Method for producing cell spheroids

Abstract

A method for producing undifferentiated cell spheroids, comprising a step of culturing undifferentiated cells on the cell-adhesive surface of a cell culture sheet; a method for maintaining the undifferentiated state of cell spheroids, comprising a step of culturing undifferentiated cell spheroids on the cell-adhesive surface of a cell culture sheet; and cell spheroids obtained by the method.

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 above methods.

Background Art

[0002] In recent years, the use of undifferentiated cells such as iPS cells in regenerative medicine has been increasing, and in clinical applications, studies on techniques for culturing cells while maintaining the undifferentiated state have been actively conducted.

[0003] For example, since the cell culture carrier of Patent Document 1 has specific pores, when seeding undifferentiated cells of stem cells such as ES cells and iPS cells, suction is applied from the back side of the carrier and / or pressure is applied from the front side, and the cells seeded outside the well are induced into the well and efficiently aggregated, so that the cells can be grown while maintaining the undifferentiated state to form spheroids.

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

[0005] Also, in Patent Document 2, somatic cells subjected to reprogramming treatment are seeded in a culture vessel coated with fibronectin and cultured in a serum-free medium without using feeder cells, so that they can be efficiently induced into iPS cells, and the undifferentiated nature and pluripotency of the iPS cells can be maintained for a long period under such culture conditions. When forming embryoid bodies thereafter, a cell low-attachment plate is used as in Non-Patent Document 1.

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

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Non - Patent Documents

[0008]

Non - Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0009] However, although the cell culture carrier of Patent Document 1 can form spheroids, since it is a porous body produced by firing ceramics, it becomes opaque and it is difficult to directly observe the inside of the container. Also, in the containers of Non - Patent Document 1 and Patent Document 2, the surface of the container is not adhesive, and it is difficult to obtain a large amount of cells in order to prepare one spheroid in one well. Furthermore, in the method of Patent Document 3, in order to realize the gravity environment by adjusting the rotation speed of the rotary bioreactor, complicated operations are required, and there are concerns about a decrease in cell function due to shear stress on the cells caused by rotation. Therefore, there is still room for improvement in all of them, and further improved technologies are required.

[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 above methods. Another aspect of the present invention aims to provide a novel method for producing cell spheroids that predominantly express integrin, and integrin-expressing cell spheroids obtained by the above method.

Means for Solving the Problems

[0011] Generally, in order to culture undifferentiated cells such as iPS cells, it is considered necessary to culture them on feeder cells or on a substrate coated with a biological component such as collagen. However, as a result of intensive studies to achieve the above object, the present inventors have found that by culturing on a culture surface having a specific surface, not only undifferentiated cell spheroids can be produced, but also the maintenance of undifferentiatedness becomes possible, and one aspect of the present invention has been completed. In addition, the present inventors have found that by culturing on a culture surface having a specific surface, cell spheroids that predominantly express integrin can be produced as compared with the case of suspension culture on a cell non-adhesive surface, and another aspect of the present invention has been completed.

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

[14] . [1] A method for producing undifferentiated cell spheroids, comprising the 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 the 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], 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 contains a polyimide resin. 〔6〕The method according to any one of 〔1〕 to 〔5〕 above, which is carried out by culturing on a flat cell-adhesive surface. 〔7〕The method according to any one of 〔1〕 to 〔6〕 above, which is carried out by culturing on a cell culture sheet having a plurality of recesses with a pore diameter of the opening of 2000 μm or less in diameter, the inner surface of the recess having a cell-nonadhesive surface, and the bottom surface of the recess having a cell-adhesive surface. 〔8〕The method according to any one of 〔1〕 to 〔7〕 above, wherein the culturing 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 with the spheroid obtained by suspension culture on a cell-nonadhesive surface, the expression level of the undifferentiated marker per cell is 3 times or more as much as the relative gene expression level at the mRNA level. An undifferentiated cell spheroid. 5 〔13〕Compared with the spheroid obtained by suspension culture on a cell-nonadhesive surface, the expression level at the protein level of integrin is 1.2 times or more as much. A cell spheroid. 〔14〕The spheroid according to any one of 〔11〕 to 〔13〕 above, wherein the diameter of the spheroid is 10 to 1500 μm.

Advantages of the Invention

[0013] According to the present invention, a spheroid maintaining an undifferentiated state can be easily and efficiently produced.

Brief Description of the Drawings

[0014]

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Figure 13

BEST MODE FOR CARRYING OUT 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. The undifferentiated cell spheroids mean cell aggregates that can be differentiated and induced into target cells by applying cell growth factors, differentiation inducers, and the like. 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 "cell-adhesive surface" in the present invention refers to, for example, a surface on which cells adhere with a certain number of adhesion points when the cells sediment on the surface in a solution used for culture. Or, it refers to a surface that adheres in such a way that it can be fixed to a degree that allows peeling by a liquid flow such as pipetting. Further, it is not a surface on which cells adhere and are maintained or proliferated two-dimensionally, but a surface that adheres to such an extent that it can form a three-dimensional or three-dimensional tissue structure such as a layered or spheroid shape. By being such a surface, cells or undifferentiated cells adhere moderately on the sheet, and further, dense cell-to-cell communication becomes possible due to appropriate cell-to-cell interactions, and not only proliferate while maintaining the undifferentiated state, but also form spheroids (cell aggregates) (and thus form adherent spheroids that can be peeled off without using drugs such as enzymes at the time of peeling and produce spheroids with adhesiveness). It is presumed to be possible. In addition, cells cultured on such a cell-adhesive surface receive some stimulus different from the normal floating state and highly express integrins that affect cell movement, proliferation, differentiation, and survival. In spheroids with high expression of integrins, cell death is suppressed to the inside of the spheroids by the action of integrins. Such spheroids are presumed to contribute to tissue regeneration because adhesion, spreading, proliferation, and differentiation at the transplantation site can be efficiently performed. Also, high expression of integrins is said to strongly contribute to the suppression of stem cell differentiation, and when integrins are highly expressed when culturing undifferentiated cells, those cells tend to maintain their undifferentiated state. However, the above speculation does not limit the present invention.

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

[0018] The substance showing cell adhesion is not particularly limited as long as it can adhere to the cells used in culture or can bind to cell surface molecules such as proteins and sugar chains present on the cell membrane of the cells used, and can be used. It may be hydrophilic or hydrophobic, but from the viewpoints of cell adhesion, spheroid formation ability, etc., hydrophilic (especially hydrophilic but not superhydrophilic) or hydrophobic (especially hydrophobic but not superhydrophobic) ones are preferred, and hydrophobic ones are more preferred. Also, the degree of adhesion of the substance showing cell adhesion may be such that the cells do not jump out of the concave portion. Examples of such substances include substances obtained from or synthesized by a living body. For example, proteins (such as collagen, fibronectin, laminin, etc.) and synthetic resins (such as fluororesins, polyimide resins, polysulfone, polyethersulfone, polydimethylsiloxane, mixtures thereof, etc.) are included. When selecting a synthetic resin, a cell culture sheet with excellent handleability can be obtained from the strength and heat resistance of the synthetic resin itself. Also, from the viewpoints of biocompatibility, improvement in the uniformity of spheroids, ease of medium exchange operation by moderately adhering to various cells, obtaining adherent spheroids, or improvement in the uniformity of the obtained spheroids, it is preferable to select a synthetic resin such as a polyimide resin. By selecting a non-biological-derived component such as a polyimide resin, the spheroids obtained by the cell culture sheet containing the polyimide resin can be easily applied to fields such as regenerative medicine and drug discovery.

