are not limited to, β1, β2, β3, β4, β5, and β6. Integrins in integrin-expressing cell spheroids can be analyzed by conventional methods (e.g., ELISA) as long as the protein expression of at least one of the various α-chain and β-chain subunits is detected. From the viewpoint of adhesiveness to wound sites, the integrin to be detected is preferably at least one of integrin αv, integrin β3, and integrin β6. Furthermore, in the case of human adipose-derived stem cells, from the viewpoint of maintaining undifferentiated state, the integrin to be detected is preferably at least one of integrin αv, integrin α5, integrin α8, integrin αIIb, and integrin α11, with integrin αv being more preferred. For example, integrin-expressing cell spheroids obtained by another embodiment of the present invention exhibit, compared to spheroids cultured in suspension on a non-cell-adhesive surface, an integrin expression level at the protein level that is, for example, 1.2-fold or more, preferably 1.5-fold or more, more preferably 2-fold or more, and even more preferably 3-fold or more. The upper limit of the integrin expression level is not particularly limited, but may be, for example, about 10-fold. 1 × 10 at the protein level 5 The expression level of integrin-expressing cell spheroids per cell is not particularly limited, but when detecting integrin αv, it may be, for example, 65 pg or more, preferably 70 pg or more, and more preferably 75 pg or more. The upper limit is not particularly limited, but may be, for example, 200 pg.

[0096] In one aspect of the present invention, undifferentiated cell spheroids can be produced, and it has been found that the undifferentiated state can be maintained by maintaining and culturing the resulting spheroids on a cell adhesive surface. Therefore, the present invention also provides a method for maintaining the undifferentiated state of cell spheroids, comprising culturing undifferentiated cell spheroids on the cell adhesive surface of a cell culture sheet. In another aspect of the present invention, cell spheroids that express integrins can be produced. The cell culture sheet, medium, culture conditions, etc. used in the method for maintaining the undifferentiated state and the method for producing integrin-expressing cell spheroids can be specified in the same manner as in the method for producing undifferentiated cell spheroids of the present invention.

[0097] The obtained undifferentiated cell spheroids contain a large number of stable cells that retain their undifferentiated state, and therefore can be used in research on the development and regeneration of various tissues and organs, and can also be used in regenerative medicine because they can be induced to differentiate into various cells. Furthermore, while cell culture vessels coated with gelatin or the like are generally required to induce differentiation of undifferentiated cells, in one embodiment of the present invention, differentiation can be efficiently induced by adding a differentiation inducer or differentiation promoter to the obtained undifferentiated cell spheroids and culturing them directly on a cell-adhesive surface. Furthermore, the obtained integrin-expressing cell spheroids have excellent cell adhesiveness due to their high expression of integrins, allowing them to adhere well to wound sites, and cell death is suppressed.In particular, in the case of undifferentiated cells, they maintain their undifferentiated state well, making it possible to produce cell spheroids that are expected to have a high therapeutic effect in regenerative medicine. [Example]

[0098] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these. In the following examples, room temperature means 20 to 30°C.

[0099] Manufacturing Example 1 <Preparation of a layer with a cell adhesive surface (preparation of a fluorinated polyimide film)> A 100 mL three-neck flask was charged with 2.976 g (10.2 mmol) of 1,4-bis(aminophenoxy)benzene, 4.524 g (10.2 mmol) of 4,4'-hexafluoroisopropylidenediphthalic anhydride, and 42.5 g of N-methylpyrrolidone. The mixture was stirred at room temperature under a nitrogen atmosphere and then held for 5 days to obtain a fluorine-containing polyamic acid resin composition (solids concentration 15.0 mass%, 6FDA / TPEQ polyamic acid). The polyamic acid resin composition had a weight-average molecular weight of 180,000 and a viscosity of 14 Pa·s. The weight-average molecular weight of the polyamic acid and the weight-average molecular weight of the fluorine-containing polyimide after baking were substantially identical.

[0100] The fluorine-containing polyamic acid resin composition obtained above was applied to a glass substrate using a die coater so that the thickness of the fluorine-containing polyimide film after baking would be 40 μm, forming a coating film.The coating film was then baked at 360°C for 1 hour in a nitrogen atmosphere.The baked product was then peeled off from the glass substrate to obtain a fluorine-containing polyimide film.The static water contact angle of this fluorine-containing polyimide film was 80.9°, and the sliding angle was 23.4°.