[0019] Examples of the polyimide resin include polyimide resins containing structural units represented by the following formula (I). From the viewpoint of good spheroid formation, resins having 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 a polyamic acid obtained by polymerizing one or more acid dianhydrides and one or more diamines. The polyimide resin may contain a polyamic acid as a part of its chemical structure. As a method for producing the polyimide resin, it may be produced by a known method. For example, a two-step synthesis method can be used. The two-step synthesis method of the polyimide resin is a method of 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 the polyamic acid derivative include polyamic acid salts, polyamic acid alkyl esters, polyamic acid amides, polyamic acid derivatives from bismethylidene pyromellitide, polyamic acid silyl esters, polyamic acid isoimides, and the like. Examples of the polyimide include polyimides composed of acid anhydrides such as pyromellitic dianhydride, biphenyltetracarboxylic dianhydride, benzophenone tetracarboxylic dianhydride, and diamines such as oxydiamine, paraphenylenediamine, metaphenylenediamine, benzophenone diamine. Examples of the resin having a fluorine atom include fluorine-containing polyimide resins containing structural units represented by the following formula (I) such as 4,4'-hexafluoroisopropylidene diphthalic anhydride (6FDA) / 1,4-bis(aminophenoxy)benzene (TPEQ) copolymer, 6FDA / 4,4'-oxydiphthalic anhydride (ODPA) / TPEQ copolymer, 4,4'-(4,4'-isopropylidene diphenoxy)diphthalic acid (BPADA) / 2,2-bis[4-(4-aminophenoxy)phenyl]hexafluoropropane (HFBAPP), 6FDA / 2,2-bis(4-(4-aminophenoxy)phenyl)propane (BAPP) copolymer; ethylene-tetrafluoroethylene copolymer, and the like.

[0020]

Chemical formula

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

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

[0023] Examples of the divalent organic group represented by X 0 specifically include an alkylene group, an arylene group, an aryleneoxy group, an arylenethio group, etc. Further, a condensed ring type divalent hydrocarbon group, a heterocyclic condensed ring type divalent hydrocarbon group, and oxy groups and thio groups thereof may also be used. Among these, an alkylene group, an aryleneoxy group, and an arylenethio group are preferable, an alkylene group and an aryleneoxy group are more preferable, and these may be substituted with fluorine atoms. The number of carbon atoms of the above alkylene group is, for example, 1 to 12, preferably 1 to 6.

[0024] X 0 Examples of the alkylene group substituted with a fluorine atom, which is an example of X, include, for example, -C(CF 3 ) 2 -, -C(CF 3 ) 2 -C(CF 3 ) 2 - and the like. Among the above-described alkylene groups that are examples of X 0 , -C(CF 3 ) 2 - is preferable.

[0025] X 0 Examples of the arylene group that is an example of X include, for example, the following.

[0026]

Chemical formula

[0027] X 0 Examples of the aryloxy group that is an example of X include, for example, the following.

[0028]

Chemical formula

[0029] X 0 Examples of the arylthio group that is an example of X include, for example, the following.

[0030]

Chemical formula

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

[0032] X 0 In the above examples of the arylene group, aryleneoxy group, and arylthio group, each is independently optionally substituted with a group selected from the group consisting of a halogen atom (e.g., a fluorine atom, chlorine atom, bromine atom, iodine atom, preferably a fluorine atom or a chlorine atom, more preferably a fluorine atom), a methyl group, and a trifluoromethyl group. These substituents may be plural, and in that case, the types of substituents may be the same or different from each other. Preferred substituents for the arylene group, aryleneoxy group, and arylthio group are a fluorine atom and / or a trifluoromethyl group, preferably a fluorine atom. When Y does not contain a fluorine atom, the arylene group, aryleneoxy group, and arylthio group are preferably substituted with at least one fluorine atom.

[0033] In the above formula (I), the divalent organic group represented by Y is not particularly limited, and examples thereof include a divalent organic group having an aromatic ring. Specifically, 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 bonded can be mentioned. Specifically, the following groups can be exemplified.

[0034]

Chemical formula

[0035]

Chemical formula

[0036]

Chemical formula

[0037]

Chemical formula

[0038] The divalent organic group having the aromatic ring described above, which is an example of Y, may be substituted, if substitutable, with a group selected from the group consisting of a halogen atom (for example, a fluorine atom, a chlorine atom, a bromine atom, an iodine atom, preferably a fluorine atom or a chlorine atom, more preferably a fluorine atom), a methyl group, and a trifluoromethyl group. A plurality of these substituents may be present, and in that case, the types of the substituents may be the same or different from each other. A preferred substituent substituting the divalent organic group having an aromatic ring is particularly X 0 When the fluorine atom is not included in X, it is preferably a fluorine atom and / or a trifluoromethyl group, 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, 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 from each other, and each independently is selected from a hydrogen atom, a fluorine atom, a chlorine atom, a bromine atom or an iodine atom. When at least one of X 0 and Y does not contain a fluorine atom, at least one of Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , and Z 6 is preferably a fluorine atom.

[0041] From the viewpoint of spheroid formation, in a preferred embodiment of the present invention, in the above formula (I), the divalent organic group represented by X 0 is -C(CF 3 ) 2- selected from the group consisting of 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 The divalent organic group represented by is -C(CF 3 ) 2 - selected from the group consisting of 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] The polyimide resin composed of the structural unit represented by the above formula (I) can be obtained by a method of baking a polyamic acid obtained by polymerization of an acid dianhydride and a diamine. The imidization rate of the above "polyimide resin composed of the structural unit represented by formula (I)" does not have to be 100%. That is, the polyimide resin composed of the structural unit represented by formula (I) may consist only of the structural unit represented by the above formula (I), but within a range where the object and effect of the present invention are not impaired, a part of the structural unit in which the cyclic imide structure remains as an amic acid without dehydration ring closure may be included.

[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 the reaction between the acid dianhydride and diamine as raw materials can proceed efficiently and it is inert to these raw materials. For example, 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, methanol, etc.; non-polar solvents such as toluene and xylene, etc. Among them, it is preferable to use a polar solvent. These organic solvents may be used alone or as a mixture of two or more. The reaction mixture after the amidation reaction may be directly subjected to thermal imidization. The concentration of the polyamic acid in the solution of the polyamic acid is not particularly limited, but from the viewpoints of the polymerization reactivity of the resulting resin composition, the viscosity after polymerization, and the ease of handling in subsequent film formation and firing, it is preferably 5% by weight or more, more preferably 10% by weight or more, preferably 50% by weight or less, and more preferably 40% by weight or less. The viscosity of the resin composition is not particularly limited, but it can be measured according to a known measurement method. For example, it is in 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. The polyimide obtained by thermal imidization has no possibility of residual catalyst and is more preferable for cell culture applications.

[0045] When imidizing by thermal imidization, for example, the polyamic acid is fired in air, or more preferably in an inert gas atmosphere such as nitrogen, helium, argon, or in vacuo, preferably at a temperature of 50 to 400 ° C, more preferably 100 to 380 ° C, preferably for a time of 0.1 to 10 hours, more preferably 0.2 to 5 hours, to perform an imidization reaction, whereby a resin composition containing polyimide can be obtained.

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

[0047] In the case of imidizing by chemical imidization, the polyamic acid can be directly imidized by using a dehydrative cyclization reagent described below in a suitable solvent.

[0048] The dehydrative cyclization reagent can be used without particular limitation as long as it has the effect of chemically dehydrating and cyclizing the polyamic acid to form polyimide. As such a dehydrative cyclization reagent, it is preferable to use a tertiary amine compound alone, or to use a combination of a tertiary amine compound and a carboxylic anhydride, in terms of being able to efficiently promote imidization.