[0101] The methods for measuring the above physical properties are as follows. (Measurement of weight average molecular weight) Apparatus: Tosoh Corporation HCL-8220GPC Column: TSKgel Super AWM-H Eluent (LiBr·H2O, NMP containing phosphoric acid): 0.01 mol / L Measurement method: A 0.5% by weight solution is prepared using an eluent, and the molecular weight is calculated based on a calibration curve prepared using polystyrene. (Viscosity measurement) Equipment: Viscometer TV-22 (AS ONE) Setting: VI RANGE: H ROTOR No. 6 SPEED: 10 rpm Viscometer calibration standard solution: Nippon Grease Co., Ltd. JS 14000 Measurement method: After calibrating with a standard viscometer calibration solution, measure using 0.3 g of varnish. (Measurement temperature: 23°C) (Static water contact angle measurement) Equipment: Automatic contact angle meter (Kyowa Interface Science: DM-500) Measurement method: Measure the adhesion angle of the droplet immediately after dropping 2 μL of water onto a surface (non-cell adhesive surface or cell adhesive surface) or a film (a film made of a substance that exhibits cell non-adhesive or cell adhesive properties) (measurement temperature: 25°C). (Measurement of fall angle) Equipment: Automatic contact angle meter (Kyowa Interface Science: DM-500) Measurement method: After dropping 25 μL of water onto a surface (non-cell adhesive surface or cell adhesive surface) or a film (a film made of a substance that exhibits cell non-adhesive or cell adhesive properties), the sheet is continuously tilted, and the angle at which the water flows down is taken as the sliding angle (measurement temperature: 25°C).

[0102] <Preparation of cell culture vessel> The obtained fluorine-containing polyimide film was placed on the bottom of each well of a 24-well culture plate to complete a vessel for cell culture.

[0103] Example 1 Human iPS cells, strain 201B7, provided by Kyoto University, were used.

[0104] <Cell expansion culture> The cells established in the feeder cell culture system were adapted to a feeder-free culture system using StemFit® AK02N (Ajinomoto Co., Inc.) and iMatrix-511® (Nippi Co., Ltd.). Cell detachment was performed by soaking the cells in PBS containing 0.5 mM EDTA and TrypLE Select CTS. TM (Thermo Fisher Scientific) in a 1:1 aqueous solution.

[0105] <Creation of Embryoid Bodies (EBs)> The iPS cells recovered with the above-mentioned detachment agent were suspended in StemFit (registered trademark) AK02N medium and placed in the cell culture vessel prepared in Production Example 1 at a density of 5.4 × 104 cells / 500μL / well (2.7×10 4 cells / cm 2 ) was used. At the time of seeding, medium containing 10 μL Y-27632 was used. One day after seeding, half of the medium was replaced, and four days later, the entire medium was replaced.

[0106] Comparative Example 1 EBs were prepared in the same manner as in Example 1, except that a PrimeSurface (registered trademark) 96U plate (manufactured by Sumitomo Bakelite) was used instead of the cell culture vessel used in Example 1. The seeding volume was 100 μL / well.

[0107] Test Example 1 Microscopic Observation The morphology of the EBs was observed under a microscope on days 1, 3, 5, and 7 of culture. The results of Example 1 are shown in FIG. 7, and the results of Comparative Example 1 are shown in FIG.

[0108] Test Example 2 Evaluation of the undifferentiated potential of EBs Seven days after seeding the iPS cells, EBs were collected and RNA was extracted using the RNeasy Mini Kit (Qiagen). Subsequently, cDNA was synthesized using ReverTra Ace (registered trademark) qPCR RT Master Mix (Toyobo). POU5F1, an undifferentiated marker, was detected using TaqMan TM Real-time PCR measurements were performed (n=3) using primers and probes for POU5F1 in the Gene Expression Assay (FAM) (Thermo Fisher Scientific). The StepOnePlus (Thermo Fisher Scientific) instrument was used. GAPDH was selected as the housekeeping gene. The data obtained were normalized with GAPDH, and relative gene expression levels were calculated using the ΔΔCt method. The results are shown in Figure 9.

[0109] From the above results, in Example 1, iPS cells began to form EBs on the first day of culture, and multiple EBs were formed on the third day of culture. On the fifth day of culture, the EBs were observed to have grown in size to approximately 400 μm (FIG. 7). On the other hand, in Comparative Example 1, EBs (approximately 500 μm in size) were formed on the first day of culture, but they did not adhere to the bottom of the well, but sank to the bottom in a non-adherent state. Furthermore, on the fifth day, the EBs grew in size to approximately 600 μm, and at the same time, their shape changed from round to irregular (FIG. 8).