[0049] Examples of the tertiary amine compound 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]nona-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'-tetraethylethylenediamine, etc. Among these, pyridine, DABCO, and N,N,N',N'-tetramethyldiaminomethane are particularly preferred, and DABCO is more preferred. The tertiary amine may be only one kind or two or more kinds.

[0050] Examples of the carboxylic acid anhydride include acetic anhydride, trifluoroacetic anhydride, propionic anhydride, butyric anhydride, isobutyric anhydride, succinic anhydride, maleic anhydride, etc. Among these, acetic anhydride and trifluoroacetic anhydride are particularly preferred, and acetic anhydride is more preferred. The carboxylic acid anhydride may be only one kind or two or more kinds.

[0051] As the solvent for dissolving the polyamic acid in chemical imidization, a polar solvent with excellent solubility is suitable. For example, tetrahydrofuran, N,N-dimethylacetamide, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, etc. are mentioned. Among these, in particular, it is preferable that it is one or more selected from the group consisting of N,N-dimethylacetamide, N,N-dimethylformamide, and N-methylpyrrolidone from the viewpoint of uniform reaction. When these solvents are used as the solvent for the amidation reaction, the polyamic acid can be used directly for chemical imidization without separating it from the reaction mixture after the amidation reaction.

[0052] The weight average molecular weight of the polyimide resin is, for example, from 5,000 to 2,000,000, preferably from 8,000 to 1,000,000, and more preferably from 20,000 to 500,000. In the present specification, the weight average molecular weight of the resin can be measured according to a known measurement method. When the weight average molecular weight is within the above range, the synthesis and handling of the polyimide resin, film formation, and spheroid formation properties are better.

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

[0054] The substance exhibiting cell adhesiveness or the cell-adhesive surface [substance or cell-adhesive surface exhibiting hydrophobic (especially non-superhydrophobic) cell adhesiveness] may have a static water contact angle of 70° or more, a roll-off angle of 15° or more, or both a static water contact angle of 70° or more and a roll-off angle of 15° or more. When the cell-adhesive surface satisfies such conditions, spheroid formation is further promoted. From the viewpoint of spheroid formation properties, the static water contact angle is more preferably more than 75°, further preferably 77° or more, still further preferably 79° or more, and particularly preferably 80° or more (for example, more than 80°). The upper limit of the static water contact angle is, for example, less than 150°, preferably 120° or less (for example, less than 120°), more preferably 110° or less, still further preferably 100° or less (for example, 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 the substance exhibiting cell adhesiveness or the cell-adhesive surface [substance or cell-adhesive surface exhibiting hydrophilic (especially non-superhydrophilic) cell adhesiveness] may be preferably 65° or less, more preferably 55° or less, and still further preferably 50° or less. The lower limit value may be 0° or more, preferably 5° or more, and more preferably 10° or more. From the perspective of spheroid formation, the contact angle of a substance exhibiting cell adhesion or a cell-adhesive surface is preferably higher in the order of 18° or more, 19° or more, 20° or more, 22° or more, 24° or more, 26° or more, 28° or more, 30° or more. The upper limit value of the contact 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] In the cell culture sheet, the cell-adhesive surface may be all or part of the sheet surface, may be partitioned, or may be flat as long as the surface in contact with the cells is the cell-adhesive surface. For example, the entire surface of the cell culture sheet may be a flat cell-adhesive surface. Also, when the cell culture sheet has irregularities on its surface and the cell culture surface is partitioned, it is sufficient that the portion where 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, there may be some irregularities as long as the seeded cells can be stably adhered, and the cells may be adhered over the entire surface regardless of the irregularities.

[0057] The cell culture sheet with a flat cell-adhesive surface may be formed by physically or chemically fixing or disposing a substance exhibiting cell adhesion on the substrate surface, or the sheet itself may be made of a substance exhibiting cell adhesion.

[0058] The immobilization of substances on the substrate surface can be achieved by drying a solution containing these substances on the substrate surface, melting and pressing the substances, curing the substances applied to the substrate with energy rays such as UV, causing a chemical reaction (e.g., a condensation reaction between functional groups such as carboxyl groups and amino groups) between the functional groups of the substances and the functional groups on the substrate to form a covalent bond, or binding the thiol groups of the substances to a metal (platinum, gold, etc.) thin film pre-formed on the substrate. The thickness during immobilization is not particularly limited, and examples include 0.01 to 1000 μm. Also, for sheet formation, for example, on a release sheet with a surface subjected to a peeling treatment (e.g., an organic polymer film such as a polyethylene substrate, ceramics, metal, glass, etc.), the substance is coated to an appropriate thickness by methods such as casting, spray coating, dip coating, spin coating, roll coating, etc., and then heated to form a layer made of such a substance into a sheet shape.

[0059] When the cell culture sheet has irregularities on its surface, it is preferable that cells adhere at the bottom of the recess, and an embodiment in which the bottom is composed of a substance exhibiting cell adhesiveness can be cited. The inner surface of the recess is not particularly limited, and for example, it may be composed of a substance exhibiting non-cell adhesiveness. Also, a cell culture sheet in which a fine pattern is formed between a cell-adhesive surface and a non-cell-adhesive surface may be used. Examples of such a cell culture sheet include a sheet having a layer of a substance exhibiting cell adhesiveness and a fine pattern of a substance exhibiting non-cell adhesiveness provided on the surface of the layer (i.e., a sheet having a mask of a fine pattern of a substance exhibiting non-cell adhesiveness on a layer of a substance exhibiting cell adhesiveness). A cell culture sheet having shallow recesses formed by a fine pattern is presumed to contribute to the functional expression of cells more than a container for culturing cells individually in each well or each cavity such as a microtiter plate or a microplate separated by conventional 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, while the culture medium in the portion above the recesses is shared across the entire sheet, and the soluble factors are shared among all the seeded cells, resulting in cell-cell interactions and improved functional expression of the cells. The formation of the fine pattern may be carried out, for example, by a microcontact printing method, a spin coating method, a casting method, a roll coating method, a die coating method, a gravure coating method, a spray coating method, a bar coating method, a flexographic printing method, a dip coating method, an inkjet method, and a patterning method in which a desired substance is injected into the gaps formed by the uneven structure on the surface of the base material by capillary force generated in the gaps. The substance exhibiting cell non-adhesiveness is not particularly limited as long as it does not bind to cell surface molecules such as proteins and sugar chains present on the cell membrane of the cells to be used, and can be used whether it has biocompatibility or not. Also, it may be hydrophobic or hydrophilic, for example, super water-repellent (super-hydrophobic) or super hydrophilic. From the viewpoints of cell non-adhesiveness, spheroid uniformity, and formability, etc., a substance exhibiting hydrophobicity (especially super-hydrophobicity) [for example, more hydrophobic (especially super-hydrophobic) than a hydrophobic or hydrophilic (especially hydrophobic) cell-adhesive surface or the resin constituting the surface (furthermore its contact angle)] is particularly preferable, but a substance exhibiting hydrophilicity (especially super-hydrophilicity) [for example, more hydrophilic (especially super-hydrophilic) than a hydrophilic or hydrophobic (for example, hydrophobic) cell-adhesive surface or the resin constituting the surface (furthermore its contact angle)] is also preferable. As an example of such a substance, for example, compounds containing polyethylene glycol and its derivatives, MPC (2-methacryloyloxyethyl phosphorylcholine) and its derivatives, HEMA (hydroxyethyl methacrylate) and its derivatives, etc., or polymers of these compounds, compounds containing SPC (segmented polyurethane) and its derivatives, etc., proteins obtained from living organisms (such as albumin), sugar chains to which cells do not adhere (such as agarose, cellulose), etc. can be appropriately selected and used according to the cell type. Among them, from the viewpoints of adhesiveness to synthetic resins, simplifying the manufacturing process of cell culture sheets, or improving the uniformity of the obtained spheroids, MPC and its derivatives or polymers thereof are preferred. In addition, as the substance showing cell non-adhesiveness, a modified one may be appropriately used according to handling properties, the desired degree of hydrophobicity (e.g., superhydrophobicity) · hydrophilicity (e.g., superhydrophilicity), etc. For example, by subjecting a hydrophilic substance to crosslinking treatment or the like, both hydrophilicity and low solubility in water may be achieved. Also, the raw material substance (e.g., hydrophobic or hydrophilic) may be appropriately subjected to hydrophobization treatment or hydrophilization treatment (e.g., introduction of a hydrophobic group or a hydrophilic group) to obtain a material with the desired hydrophobicity or hydrophilicity.

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

[0061] From the viewpoint of making the size of the formed spheroids uniform or improving the circularity, the cell non-adhesive surface can be judged, for example, using the static water contact angle described later as an index for its surface characteristics. For example, when the cell non-adhesive surface is a hydrophobic surface formed of the above-described substances, the static water contact angle is preferably 90° or more, more preferably 93° or more, and even more preferably 95° or more. Also, 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 cell non-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. Also, the static water contact angle may be 0° or more, preferably 5° or more, and more preferably 10° or more. For example, in the case of MPC with high hydrophobicity (or a surface formed of the MPC), a static water contact angle such as 90° or more, 100° or more can be realized. Note that such a static water contact angle may be a value on the cell non-adhesive surface or a value in a substance exhibiting cell non-adhesion (or a substance constituting the cell non-adhesive surface). Also, the static water contact angle may be measured, for example, by the method described later or the like.

[0062] From the viewpoint of improving the size uniformity and circularity of the obtained spheroids, it is preferable to balance the degree of adhesion between the cell adhesive surface and the cell non-adhesive surface. Therefore, even if the cell non-adhesive surface shows adhesiveness to cells, it may have lower adhesiveness than the cell adhesive surface. For example, when using the above static water contact angle as an index, the difference (or its absolute value) in the static water contact angle on the cell non-adhesive surface (or a substance exhibiting cell non-adhesion) is preferably 3° or more (for example, 5° or more), more preferably 10° or more (for example, 12° or more), and even more preferably 15° or more. Also, the upper limit of the above difference (or its absolute value) can be appropriately selected according to the combination of hydrophobicity / hydrophilicity of the cell adhesive surface (or a substance exhibiting cell adhesiveness) and the cell non-adhesive surface (or a substance exhibiting cell non-adhesion), etc., and is not particularly limited, but may be, for example, 100°, 90°, 80°, 70°, 60°, 50°, 40°, 30°, etc.

[0063] As a specific embodiment in which the sheet for cell culture has irregularities on its surface, for example, there can be mentioned a sheet for cell culture having a plurality of recesses with an aperture diameter of 1000 μm or less, the inner surface of the recess having a cell non-adhesive surface, and the bottom surface of the recess having a cell adhesive surface.

[0064] Here, regarding the sheet for cell culture having irregularities on its surface, its structure will be described using an example of a cross-sectional view of the sheet cut in a direction perpendicular to the recess. As shown in the schematic diagram of FIG. 1, there are a plurality of recesses 11 on the sheet surface 12. The recess 11 is composed of an inner surface 11a and a bottom surface 11b, and spheroids are formed within the recess 11.

[0065] The number of recesses 11 on the sheet surface 12 cannot be uniformly set depending on the sheet area, the type of cells to be cultured, etc., and can be appropriately set according to the common general knowledge in the art. For example, the lower limit number per unit area (cm 2 ) can be exemplified as 1, 10, 30, 50, etc., and the upper limit number can be exemplified as 1000, 500, 300, 200, 100, etc. Also, the total number of recesses on the sheet surface can be appropriately set, for example, as 10 or more, 100 or more, 1000 or more, 10000 or more, 50000 or more, etc.

[0066] The shape of the opening of the recess is not limited to a circular shape, and may be, for example, a polygon or an ellipse. The aperture diameter of the opening may be, for example, 2000 μm or less in diameter, and can be appropriately set according to the common general knowledge in the art depending on the size of the cells to be cultured and the size of the desired spheroids. In the present invention, the aperture diameter means the diameter (maximum length) of a circle formed so as to enclose the target location regardless of the shape of the target location, and the aperture diameter of the opening is, for example, the length indicated by D(11) in FIG. 1. Examples of the aperture diameter of the opening include a range of 10 to 2000 μm, 10 to 1000 μm, 10 to 800 μm, 10 to 500 μm.

[0067] The shape of the bottom surface of the recess is not limited to circular, and may be, for example, polygonal or elliptical, and may be the same as or different from the shape of the opening. Also, the bottom surface may be flat (flat bottom) or curved. As the area of the bottom surface, per recess, 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 etc. are exemplified. Also, the pore diameter (length) of the bottom surface may be the same as or different from the pore diameter of the opening, and the pore diameter of the bottom surface may be smaller than the pore diameter of the opening or larger than the pore diameter of the opening. The pore diameter of the bottom surface is, for example, in FIG. 1, the length indicated by DB(11b). As the pore diameter of the bottom surface, for example, ranges within 10~2000μm, 10~1000μm, 10~800μm, 10~500μm, 10~400μm, 10~300μm are exemplified. For example, when the pore diameter of the bottom surface is the same as the pore diameter of the opening, the shape of the recess is a cylindrical shape. When the pore diameter of the bottom surface is smaller than the pore diameter of the opening, the shape of the recess is a tapered shape toward the bottom surface side of the recess.

[0068] The ratio of the pore diameter of the bottom surface to the pore diameter of the opening (pore diameter of the bottom surface / pore diameter of the opening) is not particularly limited, but from the viewpoint of ease of seeding and recovery of cells, 5 / 1~1 / 5, 3 / 1~1 / 3, 1 / 1~1 / 2 are exemplified.

[0069] Also, the distance (gap) between the opening and an adjacent opening is, for example, in FIG. 1, the length indicated by D(12). Although not particularly limited, depending on the desired large-scale culture, for example, 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, 100 μm or less, etc. are exemplified, and any finite value is acceptable.

[0070] The depth of the recess can be appropriately set according to common general knowledge in the art depending on the size of the cells to be cultured. The depth of the recess is, for example, in FIG. 1, the length indicated by D(11a). For example, ranges such as 10 - 1500 μm, 10 - 1000 μm, 10 - 800 μm, 10 - 500 μm, 10 - 300 μm are exemplified. Also, when forming the recess with a fine pattern, the depth of the recess can be less than 10 μm. The lower limit of the depth of the recess in this case may be the minimum thickness at which the cells can recognize non-adhesion. For example, it can be a depth such as 100 nm - 500 nm.

[0071] The ratio of the pore diameter of the opening to the depth of the recess (pore diameter of the opening / depth of the recess) is not particularly limited, but from the viewpoint of ease of seeding and recovering cells, 5 / 1 - 1 / 5, 3 / 1 - 1 / 3, 2 / 1 - 1 / 1 are exemplified. When within the above range, it is difficult for the cells to jump out of the recess, and operations such as cell recovery and defoaming treatment become easier.