[0110] Furthermore, it was revealed that the amount of POU5F1, an undifferentiation marker, in the EBs produced in Example 1 was significantly higher than that in the non-adherent EBs produced in Comparative Example 1 (FIG. 9). Specifically, it was found that the amount of POU5F1 in the EBs produced in Example 1 was 6.7 times higher than that in the EBs produced in Comparative Example 1 (P < 0.01).

[0111] Manufacturing Example 2 <Preparation of a layer having a cell-non-adhesive surface> After peeling off the release tape on one side of double-sided tape (25 μm thick), the tape was attached to a transparent PET film (250 μm thick). A CO2 laser was used to form through-holes in a staggered pattern with a diameter of 300 μm and a pitch of 500 μm (400 through-holes formed / cm). 2 (24,000 particles / sheet, laser incident side pore diameter 500 μm, laser emission side pore diameter 300 μm) Then, using a spin coater (Mikasa: MS-A150), the surface of the PET film was coated with an MPC polymer solution (0.5% ethanol solution, hydrophobic MPC polymer) to a thickness of 0.05 μm, and dried in a dryer at 50°C for 2 hours to obtain a layer with a cell-nonadhesive surface [static water contact angle of 107.5° on the PET film coating layer (MPC polymer coating layer) side].

[0112] <Preparation of cell culture sheets and culture vessels> Next, a layer having a cell-adhesive surface prepared in the same manner as in Production Example 1 was attached to the side of the double-sided tape on which the other release tape had been removed, to prepare a cell culture sheet (sheet thickness: 315 μm). Figure 10a shows a photograph of the prepared cell culture sheet observed from the bottom side of the wells. The obtained cell culture sheet was placed in a culture plate to complete a container for cell culture. Figure 10b shows a photograph of the entire cell culture container observed from the bottom side of the wells.

[0113] Example 2 The cells used were human adipose-derived stem cells (AdSCs), purchased from the manufacturer (Lonza, PT-5006).

[0114] <Cell expansion culture> The frozen cells were thawed in a 37°C water bath and added to 9 mL of KBM ADSC-2 medium (basal medium, Kohjin Bio) containing 5% FBS and 1% antibiotics. After centrifugation at 210 × g for 5 minutes, the supernatant was removed and the cells were dispersed in 1 mL of basal medium. The cells were placed in a culture flask (culture area 225 cm). 2 ) to 1.0×10 6 Two dishes containing 30 mL of cells per dish were prepared and cultured (expanded) in a 5% (v / v) CO 2 incubator at 37°C.

[0115] <Defoaming treatment> Separately, the culture vessel was degassed. Specifically, approximately 1 mL of PBS was added to the vessel, which was then pipetted and placed in a 5% (v / v) CO2 incubator at 37°C for 15 minutes. After pipetting again, the PBS was aspirated, and 0.2 mL of KBM ADSC-2 medium containing 1% antibiotics was added. The vessel was then placed in a 5% (v / v) CO2 incubator at 37°C overnight.

[0116] <Spheroid production> The medium was removed from the culture dish, and 5 mL of cell detachment solution Accutase (Promocell) was added. The cells were then detached by placing them in a 37°C, 5% (v / v) CO2 incubator for approximately 5 minutes. The detachment solution was then collected, washed with 10 mL of PBS, and transferred to a tube. The cells were centrifuged at 210 × g for 5 minutes, suspended in 4 mL of KBM ADSC-2 medium containing 1% antibiotics, and the cell number was counted. After that, 1.0 × 10 6 The concentration was adjusted to 100 cells / mL.

[0117] The culture vessel was left overnight in a 37°C 5% (v / v) CO2 incubator to degas the culture medium, and then 500 cells / well were seeded. After 15 minutes in a safety cabinet, the vessel was placed in a 37°C 5% (v / v) CO2 incubator and left for 4 hours. Next, KBM ADSC-2 medium containing 1% antibiotics was added, and the vessel was placed in a 37°C 5% (v / v) CO2 incubator again and cultured for 3 days.

[0118] Comparative Example 2 Spheroids were produced in the same manner as in Example 2, except that an ELPLASIA (manufactured by Kuraray) was used instead of the cell culture vessel used in Example 2. The spheroids did not adhere to the bottom of the well, but sunk to the bottom of the well in a non-adherent state.

[0119] Reference example 1 Instead of the cell culture vessel used in Example 2, a 24-well plate for cell culture was used, and KBM ADSC-2 medium containing 1% antibiotics was added to each well. AdSCs were then seeded and cultured in a 5% (v / v) CO2 incubator at 37°C for 3 days.