[0072] The thickness of the bottom surface of the recess is not particularly limited and can be appropriately set according to common general knowledge in the art.

[0073] The concave portion has a cell non - adhesive surface on its inner surface and a cell - adhesive surface on its bottom surface. Specifically, for example, as shown in FIG. 1, the inner surface 11a has a cell non - adhesive surface and the bottom surface 11b has a cell - adhesive surface. Also, in the cell culture sheet, the sheet surface which is also the edge portion of the concave portion 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 cell non - adhesive surface as the inner surface of the concave portion or a different cell non - adhesive surface. In the case of the same cell non - adhesive surface, the sheet surface and the inner surface of the concave portion have a continuous surface. The ratio of the surface showing cell adhesiveness occupying the bottom surface of the concave portion is not particularly limited, but preferably occupies 90% or more, 95% or more, 99% or more, substantially the entire bottom surface of the bottom surface of the concave portion. The ratio of the cell non - adhesive surface occupying the inner surface of the concave portion is not particularly limited, but is preferably such that it reduces the adhesiveness of the 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 ratio of the surface showing cell non - adhesiveness occupying the sheet surface which is also the edge portion of the concave portion is not particularly limited, but preferably occupies 90% or more, 95% or more, 99% or more, substantially the entire edge portion of the edge portion of the concave portion. The surface showing cell non - adhesiveness preferably occupies 90% or more, 95% or more, 99% or more, substantially the entire edge portion and the inner surface of the sum of the edge portion of the concave portion and the inner surface of the concave portion. For example, as shown in the schematic diagram of FIG. 2, a substance 21 showing cell non - adhesiveness may be fixed to the sheet surface 12 and the inner surface 11a of the concave portion 11. The sheeting and immobilization of the substance can be carried out with reference to the section on the cell culture sheet which is a flat cell - adhesive surface.

[0074] The cell culture sheet having unevenness on its surface has a plurality of the above-described concave structures, but may be a layered structure including a layer containing the bottom surface of the concave portion and a layer containing the inner surface of the concave portion. Here, the layer containing the inner surface of the concave portion is a layer having through-holes in the layer itself. Therefore, as one aspect of the sheet, an aspect of a laminate of a layer having a cell non-adhesive surface with through-holes and a layer having a cell adhesive surface can be cited. For example, as shown in the schematic diagram of FIG. 3, a region including the bottom surface 11b of the concave portion 11 may be formed of a layer made of a substance 22 showing cell adhesiveness, and a layer having a cell non-adhesive surface with through-holes may be formed thereon. For reference, FIGS. 4 and 5 schematically show the spheroid formation state in the concave portion of the cell culture sheet.

[0075] From the viewpoint of forming through-holes or simplifying the production of the cell culture sheet, the layer having a cell non-adhesive surface with through-holes is preferably formed by fixing a substance showing cell non-adhesiveness to a layered base material.

[0076] As the layered base material, any known material in the technical field can be used. For example, plate-like bodies made of synthetic resins such as polystyrene, polyethylene, polypropylene, polycarbonate, polyamide, polyacetal, polyester (such as polyethylene terephthalate), polyurethane, polysulfone, polyacrylate, polymethacrylate, polyvinyl, polycycloolefin, polyether ketone, polyether ether ketone, polyimide, and silicon, synthetic rubbers such as EPDM (Ethylene Propylene Diene Monomer) and natural rubber, and metal materials such as glass, ceramic, and stainless steel can be mentioned. Being a transparent base material is also one of the preferred forms.

[0077] From the viewpoint of workability, it is preferable to perform the fixing of the substance showing cell non-adhesiveness to the layered base material after forming the through-holes described below.

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

[0079] The formation of the through-hole is not particularly limited as long as a through-hole having the above-described size can be formed and can be carried out. For example, it can be formed by drilling (such as a drill), photolithography (laser (for example, CO 2 laser, excimer laser, semiconductor laser, YAG laser, etc.), etching, embossing, etc. In the above processing, the processing may be such that the shape of the through-hole becomes a tapered shape. In that case, the periphery of the end portion is deformed, for example, as shown in FIG. 5, a structure in which the layer thicknesses of the edge portion of the opening and the portion located in the intermediate region between the opening and the adjacent opening are different may be formed.

[0080] The thickness of the layer having the 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 above-described recess.

[0081] The cell culture sheet includes an embodiment in which an adhesive layer (adhesive layer) is further included between the layer having the cell adhesive surface and the layer having the cell non-adhesive surface described above. For example, FIG. 6 shows an example thereof, and an embodiment including an adhesive layer 23 between a layer made of a substance 22 exhibiting cell adhesiveness and a portion where a substance 21 exhibiting cell non-adhesiveness is immobilized is shown.

[0082] As the subsequent layer, any layer known in the relevant technical field can be used. For example, acrylic resins, silicone resins, synthetic rubbers, natural rubbers, etc. can be mentioned, and preferably an adhesive layer with low elution properties can be used. Commercially available double-sided tapes, etc. may also be used.

[0083] The thickness of the adhesive layer is not particularly limited and can be appropriately set as long as the effects of the present invention are not impaired. For example, 0.5 to 100 μm is exemplified.

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

[0085] When laminating each layer, it may be a method of laminating each previously prepared layer in order, or a method of separately forming a layer on a previously prepared layer, or a combination thereof. Specifically, for example, on a release sheet whose surface has been subjected to a peeling treatment (for example, an organic polymer film such as a polyethylene substrate, ceramics, metal, glass, etc.), a substance exhibiting cell adhesiveness is applied to an appropriate thickness by methods such as casting, spray coating, dip coating, spin coating, roll coating, etc. and heated to form a layer having a cell adhesive surface into a sheet shape. On the other hand, after forming through-holes in the substrate of the layer having a cell non-adhesive surface, a substance exhibiting cell non-adhesiveness can be coated on the surface to prepare a layer having a cell non-adhesive surface in advance. Then, after peeling the release sheet of the layer having a cell adhesive surface formed above, it can be manufactured by laminating a separately prepared layer having a cell non-adhesive surface. In the lamination of the layer having a cell non-adhesive surface and the layer having a cell adhesive surface, the above-mentioned adhesive layer (adhesive layer) may be used for lamination, or it may be laminated by welding (high-frequency welding, ultrasonic welding, etc.), pressure bonding (thermal pressure bonding, etc.).

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

[0087] From the viewpoint of directly installing and using the thus obtained sheet for cell culture in a known cell culture device, regardless of the presence or absence of surface irregularities, it may be appropriately sized according to the size of the target device. A culture medium containing cells may be placed on one surface thereof to perform cell culture. The sheet may be housed and fixed in various cell culture containers such as a culture plate, each well of the plate, a culture dish (culture dish), a flask, a culture bag, etc., and a culture medium containing cells may be added to the container so as to cover a part or the entire surface of the fixed sheet to perform cell culture.

[0088] The undifferentiated cells cultured on the cell adhesion surface of the sheet for cell culture are not particularly limited as long as they are undifferentiated cells having the ability to differentiate. Specifically, for example, 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, placental tissue, umbilical cord tissue (umbilical cord blood), peripheral blood, etc.) of humans or animals other than humans (monkeys, pigs, dogs, rabbits, rats, mice, etc.), established cell lines, or cells obtained by genetic manipulation or the like of these can be used. In addition, the cells expressing integrin are not particularly limited as long as they are cells capable of expressing integrin, and may be undifferentiated cells. Examples of such cells can be the same cells as described above.

[0089] More specifically, stem cells and progenitor cells can be mentioned. 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. can be used. As such cells, one type of cell can be used alone, or two or more types of cells can be mixed and used at an arbitrary ratio.

[0090] The medium and culture conditions used in the culture of undifferentiated cells or cells expressing integrin can be appropriately set according to the cells to be used. In the present invention, since the cells are cultured on the above-described cell-adhesive surface, it is not necessary to culture them on feeder cells or on a substrate coated with a biological component such as collagen, and they can be cultured in the absence of feeder cells or components that act on cell adhesion (for example, collagen, hyaluronic acid, Matrigel, laminin, fibronectin, gelatin, etc.). Note that "feeder cells" refer to cells that can prepare a culture environment for undifferentiated cells or cells expressing integrin to be cultured. For example, as feeder cells when the cells to be cultured are human ES cells, mouse fibroblasts can be mentioned. For example, undifferentiated cells or cells are placed on the above-described cell-adhesive surface at 5×10 3 ~3×10 4 cells / cm 2Seeding at a density of, and culturing for about 1 to 7 days in a serum-free medium (for example, StemFit (registered trademark) medium) can form undifferentiated cell spheroids of one aspect of the present invention or integrin-expressing cell spheroids of another aspect of the present invention. When using the cell culture sheet or cell culture container, defoaming treatment may be performed in advance as necessary. The defoaming 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 performed. Preferred are spraying, pipetting, and temperature changes. Also, at the time of cell seeding, a medium containing a Rock inhibitor may be used from the viewpoint of suppressing cell death due to dispersion during seeding. If necessary, cells that have been expanded in advance and then treated with a detachment agent may be used. The detachment agent is not particularly limited, but for example, trypsin, TrypLE TM , Accutase TM and the like can be used.

[0091] The undifferentiated cell spheroids and integrin-expressing cell spheroids thus obtained are not particularly limited in diameter, but are, for example, 10 to 1500 μm, 10 to 1000 μm, 10 to 800 μm, 10 to 600 μm, 10 to 500 μm. Here, the diameter of the spheroid can be measured by a conventional method (for example, image analysis software, particle size distribution meter), and can be expressed as, for example, the hydrodynamic diameter or the equivalent circular diameter. Also, the shape of the spheroid may be spherical or dome-shaped (hemispherical). In the case of a spherical shape, 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 spheroids and integrin-expressing cell spheroids is not particularly limited, and per spheroid, for example, 1×10 1 or more, 1×10 2 or more, 1×10 3 or more, 1×10 4 or more, 1×10 5 or more, 1×10 6 or more, 1×10 7 or more, 1×108 one or more, 1×10 9 It may contain more than that. Also, its upper limit is not particularly set. For example, it may be 1×10 10 or less.

[0093] Although it is not generally determined by the constituent cells of undifferentiated cell spheroids and integrin-expressing cell spheroids, in the present invention, since spheroids are formed on the above-described cell-adhesive surface, the risk of contamination is reduced. For example, it becomes possible to form spheroids that do not contain components derived from serum. General cells adhere to each other and the extracellular matrix in the body, but are always in a floating state in a container for suspension culture. Cells that cannot adhere may undergo apoptosis, and humoral factors contained in serum are required to suppress this. On the other hand, in the method of the present invention, since cells can adhere in the same manner as in the body, humoral factors for suppressing apoptosis are not required. Also, in the present invention, in addition to forming spheroids on the above-described cell-adhesive surface, since the spheroids can be detached and recovered without enzyme treatment, the spheroids themselves have adhesiveness and can be recovered while containing the extracellular matrix. In another aspect according to the present invention, since integrin is expressed, it has excellent cell adhesiveness and can adhere well to a wound site.

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

[0095] Integrin is a protein present on the cell membrane surface and is a cell adhesion molecule, which is a heterodimer consisting of two subunits, an α-chain and a β-chain. Examples of the α-chain of integrin include, but are not limited to, α1, α2, α3, α4, α5, α6, α7, α8, α9, α10, α11, αv, αIIb, etc., and examples of the β-chain include, but are not limited to, β1, β2, β3, β4, β5, β6, etc. Integrins in integrin-expressing cell spheroids only need to detect the expression of at least one protein level among various subunits of the α-chain and β-chain, and can be analyzed by conventional methods (for example, ELISA method, etc.). From the perspective of adhesiveness to the wound site, as the integrin to be detected, at least one of integrin αv, integrin β3, and integrin β6 is preferable. Also, in the case of human adipose-derived stem cells, from the perspective of maintaining undifferentiated state, as the integrin to be detected, at least one of integrin αv, integrin α5, integrin α8, integrin αIIb, and integrin α11 is preferable, and integrin αv is more preferable. For example, the integrin-expressing cell spheroids obtained by another aspect of the present invention have an expression level at the protein level of integrin, for example, 1.2 times or more, preferably 1.5 times or more, more preferably 2 times or more, and even more preferably 3 times or more, compared with the spheroids cultured in suspension on a cell non-adhesive surface. Also, the upper limit is not particularly limited, but for example, about 10 times can be mentioned. 1×10 at the protein level 5 The expression level of integrin-expressing cell spheroids per cell is not particularly limited. However, when detecting integrin αv, for example, it may be 65 pg or more, preferably 70 pg or more, and more preferably 75 pg or more. Also, the upper limit is not particularly limited, but for example, 200 pg can be mentioned.

[0096] In one aspect of the present invention, undifferentiated cell spheroids can be produced. It has also been found that the obtained spheroids can be maintained undifferentiated by culturing them on a cell-adhesive surface. Therefore, the present invention also provides a method for maintaining the undifferentiated state of cell spheroids, which includes a step of culturing undifferentiated cell spheroids on the cell-adhesive surface of a cell culture sheet. In another aspect of the present invention, cell spheroids expressing integrin can be produced. Note that 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 defined in the same manner as the method for producing undifferentiated cell spheroids of the present invention.

[0097] The obtained undifferentiated cell spheroids contain a large number of stable cells retaining undifferentiated properties, so they can be used in research on various tissue and organ development and regeneration, etc. Also, since they can be induced to differentiate into various cells, they can be used in applications to regenerative medicine. Generally, when inducing the differentiation of undifferentiated cells, a cell culture container coated with gelatin or the like is required. However, in one aspect of the present invention, by adding a differentiation inducer, a differentiation induction promoter, etc. to the obtained undifferentiated cell spheroids and culturing them as they are on a cell-adhesive surface, it is also possible to efficiently perform differentiation induction. In addition, the obtained integrin-expressing cell spheroids highly express integrin, so they have excellent cell adhesiveness, can adhere well to the wound site, and cell death is suppressed. In particular, when they are undifferentiated cells, they maintain their undifferentiated state well, so it is possible to produce cell spheroids that are expected to have a high therapeutic effect in regenerative medicine.

Examples

[0098] Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto. In the following examples, room temperature means 20 to 30°C.

[0099] Production Example 1 <Preparation of a layer having a cell adhesion surface (preparation of a fluorine-containing polyimide film)> Into a 100 mL three-necked flask, 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 were charged. After stirring at room temperature under a nitrogen atmosphere and holding for 5 days, a fluorine-containing polyamic acid resin composition (solid content concentration: 15.0% by mass, 6FDA / TPEQ polyamic acid) was obtained. The weight average molecular weight of the polyamic acid resin composition was 180,000, and the viscosity was 14 Pa·s. The weight average molecular weight of the polyamic acid and the weight average molecular weight of the fluorine-containing polyimide after firing were substantially the same.