[0120] Test Example 3: Protein Array After 3 days of culture, the culture supernatant was collected. Proteins contained in this supernatant were evaluated using RayBio human antibody array kits (L-507, L493, RayBiotech). Measurements were performed at Cosmo Bio. The obtained values ​​were normalized to the value of the positive control according to the protocol, and further normalized by the cell number ( / 1 × 10 5 According to the protocol, a significant difference between samples was determined to exist when the difference in values ​​between the samples was 1.5 times or more or 0.65 times or less.

[0121] The amounts of undifferentiation markers Nanog, Oct 3 / 4, SSEA-4, and SOX2 in the spheroids prepared in Example 2 were found to be significantly higher than those in the non-adhesive spheroids prepared in Comparative Example 2 and the two-dimensionally cultured cells prepared in Reference Example 1 (FIG. 11). Specifically, the amounts of Nanog, Oct 3 / 4, SSEA-4, and SOX2 in the spheroids prepared in Example 2 were found to be 2.7, 2.8, 1.8, and 4.7 times higher, respectively, than those in the spheroids prepared in Comparative Example 2 (Table 1). Furthermore, the amounts of Nanog, Oct 3 / 4, SSEA-4, and SOX2 in the spheroids prepared in Example 2 were found to be 3.0, 2.0, 1.8, and 2.4 times higher, respectively, than those in the two-dimensional cells prepared in Reference Example 1 (Table 2).

[0122] [Table 1]

[0123] [Table 2]

[0124] Test Example 4 Quantification of integrin αv The cells used were the same as those used in Example 2.

[0125] <Cell preparation> The frozen cells were thawed in a 37°C incubator and added to 9 mL of KBM ADSC-2 medium (basal medium, Kohjin Bio) containing 5% FBS and 1% antibiotics. After centrifugation at 210 × g for 5 minutes, the supernatant was removed and the cells were dispersed in 1 mL of basal medium to obtain a cell suspension. Basal medium was added to an 800 mL cell culture flask with a filter cap (Sumitomo Bakelite), and the cell count was increased to 1.0 × 10 6 The cell suspension was added in an amount equivalent to the number of cells per flask, bringing the total volume to 30 mL. The flasks with filter caps were placed in a 5% (v / v) CO2 incubator at 37°C for cell culture (expansion culture). After cell culture, the medium was removed from the flasks, and the cell detachment solution Accutase was added. TM 5 mL of PBS (Promocell) was added, and the flask was left to stand at room temperature for approximately 5 minutes to detach the cells. The detachment solution containing the cells was then collected and transferred to a tube using basal medium so that the total volume was 15 mL. The tube was centrifuged at 210 × g for 5 minutes, after which the supernatant was removed, and the remaining cells were suspended in 1 mL of KBM ADSC-2 medium containing 1% antibiotics. The number of cells was counted, and the concentration of the cell suspension was adjusted to 1 × 10 6 Adjusted to cells / mL.

[0126] <Defoaming treatment> Separately, the cell culture vessel containing the medium was degassed. Specifically, approximately 1 mL of PBS was added to the cell culture vessel, which was then pipetted and placed in a 5% (v / v) CO2 incubator at 37°C for 15 minutes. After removing any bubbles that had formed in the PBS by pipetting, 0.2 mL of KBM ADSC-2 medium containing 1% antibiotics was added to the cell culture vessel, which was then placed in a 5% (v / v) CO2 incubator at 37°C overnight.

[0127] Example 3 The cell culture vessel used was the same as that used in Example 2.

[0128] After degassing, the medium was removed from the cell culture vessel, and cells were seeded at 500 cells / well. The cell culture vessel was left in a safety cabinet for 15 minutes and then placed in a 5% (v / v) CO2 incubator at 37°C for 4 hours. Next, KBM ADSC-2 medium containing 1% antibiotics was added to the cell culture vessel, and the vessel was placed in a 5% (v / v) CO2 incubator at 37°C for 3 days.

[0129] Spheroids formed in the cell culture vessel were collected by pipetting on the third day of culture. The cell extraction buffer included in the ELISA kit (Abcam) was added to the spheroids and cooled on ice for 20 minutes to obtain a cell extract. The cell extract was then centrifuged at 12,000 × g for 20 minutes, and the supernatant was collected. Integrin αv contained in the supernatant was quantified by ELISA (pg / 1 × 10 5 cells) (n=3).