[0100] The fluorine-containing polyamic acid resin composition obtained above was applied onto a glass substrate using a die coater so that the thickness of the fluorine-containing polyimide film after firing would be 40 μm to form a coating film. Next, the coating film was fired at 360 °C for 1 hour under a nitrogen atmosphere. Thereafter, the fired product was 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 falling angle was 23.4°.

[0101] The measurement methods for the physical properties described above are as follows. (Measurement of weight average molecular weight) Apparatus: HCL-8220GPC manufactured by Tosoh Corporation Column: TSKgel Super AWM-H Eluent (LiBr·H 2 O, NMP containing phosphoric acid): 0.01 mol / L Measurement method: A 0.5 wt% solution is prepared with the eluent, and the molecular weight is calculated based on a calibration curve prepared with polystyrene. (Measurement of viscosity) Apparatus: Viscometer TV-22 manufactured by AS ONE Settings: VI RANGE: H ROTOR No.6 SPEED: 10 rpm Standard solution for viscometer calibration: JS 14000 of Nippon Grease Co., Ltd. Measurement method: After calibration with a standard solution for viscometer calibration, measure using 0.3 g of varnish. (Measurement temperature: 23 °C) (Measurement of static water contact angle) Apparatus: Automatic contact angle meter (manufactured by Kyowa Interface Science Co., Ltd.: DM-500) Measurement method: Measure the adhesion angle of the liquid droplet immediately after dropping 2 μL of water onto the surface (cell non-adhesive surface or cell adhesive surface) or film (film formed of a substance showing cell non-adhesiveness or cell adhesiveness). (Measurement temperature: 25 °C) (Measurement of falling angle) Apparatus: Automatic contact angle meter (manufactured by Kyowa Interface Science Co., Ltd.: DM-500) Measurement method: After dropping 25 μL of water onto the surface (cell non-adhesive surface or cell adhesive surface) or film (film formed of a substance showing cell non-adhesiveness or cell adhesiveness), continuously tilt the sheet, and take the angle at the time of flowing down as the falling angle. (Measurement temperature: 25 °C)

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

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

[0104] <Expansion culture of cells> Cells established in a feeder cell culture system were acclimated to a feeder-free culture system using StemFit® AK02N (Ajinomoto Co., Inc.) and iMatrix-511® (Nippi Co., Ltd.). Cell detachment was performed with an aqueous solution prepared by mixing PBS containing 0.5 mM EDTA and TrypLE Select CTS TM (Thermo Fisher Scientific Inc.) at a ratio of 1:1.

[0105] <Preparation of embryoid bodies (EBs)> The iPS cells recovered with the above-mentioned releasing agent were suspended in StemFit (registered trademark) AK02N medium, and 5.4×10 4 cells / 500 μL / well (2.7×10 4 cells / cm 2 ) were seeded. At the time of seeding, a medium supplemented with 10 μL of Y-27632 was used. One day after seeding, half of the medium was replaced, and four days later, all of the medium was replaced.

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

[0107] Test Example 1 Microscopic Observation On the 1st, 3rd, 5th, and 7th days of culture, the morphology of the EBs was observed under a microscope. The results of Example 1 are shown in Fig. 7, and the results of Comparative Example 1 are shown in Fig. 8.

[0108] Test Example 2 Evaluation of the Undifferentiated Potential of EBs Seven days after seeding the iPS cells, the EBs were recovered, and RNA was extracted using the RNeasy Mini Kit (Qiagen). Subsequently, cDNA synthesis was performed using ReverTra Ace (registered trademark) qPCR RT Master Mix (Toyobo). Real-time PCR measurement was performed using primers and probes for POU5F1, which is an undifferentiated marker, against POU5F1 of TaqMan TM Gene Expression Assay (FAM) (Thermo Fisher Scientific) (n = 3). The StepOnePlus (Thermo Fisher Scientific) was used as the apparatus. GAPDH was selected as the housekeeping gene. The obtained data were corrected with GAPDH, and the relative gene expression levels were determined by the ΔΔCt method. The results are shown in Fig. 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 size of the EBs was observed to increase to about 400 μm (Figure 7). On the other hand, in Comparative Example 1, EBs (about 500 μm in size) were formed on the first day of culture but did not adhere to the bottom of the well and sank in a non-adherent state on the bottom of the well. Also, on the fifth day, the size increased to about 600 μm, and at the same time, the morphology changed from circular to irregular (Figure 8).

[0110] Also, it was revealed that the amount of POU5F1, an undifferentiated marker in the EBs prepared in Example 1, was significantly higher than that in the non-adherent EBs prepared in Comparative Example 1 (Figure 9). Specifically, it was found that the EBs prepared in Example 1 were 6.7 times more than those prepared in Comparative Example 1 (P < 0.01).

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

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

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

[0114] <Expansion culture of cells> The frozen cells were thawed in a 37°C constant temperature water bath and added to 9 mL of KBM ADSC-2 medium (basal medium, manufactured by Cosmo Bio Co., Ltd.) containing 5% FBS and 1% antibiotic. Next, after centrifugation at 210×g for 5 minutes, the supernatant was removed and dispersed in 1 mL of the basal medium. Two culture flasks (culture area 225 cm 2 ) were prepared by adding to obtain 1.0×10 6 cells / 30 mL medium / dish, and cultured (expansion cultured) in a 37°C 5% (v / v) CO 2 incubator.

[0115] <Defoaming treatment> Separately, the culture container was defoamed. Specifically, about 1 mL of PBS was added to the container, pipetting was performed, and the container was left standing in a 37°C 5% (v / v) CO 2 incubator for 15 minutes. Next, after pipetting again, the PBS was aspirated with an aspirator, 0.2 mL of KBM ADSC-2 medium containing 1% antibiotic was added, and the container was left standing in a 37°C 5% (v / v) CO 2 incubator overnight.

[0116] <Preparation of spheroids> The medium was removed from the culture dish, 5 mL of the cell detachment solution Accutase (manufactured by PromoCell) was added, and then the cells were detached by holding them in a 5% (v / v) CO 2 incubator at 37°C for about 5 minutes. Next, the detachment solution was collected, 10 mL of PBS was added for washing, and the mixture was transferred to a tube. Centrifugation was performed at 210×g for 5 minutes, and the cells were suspended in 4 mL of KBM ADSC-2 medium containing 1% antibiotic, and the cell count was performed. Then, 1.0×10 6 cells / mL was prepared.

[0117] 5% (v / v) CO at 37°C 2 The medium in the culture vessel that had been left standing overnight in a 5% (v / v) CO incubator at 37°C to perform degassing treatment was removed, and the cells were seeded at 500 cells / well. After leaving them standing in a safety cabinet for 15 minutes, 5% (v / v) CO at 37°C 2 They were placed in an incubator and left standing for 4 hours. Next, KBM ADSC-2 medium containing an additional 1% antibiotic was added, and they were again placed in a 5% (v / v) CO 2 incubator and cultured for 3 days.

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

[0119] Reference Example 1 Instead of the cell culture vessel used in Example 2, a 24-well plate for cell culture was used. KBM ADSC-2 medium containing 1% antibiotic was added to each well, AdSCs were seeded, and they were placed in a 5% (v / v) CO 2 incubator and cultured for 3 days.

[0120] Test Example 3 Implementation of Protein Array The culture supernatant was collected 3 days after culturing. The proteins contained in this supernatant were evaluated using RayBio human antibody array kits (L-507, L493, RayBiotech). The measurement was carried out by Cosmo Bio Co., Ltd. The obtained values were normalized with the values of positive controls according to the protocol, and further normalized by the number of cells ( / 1×10 5 cells). Also, according to the protocol, when the difference in numerical values between samples was 1.5 times or more, or 0.65 times or less, a significant difference was considered to exist between those samples.

[0121] It was revealed that the amounts of Nanog, Oct 3 / 4, SSEA-4, and SOX2, which are undifferentiated markers in the spheroids prepared in Example 2, were significantly higher than those in the non-adherent spheroids prepared in Comparative Example 2 and the two-dimensional cultured cells prepared in Reference Example 1 (Figure 11). Specifically, it was found that the amounts of Nanog, Oct 3 / 4, SSEA-4, and SOX2 in the spheroids prepared in Example 2 were 2.7, 2.8, 1.8, and 4.7 times higher, respectively, than those in the spheroids prepared in Comparative Example 2 (Table 1). Also, it was found that the amounts of Nanog, Oct 3 / 4, SSEA-4, and SOX2 in the spheroids prepared in Example 2 were 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 same cells as those used in Example 2 were used.

[0125] <Cell Preparation> The frozen cells were thawed in a 37°C water bath and added to 9 mL of KBM ADSC-2 medium (basic medium, manufactured by Cosmo Bio Co., Ltd.) containing 5% FBS and 1% antibiotic. Subsequently, after centrifugation at 210×g for 5 minutes, the supernatant was removed and the cells were resuspended in 1 mL of the basic medium to obtain a cell suspension. The basic medium was added to an 800 mL Erlenmeyer flask with a filter cap for cell culture (manufactured by Sumitomo Bakelite Co., Ltd.), and the cell suspension was added in an amount such that the cell density became 1.0×10 6 cells / flask, and the total volume was adjusted to 30 mL. The Erlenmeyer flask with a filter cap was incubated in a 37°C 5% (v / v) CO 2 incubator for cell culture (expansion culture). After cell culture, the medium was removed from the flask, and 5 mL of Accutase TM (Promocell) was added, and the flask was left standing at room temperature for about 5 minutes to detach the cells. Subsequently, the detached cell-containing solution was collected and transferred to a tube using the basic medium to make the total volume 15 mL. After centrifuging the tube at 210×g for 5 minutes, the supernatant was removed, and 1 mL of KBM ADSC-2 medium containing 1% antibiotic was added to the remaining cells and suspended. The number of cells was counted and the concentration of the cell suspension was adjusted to 1×10 6 cells / mL.

[0126] <Defoaming treatment> Separately, defoaming treatment of the cell culture container containing the medium was performed. Specifically, about 1 mL of PBS was added to the cell culture container and pipetting was carried out, and the container was left standing in a 37°C 5% (v / v) CO 2 incubator for 15 minutes. Subsequently, the newly generated bubbles in the PBS were removed by pipetting, and then 0.2 mL of KBM ADSC-2 medium containing 1% antibiotic was added to the cell culture container, and the cell culture container was left standing overnight in a 37°C 5% (v / v) CO 2 incubator.

[0127] Example 3 As the cell culture container, the same one as used in Example 2 was used.

[0128] After removing the medium from the cell culture vessel after defoaming treatment, cells were seeded in an amount of 500 cells / well. After leaving the cell culture vessel standing in a safety cabinet for 15 minutes, it was left standing in an incubator at 37°C with 5% (v / v) CO 2 for 4 hours. Next, KBM ADSC-2 medium containing 1% antibiotic was added to the cell culture vessel, and it was placed again in an incubator at 37°C with 5% (v / v) CO 2 and cultured for 3 days.

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

[0130] Comparative Example 3 Except for using a three-dimensional culture vessel ELPLASIA (manufactured by Kuraray) as the cell culture vessel, spheroid formation and quantification of integrin αv were performed in the same manner as in Example 3. ELPLASIA is a cell culture vessel capable of suspension culture of cells, and has wells (depth: about 400 μm, shape of the opening: circular with a diameter of about 500 μm, shape of the bottom surface: U-shaped bottom) made of polystyrene.

[0131] Comparative Example 4 As the cell culture vessel, a 24-well plate for two-dimensional cell culture (shape of the opening: circular with a diameter of about 1.6 cm, shape of the bottom surface: flat bottom, manufactured by Corning) was used, and cells were seeded in an amount of 1×10 5 cells / well. After leaving the cell culture vessel standing in a safety cabinet for 15 minutes, it was left standing in an incubator at 37°C with 5% (v / v) CO 2 for 4 hours. Next, KBM ADSC-2 medium containing 1% antibiotic was added to the cell culture vessel, and it was placed again in an incubator at 37°C with 5% (v / v) CO 2 and cultured for 3 days. On the third day of culture, AccutaseTM Add 0.5 mL and let stand at room temperature for 5 minutes. After collecting the cells by pipetting, transfer them to a 1.5 mL tube (manufactured by AS ONE), centrifuge at 510×g for 5 minutes, and then discard the supernatant. Thereafter, the quantification of integrin αv was performed in the same manner as in Example 3.

[0132] <Results> The expression level of integrin αv is shown in Fig. 12. The values are mean ± standard deviation (n = 3), p is the p-value (significance probability), and n.s. indicates not significant. It was revealed that the expression level of integrin αv in the spheroids of Example 3 was significantly higher than that in Comparative Example 3 and Comparative Example 4 (Fig. 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 Fluorescent Observation of Cell Adhesion Molecules Fig. 13 shows a photograph of spheroids of human adipose-derived stem cells cultured using the cell culture container of the present invention observed under a fluorescence microscope. Since Vinculin, which is one of the proteins underlying integrin, is prominently expressed near the cytoskeleton inside the cells, it was revealed that integrin is highly expressed on the cell membrane surface and near the bottom surface of the cell culture sheet to which the cells are adhered.

Industrial Applicability

[0134] According to one aspect of the present invention, undifferentiated cell spheroids can be easily prepared, and the cell culture work itself can also be efficiently performed. Therefore, it can be suitably used in fields such as regenerative medicine, for example. According to another aspect of the present invention, integrin-expressing cell spheroids can be easily prepared, can adhere well to the wound site, and cell death can be suppressed. Therefore, for example, integrin-expressing cell spheroids that are expected to have a high therapeutic effect in regenerative medicine can be produced.

Explanation of Symbols

[0135] 1 Sheet for cell culture 11 Recess 11a Inner surface of the recess 11b Bottom surface of the recess 12 Sheet surface 21 Substance showing cell non - adhesiveness 22 Substance showing cell adhesiveness 23 Adhesive layer

Claims

**Claim 1** A method for producing undifferentiated cell spheroids, comprising the step of culturing undifferentiated cells, which are iPS cells, on a cell-adhesive surface of a cell culture sheet, wherein the cell-adhesive surface comprises a fluorine-containing polyimide resin obtained by thermal imidization of polyamic acid. **Claim 2** A method for maintaining the undifferentiated state of cell spheroids, comprising the step of culturing undifferentiated cell spheroids, which are iPS cells, on a cell-adhesive surface of a cell culture sheet, wherein the cell-adhesive surface comprises a fluorine-containing polyimide resin obtained by thermal imidization of polyamic acid. **Claim 3** The method according to claim 1 or 2, wherein the culturing is performed on a flat cell-adhesive surface. **Claim 4** The method according to any one of claims 1 to 3, wherein the culturing is performed on a cell culture sheet having a plurality of recesses with a pore diameter of the opening of 1000 μm or less in diameter, an inner surface of the recess having a cell-nonadhesive surface, and a bottom surface of the recess having a cell-adhesive surface. **Claim 5** The method according to any one of claims 1 to 4, wherein the culturing is performed in the absence of feeder cells.

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