[0130] Comparative Example 3 Spheroid formation and quantification of integrin αv were performed in the same manner as in Example 3, except that the three-dimensional culture vessel ELPLASIA (manufactured by Kuraray) was used as the cell culture vessel. ELPLASIA is a cell culture vessel that allows suspension culture of cells and has a polystyrene well (depth: approximately 400 μm, opening shape: circular with a diameter of approximately 500 μm, bottom shape: U-shaped).

[0131] Comparative Example 4 A 24-well plate for two-dimensional cell culture (opening shape: circular with a diameter of approximately 1.6 cm, bottom shape: flat bottom, Corning) was used as a cell culture vessel, and cells were cultured at a density of 1 × 10 5 The cells were seeded at a rate of 100 cells / well. The cell culture vessel was left in a safety cabinet for 15 minutes, and then left in a 5% (v / v) CO2 incubator at 37°C for 4 hours. Next, KBM ADSC-2 medium containing 1% antibiotics was added to the cell culture vessel, which was then placed in a 5% (v / v) CO2 incubator at 37°C for another 3 days of culture. On the third day of culture, Accutase TMThe cells were collected by pipetting, then transferred to a 1.5 mL tube (manufactured by AS ONE) and centrifuged at 510 × g for 5 minutes. The supernatant was then discarded. Integrin αv was then quantified using the same method as in Example 3.

[0132] <Result> The expression levels of integrin αv are shown in Figure 12. Values ​​are mean values ​​± standard deviation (n = 3), p is the p-value (probability of significance), and ns indicates not significant. It was revealed that the expression level of integrin αv in the spheroids of Example 3 was significantly higher than those of Comparative Examples 3 and 4 (Figure 12). Specifically, the expression level of integrin αv in Example 3 was 2.8 times higher than that in Comparative Example 3 and 1.9 times higher than that in Comparative Example 4.

[0133] Test Example 5 Fluorescence observation of cell adhesion molecules A photograph of human adipose-derived stem cell spheroids cultured using the cell culture vessel of the present invention, observed with a fluorescence microscope, is shown in Figure 13. Vinculin, one of the integrin scaffolding proteins, was significantly expressed near the cytoskeleton within the cells, revealing that integrins were highly expressed on the surface of the cell membrane and near the bottom of the cell culture sheet to which the cells were adhered. [Industrial Applicability]

[0134] One aspect of the present invention allows for the easy preparation of undifferentiated cell spheroids and for the efficient cell culture process itself, making it suitable for use in fields such as regenerative medicine. Another aspect of the present invention allows for the easy preparation of integrin-expressing cell spheroids, which can adhere well to wound sites and suppress cell death, making it possible to produce integrin-expressing cell spheroids that are expected to have a high therapeutic effect in regenerative medicine, for example. [Explanation of symbols]

[0135] 1 Cell culture sheet 11 Recess 11a Inner surface of recess 11b Bottom surface of recess 12 Sheet surface 21 Substances that exhibit cell non-adhesive properties 22 Substances that exhibit cell adhesive properties 23 Adhesive layer

Claims

1. A method for producing undifferentiated cell spheroids, comprising a step of culturing undifferentiated cells, which are stem cells, on the cell adhesive surface of a cell culture sheet, the cell culture sheet has a plurality of recesses with an opening pore size of 1000 μm or less, the opening pore size / depth of the recesses is 5 / 1 to 1 / 5, the inner side surfaces of the recesses have a non-cell-adhesive surface, and the bottom surfaces of the recesses have a cell-adhesive surface; A method in which the cell adhesive surface comprises a fluorine-containing polyimide resin obtained by thermal imidization of a polyamic acid.

2. A method for maintaining an undifferentiated state of cell spheroids, which are stem cells, comprising culturing the undifferentiated cell spheroids on a cell-adhesive surface of a cell culture sheet, the cell culture sheet has a plurality of recesses with an opening pore size of 1000 μm or less, the opening pore size / depth of the recesses is 5 / 1 to 1 / 5, the inner side surfaces of the recesses have a non-cell-adhesive surface, and the bottom surfaces of the recesses have a cell-adhesive surface; A method in which the cell adhesive surface comprises a fluorine-containing polyimide resin obtained by thermal imidization of a polyamic acid.

3. The method according to claim 1 or 2, wherein the cells are cultured on a flat cell-adherent surface.

4. The method according to any one of claims 1 to 3, wherein the culture is carried out in the absence of feeder cells.

5. The method according to any one of claims 1 to 4, wherein the stem cells are hematopoietic stem cells, mesenchymal stem cells, neural stem cells, tissue stem cells, embryonic stem cells or pluripotent stem cells.

6. The method according to any one of claims 1 to 5, wherein the cells are iPS cells.

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